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Nucleofection of phiC31 Integrase Protein Mediates Sequence-Specific Genomic Integration in Human Cells. Journal of molecular biology Guha, T. K., Calos, M. P. 2020
Abstract

The phage-derived phiC31 integrase is a useful tool for mediating sequence-specific genomic integration in mammalian cells, recombining donor plasmids bearing the attB recognition site with introduced genomic attP sites or endogeneous pseudo-attP sites having partial identity to attP. In most prior studies, phiC31 integrase has been introduced as plasmid DNA or mRNA. The current report examines whether phiC31 integrase functions efficiently in mammalian cells when co-nucleofected as a purified protein, along with attB-containing donor plasmids or PCR fragments. We describe preparation of phiC31 integrase protein and evidence that it can mediate genomic integration in human 293 cells, including PCR evidence for integration at an endogenous pseudo-attP site. This work demonstrates for the first time the ability of 605- and 613-amino acid versions of phiC31 integrase protein to mediate efficient, site-specific integration into the genome of human cells when co-nucleofected with full-sized attB-containing donor plasmids or linear 2.5-kb PCR fragments. This protein-mediated approach may be especially useful for integration of exogenous sequences into valuable therapeutic target cells, such as hematopoietic stem cells or T cells, that are sensitive to introduced DNA.

View details for DOI 10.1016/j.jmb.2020.04.019

View details for PubMedID 32339531
Plasmid-Mediated Gene Therapy in Mouse Models of Limb Girdle Muscular Dystrophy. Molecular therapy. Methods & clinical development Guha, T. K., Pichavant, C., Calos, M. P. 2019; 15: 294?304
Abstract

We delivered plasmid DNA encoding therapeutic genes to the muscles of mouse models of limb girdle muscular dystrophy (LGMD) 2A, 2B, and 2D, deficient in calpain3, dysferlin, and alpha-sarcoglycan, respectively. We also delivered the human follistatin gene, which has the potential to increase therapeutic benefit. After intramuscular injection of DNA, electroporation was applied to enhance delivery to muscle fibers. When plasmids encoding the human calpain3 or dysferlin cDNA sequences were injected into quadriceps muscles of LGMD2A and LGMD2B mouse models, respectively, in 3-month studies, robust levels of calpain3 and dysferlin proteins were detected. We observed a statistically significant decrease in Evans blue dye penetration in LGMD2B mouse muscles after delivery of the dysferlin gene, consistent with repair of the muscle membrane defect in these mice. The therapeutic value of delivery of the genes for alpha-sarcoglycan and follistatin was documented by significant drops in Evans blue dye penetration in gastrocnemius muscles of LGMD2D mice. These results indicated for�the first time that a combined gene therapy involving both alpha-sarcoglycan and follistatin would be valuable for LGMD2D patients. We suggest that this non-viral gene delivery method should be explored for its translational potential in patients.

View details for DOI 10.1016/j.omtm.2019.10.002

View details for PubMedID 31890729

View details for PubMedCentralID PMC6923511
Lack of RAC. Molecular therapy : the journal of the American Society of Gene Therapy Calos, M. P. 2018
View details for PubMedID 30245159
Genomic integration of the full-length dystrophin coding sequence in Duchenne muscular dystrophy induced pluripotent stem cells BIOTECHNOLOGY JOURNAL Farruggio, A. P., Bhakta, M. S., du Bois, H., Ma, J., Calos, M. P. 2017; 12 (4)
View details for DOI 10.1002/biot.201600477

View details for Web of Science ID 000400612400003
Genome Editing Techniques and Their Therapeutic Applications CLINICAL PHARMACOLOGY & THERAPEUTICS Calos, M. P. 2017; 101 (1): 42-51
View details for DOI 10.1002/cpt.542

View details for Web of Science ID 000391927400017
Knock-in Blunt Ligation Utilizing CRISPR/Cas9 bio-protocol Geisinger, J. M., Calos, M. P. 2017; 7 (05, Mar 05, 2017)
Three novel immune-deficient mouse models of muscular dystrophy PLOS Currents Muscular Dystrophy Pichavant, C., Bjornson, C. R., Gallagher, T., Neal, T. L., du Bois, H., Bhakta, M., Calos, M. P. 2017
View details for DOI 10.1371/currents.md.25e9cd5f4740329f2650972a75230188
Use of the DICE (Dual Integrase Cassette Exchange) System. Methods in molecular biology (Clifton, N.J.) Farruggio, A. P., Bhakta, M. S., Calos, M. P. 2017; 1642: 69?85
Abstract

When constructing transgenic cell lines via plasmid DNA integration, precise targeting to a desired genomic location is advantageous. It is also often advantageous to remove the bacterial backbone, since bacterial elements can lead to the epigenetic silencing of neighboring DNA. The least cumbersome method to remove the plasmid backbone is recombinase-mediated cassette exchange (RMCE). RMCE is accomplished by arranging recombinase sites in the genome and in a donor plasmid such that a recombinase can both integrate the donor plasmid and excise its bacterial backbone. When a single recombinase is used for RMCE, recombination between undesired pairings of the sites can lead to a significant number of unwanted cell lines. To reduce the frequency with which these side products occur, several variants of RMCE that increase desired outcomes have been developed. Nevertheless, an important feature lacking from these improved RMCE methods is that none have fully utilized the recombinases that have been demonstrated to be the most robust and stringent at performing genomic integration in plants and animals, i.e., the phiC31 and Bxb1 phage integrases. To address this need, we have developed an RMCE protocol using these two serine integrases that we call dual integrase cassette exchange (DICE). Our DICE system provides a means to achieve high-precision DNA integration at a desired location and is especially well suited for repeated recombination into the same locus. In this chapter, we provide our most current protocols for using DICE in feeder-free human-induced pluripotent stem cells .

View details for PubMedID 28815494
DNA-Mediated Gene Therapy in a Mouse Model of Limb Girdle Muscular Dystrophy 2B. Molecular therapy. Methods & clinical development Ma, J., Pichavant, C., du Bois, H., Bhakta, M., Calos, M. P. 2017; 7: 123?31
Abstract

Mutations in the gene for dysferlin cause a degenerative disorder of skeletal muscle known as limb girdle muscular dystrophy 2B. To achieve gene delivery of plasmids encoding dysferlin to hind limb muscles of dysferlin knockout mice, we used a vascular injection method that perfused naked plasmid DNA into all major muscle groups of the hind limb. We monitored delivery by luciferase live imaging and western blot, confirming strong dysferlin expression that persisted over the 3-month time course of the experiment. Co-delivery of the follistatin gene, which may promote muscle growth, was monitored by ELISA. Immunohistochemistry documented the presence of dysferlin in muscle fibers in treated limbs, and PCR confirmed the presence of plasmid DNA. Because dysferlin is involved in repair of the sarcolemmal membrane, dysferlin loss leads to fragile sarcolemmal membranes that can be detected by permeability to Evan's blue dye. We showed that after gene therapy with a plasmid encoding both dysferlin and follistatin, statistically significant reduction in Evan's blue dye permeability was present in hamstring muscles. These results suggest that vascular delivery of plasmids carrying these therapeutic genes may lead to simple and effective approaches for improving the clinical condition of limb girdle muscular dystrophy 2B.

View details for PubMedID 29159199

View details for PubMedCentralID PMC5684445
In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining NUCLEIC ACIDS RESEARCH Geisinger, J. M., Turan, S., Hernandez, S., Spector, L. P., Calos, M. P. 2016; 44 (8)
Abstract

The CRISPR/Cas9 system facilitates precise DNA modifications by generating RNA-guided blunt-ended double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting these breaks to insert exogenous PCR-generated sequences in a homology-independent manner without loss of additional nucleotides. This method is useful for making precise additions to the genome such as insertions of marker gene cassettes or functional elements, without the need for homology arms. We successfully utilized this method in human and mouse cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 36% in HEK293 cells without selection. We also created versions of Cas9 fused to the FKBP12-L106P destabilization domain in an effort to improve Cas9 performance. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches.

View details for DOI 10.1093/nar/gkv1542

View details for Web of Science ID 000376389000007

View details for PubMedID 26762978

View details for PubMedCentralID PMC4856974
The CRISPR Way to Think about Duchenne's NEW ENGLAND JOURNAL OF MEDICINE Calos, M. P. 2016; 374 (17): 1684-1686
View details for DOI 10.1056/NEJMcibr1601383

View details for Web of Science ID 000374843400013

View details for PubMedID 27119241
Precise Correction of Disease Mutations in Induced Pluripotent Stem Cells Derived From Patients With Limb Girdle Muscular Dystrophy. Molecular therapy : the journal of the American Society of Gene Therapy Turan, S., Farruggio, A. P., Srifa, W., Day, J. W., Calos, M. P. 2016; 24 (4): 685-696
Abstract

Limb girdle muscular dystrophies types 2B (LGMD2B) and 2D (LGMD2D) are degenerative muscle diseases caused by mutations in the dysferlin and alpha-sarcoglycan genes, respectively. Using patient-derived induced pluripotent stem cells (iPSC), we corrected the dysferlin nonsense mutation c.5713C>T; p.R1905X and the most common alpha-sarcoglycan mutation, missense c.229C>T; p.R77C, by single-stranded oligonucleotide-mediated gene editing, using the CRISPR/Cas9 gene-editing system to enhance the frequency of homology-directed repair. We demonstrated seamless, allele-specific correction at efficiencies of 0.7-1.5%. As an alternative, we also carried out precise gene addition strategies for correction of the LGMD2B iPSC by integration of wild-type dysferlin cDNA into the H11 safe harbor locus on chromosome 22, using dual integrase cassette exchange (DICE) or TALEN-assisted homologous recombination for insertion precise (THRIP). These methods employed TALENs and homologous recombination, and DICE also utilized site-specific recombinases. With DICE and THRIP, we obtained targeting efficiencies after selection of ~20%. We purified iPSC corrected by all methods and verified rescue of appropriate levels of dysferlin and alpha-sarcoglycan protein expression and correct localization, as shown by immunoblot and immunocytochemistry. In summary, we demonstrate for the first time precise correction of LGMD iPSC and validation of expression, opening the possibility of cell therapy utilizing these corrected iPSC.

View details for DOI 10.1038/mt.2016.40

View details for PubMedID 26916285
Phage Integrases for Genome Editing GENOME EDITING: THE NEXT STEP IN GENE THERAPY Calos, M. P. 2016; 895: 81-91
View details for DOI 10.1007/978-1-4939-3509-3_5

View details for Web of Science ID 000385740200006
Using phage integrases in a site-specific dual integrase cassette exchange strategy. Methods in molecular biology (Clifton, N.J.) Geisinger, J. M., Calos, M. P. 2015; 1239: 29-38
Abstract

?C31 integrase, a site-specific large serine recombinase, is a useful tool for genome engineering in a variety of eukaryotic species and cell types. ?C31 integrase performs efficient recombination between its attB site and either its own placed attP site or a partially mismatched genomic pseudo attP site. Bxb1 integrase, another large serine recombinase, has a similar level of recombinational activity, but recognizes only its own attB and attP sites. Previously, we have used these integrases sequentially to integrate plasmid DNA into the genome. This approach relied on placing a landing pad attP for Bxb1 integrase in the genome by using phiC31 integrase-mediated recombination at a genomic pseudo attP site. In this chapter, we present a protocol for using these integrases simultaneously to facilitate cassette exchange at a predefined location. This approach permits greater control and accuracy over integration. We also present a general method for using polymerase chain reaction assays to verify that the desired cassette exchange occurred successfully.

View details for DOI 10.1007/978-1-4939-1862-1_3

View details for PubMedID 25408400
Nonverbal genome modification strategies for gene therapy: Transposon, integrase, and nuclease systems Gene and Cell Therapy Therapeutic Mechanisms and Strategies Woodard, L. E., Calos, M. P. CRC Press. 2015; 4: 675?699
Recombinase-Mediated Reprogramming and Dystrophin Gene Addition in mdx Mouse Induced Pluripotent Stem Cells PLOS ONE Zhao, C., Farruggio, A. P., Bjornson, C. R., Chavez, C. L., Geisinger, J. M., Neal, T. L., Karow, M., Calos, M. P. 2014; 9 (4)
View details for DOI 10.1371/journal.pone.0096279

View details for Web of Science ID 000335504900028

View details for PubMedID 24781921
DICE, an efficient system for iterative genomic editing in human pluripotent stem cells. Nucleic acids research Zhu, F., Gamboa, M., Farruggio, A. P., Hippenmeyer, S., Tasic, B., Sch�le, B., Chen-Tsai, Y., Calos, M. P. 2014; 42 (5)
View details for DOI 10.1093/nar/gkt1290

View details for PubMedID 24304893
Serine integrase chimeras with activity in E. coli and HeLa cells. Biology open Farruggio, A. P., Calos, M. P. 2014; 3 (10): 895-903
Abstract

In recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy that has been successful with several small serine recombinases. We created chimeras derived from three characterized members of the serine integrase family, phiC31, phiBT1, and TG1 integrases, by joining their amino- and carboxy-terminal portions. We found that several phiBT1-phiC31 (BC) and phiC31-TG1 (CT) hybrid integrases are active in E. coli. BC chimeras function on native att-sites and on att-sites that are hybrids between those of the two donor enzymes, while CT chimeras only act on the latter att-sites. A BC hybrid, BC{-1}, was also active in human HeLa cells. Our work is the first to demonstrate chimeric serine integrase activity. This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

View details for DOI 10.1242/bio.20148748

View details for PubMedID 25217617

View details for PubMedCentralID PMC4197438
Generating Dystrophin plus Myogenic Cells from mdx Fibroblasts by Using a Triple-Recombinase iPS Cell-Based Strategy 16th Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT) Geisinger, J. M., Zhao, C., Farrugio, A. P., Chavez, C. L., Bjornson, C. R., Neal, T. L., Calos, M. P. NATURE PUBLISHING GROUP. 2013: S68?S69
View details for Web of Science ID 000319858400174
Translating the Genomics Revolution: The Need for an International Gene Therapy Consortium for Monogenic Diseases MOLECULAR THERAPY Tremblay, J. P., Xiao, X., Aartsma-Rus, A., Barbas, C., Blaus, H. M., Bogdanove, A. J., Boycott, K., Brauns, S., Breakefield, X. O., Bueren, J. A., Buschmann, M., Byrne, B. J., Calos, M., Cathomen, T., Chamberlain, J., Chuah, M., Cornetta, K., Davies, K. E., Dickson, J. G., Duchateau, P., Flotte, T. R., Gaudet, D., Gersbach, C. A., Gilbert, R., Glorioso, J., Herzog, R. W., High, K. A., Huang, W., Huard, J., Joung, J. K., Liu, D., Liu, D., Lochmueller, H., Lustig, L., Martens, J., Massie, B., Mavilio, F., Mendell, J. R., Nathwani, A., Ponder, K., Porteus, M., Puymirat, J., Samulski, J., Takeda, S., Thrasher, A., Vandendriessche, T., Wei, Y., Wilson, J. M., Wilton, S. D., Wolfe, J. H., Gao, G. 2013; 21 (2): 266-268
View details for DOI 10.1038/mt.2013.4

View details for Web of Science ID 000314434600002

View details for PubMedCentralID PMC3594019
Translating the genomics revolution: the need for an international gene therapy consortium for monogenic diseases. Molecular therapy : the journal of the American Society of Gene Therapy Tremblay, J. P., Xiao, X., Aartsma-Rus, A., Barbas, C., Blau, H. M., Bogdanove, A. J., Boycott, K., Braun, S., Breakefield, X. O., Bueren, J. A., Buschmann, M., Byrne, B. J., Calos, M., Cathomen, T., Chamberlain, J., Chuah, M., Cornetta, K., Davies, K. E., Dickson, J. G., Duchateau, P., Flotte, T. R., Gaudet, D., Gersbach, C. A., Gilbert, R., Glorioso, J., Herzog, R. W., High, K. A., Huang, W., Huard, J., Joung, J. K., Liu, D., Liu, D., Lochm�ller, H., Lustig, L., Martens, J., Massie, B., Mavilio, F., Mendell, J. R., Nathwani, A., Ponder, K., Porteus, M., Puymirat, J., Samulski, J., Takeda, S., Thrasher, A., Vandendriessche, T., Wei, Y., Wilson, J. M., Wilton, S. D., Wolfe, J. H., Gao, G. 2013; 21 (2): 266-268
View details for DOI 10.1038/mt.2013.4

View details for PubMedID 23369965

View details for PubMedCentralID PMC3594019
Site-Specific Recombination Using PhiC31 Integrase Site-directed Insertion of Transgenes Geisinger, J. M., Calos, M. P. Springer Science+Business Media Dordrecht. 2013: 211?239
View details for DOI 10.1007/978-94-007-4531-5_8
Efficient reversal of phiC31 integrase recombination in mammalian cells BIOTECHNOLOGY JOURNAL Farruggio, A. P., Chavez, C. L., Mikell, C. L., Calos, M. P. 2012; 7 (11)
Abstract

Over the past decade, the integrase enzyme from phage phiC31 has proven to be a useful genome engineering tool in a wide variety of species, including mammalian cells. The enzyme efficiently mediates recombination between two distinct sequences, attP and attB, producing recombinant product sites, attL and attR. The reaction proceeds exclusively in a unidirectional manner, because integrase is unable to synapse attL and attR. To date, use of phiC31 integrase has been limited to attP � attB recombination. The factor needed for the reverse reaction--the excisionase or recombination directionality factor (RDF)--was identified recently and shown to function in vitro and in bacterial cells. To determine whether the phiC31 RDF could also function in mammalian cells, we cloned and tested several vectors that permit assessment of phiC31 RDF activity in mammalian environments. In the human and mouse cell lines tested (HeLa, HEK293, and NIH3T3), we observed robust RDF activity, using plasmid and/or genomic assays. This work is the first to demonstrate attL-attR serine integrase activity in mammalian cells and validates phiC31 RDF as a new tool that will enable future studies to take advantage of phiC31 integrase recombination in the forward or reverse direction.

View details for DOI 10.1002/biot.201200283

View details for PubMedID 22933343
Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Technique Meeting of the American-Heart-Association Lan, F., Liu, J., Narsinh, K. H., Hu, S., Han, L., Lee, A. S., Karow, M., Nguyen, P. K., Nag, D., Calos, M. P., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2012: S20-?
Abstract

Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts.We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging.The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

View details for DOI 10.1161/CIRCULATIONAHA.111.084913

View details for Web of Science ID 000314150200003

View details for PubMedID 22965984

View details for PubMedCentralID PMC3481839
Recombinase Strategies To Make and Modify iPS Cells 15th Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT) Chavez, C. L., Zhao, C., Farruggio, A. P., Geisinger, J., Karow, M., Bjornson, C., Neal, T., Calos, M. P. NATURE PUBLISHING GROUP. 2012: S174?S174
View details for Web of Science ID 000303484600450
Site-specific integration with bacteriophage FC31 integrase. Cold Spring Harbor protocols Hillman, R. T., Calos, M. P. 2012; 2012 (5)
Abstract

Few nonviral techniques exist for efficient and stable eukaryotic gene transfer and fewer still are broadly useful in both cell culture and whole-organism applications. C31 integrase, a site-specific bacteriophage recombinase, is able to catalyze chromosomal transgene insertion under a diverse range of experimental and therapeutic conditions. The enzyme recognizes and catalyzes unidirectional recombination between attachment motifs found in phage and bacterial genomes (attP and attB sites, respectively). Use of C31 integrase for gene transfer requires that an attB sequence be cloned into a transgene-bearing plasmid. When this modified plasmid is introduced into cells alongside integrase-expressing plasmid, C31 integrase is able to catalyze insertion of the transgene plasmid into one of a limited pool of sites in the target genome that show sequence similarity to wild-type attP. Efficient delivery of C31 integrase and attB donor plasmid to the tissue or cells of interest remains the most challenging aspect of the system. Unlike viral methods of genome manipulation, use of C31 integrase almost always requires an additional method of stimulating cellular DNA uptake. However, the relative simplicity of the plasmid-based system means that nearly any proven method of introducing exogenous DNA into cells can be used with C31 integrase. This protocol describes the use of C31 integrase in mammalian cell culture for the creation of clonal lines showing robust and stable expression of an experimental transgene.

View details for DOI 10.1101/pdb.prot069211

View details for PubMedID 22550292
Long-Term Expression of Human Coagulation Factor VIII in a Tolerant Mouse Model Using the phi C31 Integrase System HUMAN GENE THERAPY Chavez, C. L., Keravala, A., Chu, J. N., Farruggio, A. P., Cuellar, V. E., Voorberg, J., Calos, M. P. 2012; 23 (4): 390-398
Abstract

We generated a mouse model for hemophilia A that combines a homozygous knockout for murine factor VIII (FVIII) and a homozygous addition of a mutant human FVIII (hFVIII). The resulting mouse, having no detectable FVIII protein or activity and tolerant to hFVIII, is useful for evaluating FVIII gene-therapy protocols. This model was used to develop an effective gene-therapy strategy using the ?C31 integrase to mediate permanent genomic integration of an hFVIII cDNA deleted for the B-domain. Various plasmids encoding ?C31 integrase and hFVIII were delivered to the livers of these mice by using hydrodynamic tail-vein injection. Long-term expression of therapeutic levels of hFVIII was observed over a 6-month time course when an intron was included in the hFVIII expression cassette and wild-type ?C31 integrase was used. A second dose of the hFVIII and integrase plasmids resulted in higher long-term hFVIII levels, indicating that incremental doses were beneficial and that a second dose of ?C31 integrase was tolerated. We observed a significant decrease in the bleeding time after a tail-clip challenge in mice treated with plasmids expressing hFVIII and ?C31 integrase. Genomic integration of the hFVIII expression plasmid was demonstrated by junction PCR at a known hotspot for integration in mouse liver. The ?C31 integrase system provided a nonviral method to achieve long-term FVIII gene therapy in a relevant mouse model of hemophilia A.

View details for DOI 10.1089/hum.2011.110

View details for Web of Science ID 000303046800009

View details for PubMedID 22077817

View details for PubMedCentralID PMC3327602
Site-Specific Recombinase Strategy to Create Induced Pluripotent Stem Cells Efficiently with Plasmid DNA STEM CELLS Karow, M., Chavez, C. L., Farruggio, A. P., Geisinger, J. M., Keravala, A., Jung, W. E., Lan, F., Wu, J. C., Chen-Tsai, Y., Calos, M. P. 2011; 29 (11): 1696-1704
Abstract

Induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field. iPSCs are most often produced by using retroviruses. However, the resulting cells may be ill-suited for clinical applications. Many alternative strategies to make iPSCs have been developed, but the nonintegrating strategies tend to be inefficient, while the integrating strategies involve random integration. Here, we report a facile strategy to create murine iPSCs that uses plasmid DNA and single transfection with sequence-specific recombinases. PhiC31 integrase was used to insert the reprogramming cassette into the genome, producing iPSCs. Cre recombinase was then used for excision of the reprogramming genes. The iPSCs were demonstrated to be pluripotent by in vitro and in vivo criteria, both before and after excision of the reprogramming cassette. This strategy is comparable with retroviral approaches in efficiency, but is nonhazardous for the user, simple to perform, and results in nonrandom integration of a reprogramming cassette that can be readily deleted. We demonstrated the efficiency of this reprogramming and excision strategy in two accessible cell types, fibroblasts and adipose stem cells. This simple strategy produces pluripotent stem cells that have the potential to be used in a clinical setting.

View details for DOI 10.1002/stem.730

View details for Web of Science ID 000296565500007

View details for PubMedID 21898697

View details for PubMedCentralID PMC3466168
The therapeutic potential of phiC31 integrase as a gene therapy system EXPERT OPINION ON BIOLOGICAL THERAPY Karow, M., Calos, M. P. 2011; 11 (10): 1287-1296
Abstract

The ?C31 integrase system is a phage-derived system that offers the ability to integrate plasmid DNA into the chromosomes at a subset of endogenous preferred locations associated with robust gene expression. Recent progress highlights the unique advantages of this system for in vivo gene therapy and for use in stem cells. AREAS COVERED: The ?C31 integrase system has been under development for ten years and has been demonstrated to be effective for integration of plasmids in a variety of tissues and organs for gene therapy in animal systems, as well as in isolated human cells. We focus on work with the ?C31 integrase system during the past 12-18 months. This work has centered on a series of papers involving in vivo delivery of the integrase system to the liver and a variety of studies demonstrating the utility of the integrase system in stem cells. EXPERT OPINION: We conclude that the ?C31 integrase system has significant potential for liver gene therapy, if effective DNA delivery methods for large mammals become available. The ?C31 integrase system displays an outstanding fit for use in pluripotent stem cells, and this area is expected to be the subject of intense development.

View details for DOI 10.1517/14712598.2011.601293

View details for Web of Science ID 000294492800003

View details for PubMedID 21736536
Therapeutic Applications of the PhiC31 Integrase System CURRENT GENE THERAPY Chavez, C. L., Calos, M. P. 2011; 11 (5): 375-381
Abstract

The potential use of the ?C31 integrase system in gene therapy opens up the possibilities of new treatments for old diseases. ?C31 integrase mediates the integration of plasmid DNA into the chromsomes of mammalian cells in a sequence-specific manner, resulting in robust, long-term transgene expression. In this article, we review how ?C31 integrase mediates transgene integration into the genomes of target cells and summarize the recent preclinical applications of the system to gene therapy. These applications encompass in vivo studies in liver and lung, as well as increasing ex vivo uses of the system, including in neural and muscle stem cells, in cord-lining epithelial cells, and for the production of induced pluripotent stem cells. The safety of the ?C31 integrase system for gene therapy is evaluated, and its ability to provide treatments for hemophilia is discussed. We conclude that gene therapy strategies utilizing ?C31 integrase offer great promise for the development of treatments in the future.

View details for Web of Science ID 000301945300006

View details for PubMedID 21888619
Long-term phenotypic correction in factor IX knockout mice by using phiC31 integrase-mediated gene therapy GENE THERAPY Keravala, A., Chavez, C. L., Hu, G., Woodard, L. E., Monahan, P. E., Calos, M. P. 2011; 18 (8): 842-848
Abstract

Hemophilia B, a hereditary bleeding disorder caused by a deficiency of coagulation factor IX (FIX), is an excellent candidate for gene therapy. However, to date, success in hemophilia gene therapy clinical trials has been limited due to failure to achieve or sustain therapeutic levels of factor expression. The ?C31 integrase system efficiently integrates plasmid DNA carrying a transgene and an attB site into a limited number of endogenous pseudo attP sites in mammalian genomes, leading to robust, sustained transgene expression. A strategy utilizing plasmid DNA integrated with ?C31 integrase may offer a facile and safe alternative for sustained human FIX (hFIX) expression. Hydrodynamic tail vein injection was used for delivery of plasmids encoding ?C31 integrase and hFIX to the liver of FIX knockout mice. We demonstrated prolonged therapeutic levels of hFIX in this knockout mouse model of hemophilia B over a 6-month time course when ?C31 integrase was used. Additionally, we observed sustained FIX activity in plasma and phenotypic correction of bleeding after tail clip in ?C31-treated mice. In the livers that received integrase, we also demonstrated prolonged hFIX expression in hepatocytes by immunohistochemistry and documented sequence-specific genomic integration of the hFIX plasmid. These studies suggest the possibility that a similar approach in large animals and humans could lead to a simple and successful gene therapy for hemophilia.

View details for DOI 10.1038/gt.2011.31

View details for Web of Science ID 000293779500011

View details for PubMedID 21412285
Site-specific integration of transgene targeting an endogenous lox-like site in early mouse embryos JOURNAL OF APPLIED GENETICS Ito, M., Yamanouchi, K., Naito, K., Calos, M. P., Tojo, H. 2011; 52 (1): 89-94
Abstract

Functional lox-like sequences have been identified within the yeast and mammalian genome. These hetero-specific lox sites also allow Cre recombinase to specifically target efficient integration of exogenous DNA into the endogenous pseudo-lox (?lox) sequences that occur naturally in the host genome using a Cre/loxP integrative recombination system. We investigated whether the Cre/?lox system is useful for site-specific integration of transgenes and for improving the production efficiency of transgenic animals. This is the first report on Cre-mediated integrative recombination targeting an endogenous lox-like sequence termed pseudo-loxm5 (?loxm5) in early mouse embryos. We characterized the Cre/?loxm5 system in embryonic environment. Cre-expressing plasmid and a transgene (CMV/LacZ gene) flanked by ?loxm5 and ?loxcorem5 sites were co-microinjected into the pronucleus of fertilized mouse oocytes. The injected eggs were transferred into foster mothers, and the recombination products were investigated. The results show that the ?loxm5 site is an active substrate for Cre-mediated recombination in the mouse embryonic environment. The transgenesis efficiency was up to 27% (6/22). The site-specific integration of the transgene into the endogenous ?loxm5 site was found in 50 % of the transgenic pups. Our findings demonstrated that the Cre/?loxm5 integrative recombination system is an efficient and simple strategy for targeting an endogenous lox-like site in mammalian embryos.

View details for DOI 10.1007/s13353-010-0011-3

View details for Web of Science ID 000290345300012

View details for PubMedID 21110150
Kinetics and Longevity of phi C31 Integrase in Mouse Liver and Cultured Cells HUMAN GENE THERAPY Chavez, C. L., Keravala, A., Woodard, L. E., Hillman, R. T., Stowe, T. R., Chu, J. N., Calos, M. P. 2010; 21 (10): 1287-1297
Abstract

The ?C31 integrase system provides genomic integration of plasmid DNA that may be useful in gene therapy. For example, the ?C31 system has been used in combination with hydrodynamic injection to achieve long-term expression of factor IX in mouse liver. However, a concern is that prolonged expression of ?C31 integrase within cells could potentially stimulate chromosome rearrangements or an immune response. Western blot and immunofluorescence analyses were performed to investigate the duration of ?C31 integrase expression in mouse liver. Integrase was expressed within 2 to 3 hr after hydrodynamic injection of a plasmid expressing ?C31 integrase. Expression peaked between 8 and 16 hr and fell to background levels by 24-48 hr postinjection. Analysis of the amount of integrase plasmid DNA present in the liver over time suggested that the brief period of integrase expression could largely be accounted for by rapid loss of the bulk of the plasmid DNA, as well as by silencing of plasmid expression. PCR analysis of integration indicated that ?C31 integrase carried out genomic integration of a codelivered attB-containing plasmid by 3 hr after plasmid injection. Integrase was expressed for longer times and at higher levels in transfected cultured cells compared with liver. Inhibitor studies suggested that the enzyme had a short half-life and was degraded by the 26S proteasome. The short duration of integrase expression in liver and rapid integration reaction appear to be features favorable for use in gene therapy.

View details for DOI 10.1089/hum.2010.049

View details for Web of Science ID 000282955500007

View details for PubMedID 20497035

View details for PubMedCentralID PMC2974851
Impact of Hydrodynamic Injection and phiC31 Integrase on Tumor Latency in a Mouse Model of MYC-Induced Hepatocellular Carcinoma PLOS ONE Woodard, L. E., Keravala, A., Jung, W. E., Wapinski, O. L., Yang, Q., Felsher, D. W., Calos, M. P. 2010; 5 (6)
Abstract

Hydrodynamic injection is an effective method for DNA delivery in mouse liver and is being translated to larger animals for possible clinical use. Similarly, phiC31 integrase has proven effective in mediating long-term gene therapy in mice when delivered by hydrodynamic injection and is being considered for clinical gene therapy applications. However, chromosomal aberrations have been associated with phiC31 integrase expression in tissue culture, leading to questions about safety.To study whether hydrodynamic delivery alone, or in conjunction with delivery of phiC31 integrase for long-term transgene expression, could facilitate tumor formation, we used a transgenic mouse model in which sustained induction of the human C-MYC oncogene in the liver was followed by hydrodynamic injection. Without injection, mice had a median tumor latency of 154 days. With hydrodynamic injection of saline alone, the median tumor latency was significantly reduced, to 105 days. The median tumor latency was similar, 106 days, when a luciferase donor plasmid and backbone plasmid without integrase were administered. In contrast, when active or inactive phiC31 integrase and donor plasmid were supplied to the mouse liver, the median tumor latency was 153 days, similar to mice receiving no injection.Our data suggest that phiC31 integrase does not facilitate tumor formation in this C-MYC transgenic mouse model. However, in groups lacking phiC31 integrase, hydrodynamic injection appeared to contribute to C-MYC-induced hepatocellular carcinoma in adult mice. Although it remains to be seen to what extent these findings may be extrapolated to catheter-mediated hydrodynamic delivery in larger species, they suggest that caution should be used during translation of hydrodynamic injection to clinical applications.

View details for DOI 10.1371/journal.pone.0011367

View details for Web of Science ID 000279369900016

View details for PubMedID 20614008

View details for PubMedCentralID PMC2894073
Effect of nuclear localization and hydrodynamic delivery-induced cell division on phi C31 integrase activity GENE THERAPY Woodard, L. E., Hillman, R. T., Keravala, A., Lee, S., Calos, M. P. 2010; 17 (2): 217-226
Abstract

Phage phiC31 integrase is a recombinase that can be expressed in mammalian cells to integrate plasmids carrying an attB sequence into the genome at specific pseudo attP locations. We show by immunofluoresence that wild-type phiC31 integrase is cytoplasmic and that addition of the SV40 nuclear localization signal (NLS) localized phiC31 integrase to the nucleus. Unexpectedly, the NLS depressed integration efficiency in HeLa cells and provided no benefit when used to integrate the human Factor IX (hFIX) gene into mouse liver. As breakdown of the nuclear membrane during mitosis could allow cytoplasmic integrase access to the chromosomes, we analyzed whether cell division was required for integration into liver cells in vivo. Hepatocytes were labeled with iododeoxyuridine to mark cells that underwent DNA replication during the week after hydrodynamic injection. Hydrodynamic delivery led to DNA replication in one-third of hepatocytes. Approximately three out of four cells having phiC31 integrase-mediated stable hFIX expression did not undergo replication, indicating that cell division was not required for integrase function in liver. Therefore, although the bulk of phiC31 integrase protein seems to be cytoplasmic in mammalian cells, integration can still occur in the nucleus, even without cell division.

View details for DOI 10.1038/gt.2009.136

View details for Web of Science ID 000274398000008

View details for PubMedID 19847205

View details for PubMedCentralID PMC2820593
DNA integrating vectors (transposon, integrase) A Guide to Human Gene Therapy Woodard, L. E., Calos, M. P. World Scientific Publishing Co.. 2010: 123?138
PhiC31 Integrase-Mediated Gene Transfer into Mesoangioblast Stem Cells for Amelioration of Muscular Dystrophy 12th Annual Meeting of the American Society of Gene Therapy Keravala, A., Woodard, L. E., Jung, W. E., Mosely, N. L., Calos, M. P. NATURE PUBLISHING GROUP. 2009: S203?S203
View details for Web of Science ID 000278019801105
Mutational Derivatives of PhiC31 Integrase With Increased Efficiency and Specificity MOLECULAR THERAPY Keravala, A., Lee, S., Thyagarajan, B., Olivares, E. C., Gabrovsky, V. E., Woodard, L. E., Calos, M. P. 2009; 17 (1): 112-120
Abstract

phiC31 integrase is a sequence-specific phage recombinase that can recombine two short DNA sequences called attB and attP. The enzyme can also promote genomic integration of plasmids carrying attB into native mammalian sequences having partial identity to attP. To increase the efficiency of integration, we mutated the phiC31 integrase gene and screened the mutants in human cells in an assay for higher recombination frequency between attB and attP. We report in this article the isolation of a mutant, P2 that has twice the chromosomal integration frequency of wild-type phiC31 integrase, at both a preintegrated chromosomal attP site and at endogenous pseudo attP sequences in cultured human cells. In mouse liver, P2-mediated integration provided therapeutic long-term levels of human factor IX that were double those generated by wild-type phiC31 integrase. We also describe an additional mutant, P3 that combines the mutations of P2 with further changes and possesses an elevated specificity for integration at a chromosomally placed attP site in human cells. Forty-four percent of colonies carrying integration events mediated by P3 have integration at the placed attP site. These mutant integrases are useful for gene therapy and genome modification, and they demonstrate the feasibility of engineering phiC31 integrase toward more desirable properties.

View details for DOI 10.1038/mt.2008.241

View details for Web of Science ID 000262137100017

View details for PubMedID 19002165

View details for PubMedCentralID PMC2834998
Factoring nonviral gene therapy into a cure for hemophilia A CURRENT OPINION IN MOLECULAR THERAPEUTICS Gabrovsky, V., Calos, M. P. 2008; 10 (5): 464-470
Abstract

Gene therapy for hemophilia A has fallen short of success despite several clinical trials conducted over the past decade. Challenges to its success include vector immunogenicity, insufficient transgene expression levels of Factor VIII, and inhibitor antibody formation. Gene therapy has been dominated by the use of viral vectors, as well as the immunogenic and oncogenic concerns that accompany these strategies. Because of the complexity of viral vectors, the development of nonviral DNA delivery methods may provide an efficient and safe alternative for the treatment of hemophilia A. New types of nonviral strategies, such as DNA integrating vectors, and the success of several nonviral animal studies, suggest that nonviral gene therapy has curative potential and justifies its clinical development.

View details for Web of Science ID 000259679400006

View details for PubMedID 18830922
Long-term transgene expression in mouse neural progenitor cells modified with phi C31 integrase JOURNAL OF NEUROSCIENCE METHODS Keravala, A., Ormerod, B. K., Palmer, T. D., Calos, M. P. 2008; 173 (2): 299-305
Abstract

Stem cells can potentially be utilized in combined gene/cell therapies for neural diseases. We examined the ability of the non-viral phiC31 integrase system to promote stable transgene expression in mouse neural progenitor cells (mNPCs). phiC31 integrase catalyzes the sequence-specific integration of attB-containing plasmids into pseudo attP sites in mammalian genomes, to produce long-term transgene expression. We achieved gene transfer by co-nucleofection of a plasmid carrying the luciferase marker gene and an attB site and a plasmid expressing integrase in mNPCs that had been generated in a neurosphere preparation. Luciferase expression was quantified in live cells for 8 weeks, revealing persistence of gene expression. Sequence-specific integration at a preferred pseudo attP site in the mouse genome was detected by using PCR. Furthermore, sustained transgene expression was demonstrated in genetically modified NPCs that were cultured in conditions that promoted either growth or differentiation into neurons and astrocytes. Our results demonstrate that the phiC31 integrase system produces stable transgene expression in adult mNPCs and their progeny and may be useful in strategies for combating neurodegenerative disorders.

View details for DOI 10.1016/j.jneumeth.2008.06.005

View details for Web of Science ID 000258906300016

View details for PubMedID 18606184

View details for PubMedCentralID PMC2615000
Site-specific chromosomal integration mediated by phiC31 integrase. Methods in molecular biology (Clifton, N.J.) Keravala, A., Calos, M. P. 2008; 435: 165-173
Abstract

phiC31 integrase is a site-specific recombinase from a bacteriophage that has become a useful tool in mammalian cells. The enzyme normally performs precise, unidirectional recombination between two attachment or att sites called attB and attP. We have shown that an attP site preintegrated into a mammalian chromosome can serve as a target for integration of an introduced plasmid carrying an attB site. Recombination leads to precise integration of the plasmid into the chromosome at the attP site. This reaction is useful for placing introduced genes into the same chromosomal environment, in order to minimize position effects associated with random integration. Because phiC31 integrase can also mediate integration at endogenous sequences that resemble attP, called pseudo attP sites, a selection system is used that yields integration only at the desired preintegrated attP site. This chapter provides a protocol that features a simple antibiotic selection to isolate cell lines in which the introduced plasmid has integrated at the desired attP site. A polymerase chain reaction assay is also presented to verify correct chromosomal placement of the introduced plasmid. This integration system based on phiC31 integrase supplies a simple method to obtain repeated integration at the same chromosomal site in mammalian cells.

View details for DOI 10.1007/978-1-59745-232-8_12

View details for PubMedID 18370075
Creating transgenic Drosophila by microinjecting the site-specific phi C31 integrase mRNA and a transgene-containing donor plasmid NATURE PROTOCOLS Fish, M. P., Groth, A. C., Calos, M. P., Nusse, R. 2007; 2 (10): 2325-2331
Abstract

We describe a microinjection-based phiC31 integrase mRNA-mediated method for creating transgenic Drosophila strains. This approach is more efficient than traditional methods and ensures that the transgene is targeted to a precise genomic position. The method involves targeting integration of an exogenous plasmid (containing the transgene and sequences to facilitate integration) to a preplaced recipient site in the Drosophila genome. The plasmid is coinjected into embryos with mRNA encoding the phiC31 integrase, the enzyme that catalyzes the integration reaction. Using the protocol described here, transgenic lines can be established from, on average, 46% of fertile adults obtained after injection, and all integrations should be targeted to the chosen genomic insertion site. The whole procedure, from injection to established transgenic stocks, can be completed in three generations (approximately 1 month) and can be adapted for other types of transgenesis and mRNA injections in Drosophila.

View details for DOI 10.1038/nprot.2007.328

View details for Web of Science ID 000253139900004

View details for PubMedID 17947973
The phi C31 integrase system for gene therapy CURRENT GENE THERAPY Calos, M. P. 2006; 6 (6): 633-645
Abstract

The phiC31 integrase system represents a novel technology that opens up new possibilities for gene therapy. The phiC31 integrase can integrate introduced plasmid DNA into preferred locations in unmodified mammalian genomes, resulting in robust, long-term expression of the integrated transgene. This review describes the nature of the integration reaction and the genomic integration sites used by the enzyme in human cells. Preclinical applications of the system to gene therapy to date are summarized, including in vivo use in liver, muscle, eye, and joint and ex vivo use in skin keratinocytes, muscle precursor cells, and T cell lines. The safety of this phage integrase system for gene therapy is evaluated, and its strengths and limitations are compared to other gene therapy approaches. Ongoing and planned improvements to the phage integrase system are discussed. We conclude that gene therapy strategies using phiC31 integrase and its derivatives offer great promise for success in the near term.

View details for Web of Science ID 000242752600003

View details for PubMedID 17168696
Gene transfer to rabbit retina with electron avalanche transfection INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Chalberg, T. W., Vankov, A., Molnar, F. E., Butterwick, A. F., Huie, P., Calos, M. P., Palanker, D. V. 2006; 47 (9): 4083-4090
Abstract

Nonviral gene therapy represents a promising treatment for retinal diseases, given clinically acceptable methods for efficient gene transfer. Electroporation is widely used for transfection, but causes significant collateral damage and a high rate of cell death, especially in applications in situ. This study was conducted in the interest of developing efficient and less toxic forms of gene transfer for the eye.A novel method for nonviral DNA transfer, called electron avalanche transfection, was used that involves microsecond electric plasma-mediated discharges applied via microelectrode array. This transfection method, which produces synchronized pulses of mechanical stress and high electric field, was first applied to chorioallantoic membrane as a model system and then to rabbit RPE in vivo. Gene transfer was measured by using luciferase bioluminescence and in vivo fluorescent fundus photography. Safety was evaluated by performing electroretinograms and histology.In chorioallantoic membrane, electron avalanche transfection was approximately 10,000-fold more efficient and produced less tissue damage than conventional electroporation. Also demonstrated was efficient plasmid DNA transfer to the rabbit retina after subretinal DNA injection and transscleral electron avalanche transfection. Electroretinograms and histology showed no evidence of damage from the procedure.Electron avalanche transfection is a powerful new technology for safe DNA delivery that has great promise as a nonviral system of gene transfer.

View details for DOI 10.1167/iovs.06-0092

View details for Web of Science ID 000240050700055

View details for PubMedID 16936128
A diversity of serine phage integrases mediate site-specific recombination in mammalian cells MOLECULAR GENETICS AND GENOMICS Keravala, A., Groth, A. C., Jarrahian, S., Thyagarajan, B., Hoyt, J. J., Kirby, P. J., Calos, M. P. 2006; 276 (2): 135-146
Abstract

This study evaluated the ability of five serine phage integrases, from phages A118, U153, Bxb1, phiFC1, and phiRV1, to mediate recombination in mammalian cells. Two types of recombination were investigated, including the ability of an integrase to mediate recombination between its own phage att sites in the context of a mammalian cell and the ability of an integrase to perform genomic integration pairing a phage att site with an endogenous mammalian sequence. We demonstrated that the A118 integrase mediated precise intra-molecular recombination of a plasmid containing its attB and attP sites at a frequency of approximately 50% in human cells. The closely related U153 integrase also performed efficient recombination in human cells on a plasmid containing the attB and attP sites of A118. The integrases from phages Bxb1, phiFC1, and phiRV1 carried out such recombination at their attB and attP sites at frequencies ranging from 11 to 75%. Furthermore, the A118 integrase mediated recombination between its attP site on a plasmid and pseudo attB sites in the human genome, i.e. native sequences with partial identity to attB. Fifteen such A118 pseudo att sites were analyzed, and a consensus recognition site was identified. The other integrases did not mediate integration at genomic sequences at a frequency above background. These site-specific integrases represent valuable new tools for manipulating eukaryotic genomes.

View details for DOI 10.1007/s00438-006-0129-5

View details for Web of Science ID 000240283200004

View details for PubMedID 16699779
Long-term increase in mVEGF164 in mouse hindlimb muscle mediated by phage phi C31 integrase after nonviral DNA delivery HUMAN GENE THERAPY Portlock, J. L., Keravala, A., Bertoni, C., Lee, S., Rando, T. A., Calos, M. P. 2006; 17 (8): 871-876
Abstract

Peripheral vascular disease (PVD), characterized by insufficient blood supply to extremities, can be a devastating illness. Although many gene therapy strategies for PVD using vascular endothelial growth factor (VEGF) have resulted in increased blood vessel formation, the vessels are often impermanent and regress after therapy, probably because of the short-lived VEGF expression mediated by gene therapy vectors (14 days or less). phiC31 integrase is a recombinase originally isolated from a bacteriophage of Streptomyces. This integrase performs efficient chromosomal integration of plasmid DNA into mammalian genomes that results in long-term transgene expression. In this study, gene transfer was achieved by intramuscular injection of VEGF and integrase plasmid DNAs into the tibialis anterior muscle in the mouse hindlimb, followed by electroporation of the muscle with needle electrodes. We observed VEGF levels significantly above background 40 days after injection in animals that received phiC31 integrase and the VEGF plasmid. Site-specific integration of plasmid DNA in the chromosomes of muscle tissue was verified by polymerase chain reaction at a common integration site. These results suggest the possible utility of the phiC31 integrase system to treat ischemic disease.

View details for PubMedID 16942446
PhiC31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints JOURNAL OF GENE MEDICINE Keravala, A., Portlock, J. L., Nash, J. A., Vitrant, D. G., Robbins, P. D., Calos, M. P. 2006; 8 (8): 1008-1017
Abstract

Gene transfer to synovium in joints has been shown to be an effective approach for treating pathologies associated with rheumatoid arthritis (RA) and related joint disorders. However, the efficiency and duration of gene delivery has been limiting for successful gene therapy for arthritis. The transient gene expression that often accompanies non-viral gene delivery can be prolonged by integration of vector DNA into the host genome. We report a novel approach for non-viral gene therapy to joints that utilizes phage phiC31 integrase to bring about unidirectional genomic integration.Rabbit and human synovial cells were co-transfected with a plasmid expressing phiC31 integrase and a plasmid containing the transgene and an attB site. Cells were cultured with or without G418 selection and the number of neo-resistant colonies or eGFP cells determined, respectively. Plasmid rescue, PCR query, and DNA sequence analysis were performed to reveal integration sites in the rabbit and human genomes. For in vivo studies, attB-reporter gene plasmids and a plasmid expressing phiC31 integrase were intra-articularly injected into rabbit knees. Joint sections were used for histological analysis of beta-gal expression, and synovial cells were isolated to measure luciferase expression.We demonstrated that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the transgene and attB increased the frequency of transgene expression in rabbit synovial fibroblasts and primary human RA synoviocytes. Plasmid rescue and DNA sequence analysis of plasmid-chromosome junctions revealed integration at endogenous pseudo attP sequences in the rabbit genome, and PCR query detected integration at previously characterized integration sites in the human genome. Significantly higher levels of transgene expression were detected in vivo in rabbit knees after intra-articular injection of attB-reporter gene plasmids and a plasmid expressing phiC31 integrase.The ability of phiC31 integrase to facilitate genomic integration in synovial cells and increase transgene expression in the rabbit synovium suggests that, in combination with more efficient DNA delivery methods, this integrase system could be beneficial for treatment of rheumatoid arthritis and other joint disorders.

View details for DOI 10.1002/jgm.928

View details for Web of Science ID 000240163900008

View details for PubMedID 16779871
Phage phi C31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines JOURNAL OF GENE MEDICINE Ishikawa, Y., Tanaka, N., Murakami, K., Uchiyama, T., Kumaki, S., Tsuchiya, S., Kugoh, H., Oshimura, M., Calos, M. P., Sugamura, K. 2006; 8 (5): 646-653
Abstract

X-linked severe combined immunodeficiency (SCID-X1, X-SCID) is a life-threatening disease caused by a mutated common cytokine receptor gamma chain (gammac) gene. Although ex vivo gene therapy, i.e., transduction of the gammac gene into autologous CD34(+) cells, has been successful for treating SCID-X1, the retrovirus vector-mediated transfer allowed dysregulated integration, causing leukemias. Here, to explore an alternative gene transfer methodology that may offer less risk of insertional mutagenesis, we employed the phiC31 integrase-based integration system using human T-cell lines, including the gammac-deficient ED40515(-).A phiC31 integrase and a neo(r) gene expression plasmid containing the phiC31 attB sequence were co-delivered by electroporation into Jurkat cells. After G418 selection, integration site analyses were performed using linear amplification mediated-polymerase chain reaction (LAM-PCR). ED40515(-) cells were also transfected with a gammac expression plasmid containing attB, and the integration sites were determined. IL-2 stimulation was used to assess the functionality of the transduced gammac in an ED40515(-)-derived clone.Following co-introduction of the phiC31 integrase expression plasmid and the plasmid carrying attB, the efficiency of integration into the unmodified human genome was assessed. Several integration sites were characterized, including new integration sites in intergenic regions on chromosomes 13 and 18 that may be preferred in hematopoietic cells. An ED40515(-) line bearing the integrated gammac gene exhibited stable expression of the gammac protein, with normal IL-2 signaling, as assessed by STAT5 activation.This study supports the possible future use of this phiC31 integrase-mediated genomic integration strategy as an alternative gene therapy approach for treating SCID-X1.

View details for DOI 10.1002/jgm.891

View details for Web of Science ID 000238230800010

View details for PubMedID 16508910
Integration specificity of phage phi C31 integrase in the human genome JOURNAL OF MOLECULAR BIOLOGY Chalberg, T. W., Portlock, J. L., Olivares, E. C., Thyagarajan, B., Kirby, P. J., Hillman, R. T., Hoelters, J., Calos, M. P. 2006; 357 (1): 28-48
Abstract

The site-specific integrase from bacteriophage phiC31 functions in mammalian cells and is being applied for genetic engineering, including gene therapy. The phiC31 integrase catalyzes precise, unidirectional recombination between its 30-40-bp attP and attB recognition sites. In mammalian cells, the enzyme also mediates integration of plasmids bearing attB into native sequences that have partial sequence identity with attP, termed pseudo attP sites. Here, we analyzed the features of phiC31-mediated integration into pseudo attP sites in the human genome. Sequence analysis of 196 independent integration events derived from three cell lines revealed approximately 101 integration sites: 56% of the events were recurrent integrations distributed among 19 pseudo attP sequences. Bioinformatics analysis revealed a approximately 30-bp palindromic consensus sequence motif shared by all of the repeat occurrences and most of the single occurrence sites, verifying that phiC31-mediated integration into pseudo attP sites is significantly guided by DNA sequence recognition. The most favored unique sequence in these cell lines occurred at chromosome 19q13.31 and accounted for 7.5% of integration events. Other frequent integration sites were in three specific sequences in subfamilies of ERVL and L1 repetitive sequences, accounting for an additional 17.9% of integration events. Integrations could occur in either orientation at a pseudo attP site, were often accompanied by small deletions, and typically occurred in a single copy per cell. A number of aberrant events were also described, including large deletions and chromosome rearrangements. phiC31 integrase-mediated integration only slightly favored genes and did not favor promoter regions. Gene density and expression studies suggested chromatin context effects. An analysis of the safety of integration sites in terms of proximity to cancer genes suggested minimal cancer risk. We conclude that integration systems derived from phiC31 integrase have great potential utility.

View details for DOI 10.1016/j.jmb.2005.11.098

View details for Web of Science ID 000235823900004

View details for PubMedID 16414067
Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bertoni, C., Jarrahian, S., Wheeler, T. M., Li, Y. N., Olivares, E. C., Calos, M. P., Rando, T. A. 2006; 103 (2): 419-424
Abstract

Plasmid-mediated gene therapy can restore dystrophin expression in skeletal muscle in the mdx mouse, a model of Duchenne muscular dystrophy. However, sufficient long-term expression and distribution of dystrophin remain a hurdle for translating this technology into a viable treatment for Duchenne muscular dystrophy. To improve plasmid-mediated gene therapy for muscle diseases, we studied the effects of targeted plasmid integration using a phage integrase (phiC31) that can mediate the integration of suitably modified plasmids into the mammalian genome. Using a luciferase expression plasmid, we monitored plasmid gene expression noninvasively in living mice by bioluminescence imaging. Coinjection of an integrase plasmid resulted in 5- to 10-fold higher levels of sustained luciferase expression. Likewise, plasmid-mediated dystrophin expression in mdx muscle was enhanced by integration. Using a combination of dystrophin and luciferase plasmids, we analyzed the functional benefit of dystrophin expression in the dystrophic muscle. The expression of dystrophin slowed the loss of luciferase expression associated with muscle degeneration, and that protection was enhanced by targeted integration of the dystrophin plasmid. In the presence of integrase, dystrophin expression was distributed along a much greater length of individual fibers, and this was associated with increased protection against degenerative changes. These data demonstrate the importance of both the level and distribution of dystrophin expression to achieve therapeutic efficacy, and that the efficacy can be enhanced by targeted plasmid integration.

View details for Web of Science ID 000234624100031

View details for PubMedID 16387861
Plasma-mediated transfection of RPE 16th Conference on Ophthalmic Technologies Palanker, D., Chalberg, T., Vankov, A., Huie, P., Molnar, F. E., Butterwick, A., Calos, M., Marmor, M., Blumenkranz, M. S. SPIE-INT SOC OPTICAL ENGINEERING. 2006
View details for DOI 10.1117/12.649624

View details for Web of Science ID 000237708800038
phi C31 integrase confers genomic integration and long-term transgene expression in rat retina INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Chalberg, T. W., Genise, H. L., Vollrath, D., Calos, M. P. 2005; 46 (6): 2140-2146
Abstract

Gene therapy has shown promise in animal models of retinal disease, with the most success achieved to date with viral vectors used for gene delivery. Viral vectors, however, have side effects and limitations and are difficult to manufacture. The present study was conducted in an attempt to develop a novel system for long-term gene transfer in rat retinal pigment epithelium (RPE), by using nonviral transfection methods for gene transfer and the integrase from the bacteriophage phiC31 to confer long-term gene expression by means of genomic integration.Efficient nonviral delivery of plasmid DNA to rat RPE in vivo was achieved by using subretinal injection of plasmid DNA, followed by in situ electroporation. Gene delivery was evaluated by analyzing enhanced green fluorescent protein (eGFP) expression in frozen sections. In subsequent experiments, a plasmid expressing luciferase, with or without a plasmid encoding the phiC31 integrase, was delivered to rat RPE. Luciferase expression was followed over time by using in vivo luciferase imaging.Subretinal injection followed by electroporation yielded abundant transgene expression in the rat RPE. Expression was strongest 48 hours after delivery. In the absence of phiC31 integrase, transgene expression declined to near-background levels within 3 to 4 weeks after treatment. By contrast, coinjection of the integrase plasmid led to long-term stable transgene expression throughout the 4.5-month test period. Eyes injected with phiC31 integrase showed approximately 85-fold higher long-term transgene expression in the retina than eyes without integrase.Subretinal injection of DNA followed by electroporation affords abundant transfer of plasmid DNA in rat RPE. phiC31 integrase confers robust long-term transgene expression by mediating genomic integration of the transgene. These findings suggest that phiC31 integrase may be a simple and effective tool for nonviral long-term gene transfer in the eye.

View details for DOI 10.1167/iovs.04-1252

View details for Web of Science ID 000229504600038

View details for PubMedID 15914635
In vivo correction of murine hereditary tyrosinemia type I by phi C31 integrase-mediated gene delivery MOLECULAR THERAPY Held, P. K., Olivares, E. C., Aguilar, C. P., Finegold, M., Calos, M. P., Grompe, M. 2005; 11 (3): 399-408
Abstract

Phage phiC31 integrase is a site-specific recombinase that mediates efficient integration of circular extrachromosomal DNA into the host genome. Here, the integrase system was used to transfer the fumarylacetoacetate hydrolase (FAH) gene into the liver of mice affected with hereditary tyrosinemia type 1. Approximately 3.6% of transfected hepatocytes experienced an integration event. The absolute frequency of integration was 1/1374. A higher proportion of integrase-transfected FAH+ hepatocytes displayed abnormal morphology (bizarre nuclei, enlarged cells) on day 25 after gene transfer, compared to cells not receiving integrase. The increased frequency of these abnormal cells correlated with the amount of integrase plasmid administered, suggesting some form of integrase toxicity in Fah-/- livers. The abnormal hepatocyte appearance was transient and livers analyzed after longer selection (90 days) showed 60% repopulation with only normal healthy FAH+ hepatocytes. A total of seven different integration sites (accounting for >90% of integration) were identified. Serial transplantation of integrase-corrected hepatocytes to Fah-/- recipients was successful, suggesting long-term viability of corrected cells and persistent gene expression through many rounds of cell division. The stability of transgene expression, relatively high integration frequency, and significant site specificity that characterize the phiC31 integration system suggest that it may have utility in many gene therapy settings.

View details for DOI 10.1016/j.ymthe.2004.11.001

View details for PubMedID 15727936
Site-Specific Integration with phi C31 Integrase for Prolonged Expression of Therapeutic Genes NON-VIRAL VECTORS FOR GENE THERAPY, SECOND EDITION: PART 2 Ginsburg, D. S., Calos, M. P. 2005; 54: 179-187
Abstract

Need of a site-specific integrating vector in gene therapy has become pressing, as recent work has shown that many of the current integrating vectors used preferentially integrate in the vicinity of genes. A site-specific integrating vector would reduce the risk of insertional mutagenesis posed by randomly integrating vectors, and a non-viral vector would reduce the safety and immunogenicity problems associated with viral vectors. The phiC31 integrase is a protein from Streptomyces phage phiC31 that has been developed as a non-viral site-specific gene therapy vector. The phiC31 integrase catalyzes the integration of a plasmid containing attB into pseudo attP sites in mammalian genomes. It has been shown to function in tissue culture cells as well as in mice. Vectors based on the phiC31 integrase were able to treat tyrosinemia type I in a mouse model and two forms of epidermolysis bullosa in keratinocytes from patients, demonstrating its effectiveness as a gene therapy vector. Development of phiC31 integrase-based vectors is still underway, but it has already been shown to provide long-term expression through site-specific integration.

View details for DOI 10.1016/S0065-2660(05)54008-2

View details for Web of Science ID 000280571400009

View details for PubMedID 16096012
Site-specific integration for high-level protein production in mammalian cells. Methods in molecular biology (Clifton, N.J.) Thyagarajan, B., Calos, M. P. 2005; 308: 99-106
View details for PubMedID 16082029
Nucleofection of muscle-derived stem cells and myoblasts with phi C31 integrase: Stable expression of a full-length-dystrophin fusion gene by human myoblasts MOLECULAR THERAPY Quenneville, S. P., Chapdelaine, P., Rousseau, J., Beaulieu, J., Caron, N. J., Skuk, D., Mills, P., Olivares, E. C., Calos, M. P., Tremblay, J. P. 2004; 10 (4): 679-687
Abstract

Ex vivo gene therapy offers a potential treatment for Duchenne muscular dystrophy by transfection of the dystrophin gene into the patient's own myogenic precursor cells, followed by transplantation. We used nucleofection to introduce DNA plasmids coding for enhanced green fluorescent protein (eGFP) or eGFP-dystrophin fusion protein and the phage phiC31 integrase into myogenic cells and to integrate these genes into a limited number of sites in the genome. Using a plasmid expressing eGFP, we transfected 50% of a mouse muscle-derived stem cell line and 60% of normal human myoblasts. Co-nucleofection of a plasmid expressing the phiC31 integrase and an eGFP expression plasmid containing an attB sequence produced 15 times more frequent stable expression, because of site-specific integration of the transgene. Co-nucleofection of the phiC31 integrase plasmid and a large plasmid containing the attB sequence and the gene for an eGFP-full-length dystrophin fusion protein produced fluorescent human myoblasts that were able to form more intensely fluorescent myotubes after 1 month of culture. A nonviral approach combining nucleofection and the phiC31 integrase may eventually permit safe autotransplantation of genetically modified cells to patients.

View details for DOI 10.1016/j.ymthe.2004.05.034

View details for Web of Science ID 000224964800014

View details for PubMedID 15451452
Development of a novel helper-dependent adenovirus-Epstein-Barr virus hybrid system for the stable transformation of mammalian cells JOURNAL OF VIROLOGY Dorigo, O., Gil, J. S., Gallaher, S. D., Tan, B. T., Castro, M. G., Lowenstein, P. R., Calos, M. P., Berk, A. J. 2004; 78 (12): 6556-6566
Abstract

Epstein-Barr virus (EBV) episomes are stably maintained in permissive proliferating cell lines due to EBV nuclear antigen 1 (EBNA-1) protein-mediated replication and segregation. Previous studies showed the ability of EBV episomes to confer long-term transgene expression and correct genetic defects in deficient cells. To achieve quantitative delivery of EBV episomes in vitro and in vivo, we developed a binary helper-dependent adenovirus (HDA)-EBV hybrid system that consists of one HDA vector for the expression of Cre recombinase and a second HDA vector that contains all of the sequences for the EBV episome flanked by loxP sites. Upon coinfection of cells, Cre expressed from the first vector recombined loxP sites on the second vector. The resulting circular EBV episomes expressed a transgene and contained the EBV-derived family of repeats, an EBNA-1 expression cassette, and 19 kb of human DNA that functions as a replication origin in mammalian cells. This HDA-EBV hybrid system transformed 40% of cultured cells. Transgene expression in proliferating cells was observed for over 20 weeks under conditions that selected for the expression of the transgene. In the absence of selection, EBV episomes were lost at a rate of 8 to 10% per cell division. Successful delivery of EBV episomes in vivo was demonstrated in the liver of transgenic mice expressing Cre from the albumin promoter. This novel gene transfer system has the potential to confer long-term episomal transgene expression and therefore to correct genetic defects with reduced vector-related toxicity and without insertional mutagenesis.

View details for DOI 10.1128/JVI.78.12.6556-6566.2004

View details for Web of Science ID 000221772000048

View details for PubMedID 15163748

View details for PubMedCentralID PMC416543
Site-specific integration enhances expression of DNA introduced into skeletal muscle 7th Annual Meeting of the American-Society-of-Gene-Therapy Wheeler, T. M., Jarrahian, S., Bertoni, C., Olivares, E. C., Doyle, T., Contag, C. H., Calos, M. P., Rando, T. A. NATURE PUBLISHING GROUP. 2004: S307?S307
View details for Web of Science ID 000222316600811
Construction of transgenic Drosophila by using the site-specific integrase from phage phi C31 GENETICS Groth, A. C., Fish, M., Nusse, R., Calos, M. P. 2004; 166 (4): 1775-1782
Abstract

The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosophila and other insects.

View details for Web of Science ID 000221377700016

View details for PubMedID 15126397
Phage integrases: Biology and applications JOURNAL OF MOLECULAR BIOLOGY Groth, A. C., Calos, M. P. 2004; 335 (3): 667-678
Abstract

Phage integrases are enzymes that mediate unidirectional site-specific recombination between two DNA recognition sequences, the phage attachment site, attP, and the bacterial attachment site, attB. Integrases may be grouped into two major families, the tyrosine recombinases and the serine recombinases, based on their mode of catalysis. Tyrosine family integrases, such as lambda integrase, utilize a catalytic tyrosine to mediate strand cleavage, tend to recognize longer attP sequences, and require other proteins encoded by the phage or the host bacteria. Phage integrases from the serine family are larger, use a catalytic serine for strand cleavage, recognize shorter attP sequences, and do not require host cofactors. Phage integrases mediate efficient site-specific recombination between two different sequences that are relatively short, yet long enough to be specific on a genomic scale. These properties give phage integrases growing importance for the genetic manipulation of living eukaryotic cells, especially those with large genomes such as mammals and most plants, for which there are few tools for precise manipulation of the genome. Integrases of the serine family have been shown to work efficiently in mammalian cells, mediating efficient integration at introduced att sites or native sequences that have partial identity to att sites. This reaction has applications in areas such as gene therapy, construction of transgenic organisms, and manipulation of cell lines. Directed evolution can be used to increase further the affinity of an integrase for a particular native sequence, opening up additional applications for genomic modification.

View details for DOI 10.1016/j.jmb.2003.09.082

View details for Web of Science ID 000188066900001

View details for PubMedID 14687564
Phage integrases for the construction and manipulation of transgenic mammals. Reproductive biology and endocrinology Hollis, R. P., Stoll, S. M., Sclimenti, C. R., Lin, J., Chen-Tsai, Y., Calos, M. P. 2003; 1: 79-?
Abstract

Phage integrases catalyze site-specific, unidirectional recombination between two short att recognition sites. Recombination results in integration when the att sites are present on two different DNA molecules and deletion or inversion when the att sites are on the same molecule. Here we demonstrate the ability of the phiC31 integrase to integrate DNA into endogenous sequences in the mouse genome following microinjection of donor plasmid and integrase mRNA into mouse single-cell embryos. Transgenic early embryos and a mid-gestation mouse are reported. We also demonstrate the ability of the phiC31, R4, and TP901-1 phage integrases to recombine two introduced att sites on the same chromosome in human cells, resulting in deletion of the intervening material. We compare the frequencies of mammalian chromosomal deletion catalyzed by these three integrases in different chromosomal locations. The results reviewed here introduce these bacteriophage integrases as tools for site-specific modification of the genome for the creation and manipulation of transgenic mammals.

View details for PubMedID 14613545
Site-specific genomic strategies for gene therapy CURRENT OPINION IN MOLECULAR THERAPEUTICS Portlock, J. L., Calos, M. P. 2003; 5 (4): 376-382
Abstract

Like any disease treatment, gene therapy should be safe and efficacious. Safety can be addressed by directly correcting the defective gene itself or by ensuring that genomic integration of a transgene is site-specific. Unfortunately, it has proven difficult to achieve this level of safety without a concomitant loss in efficiency of gene correction or insertion. In this review, recent research attempts to achieve efficient site-specific gene therapy, including strategies using targeted gene conversion, adeno-associated virus vectors and site-specific bacteriophage recombinases are discussed. We believe that these approaches hold promise for site-specific, safe and efficient gene therapy.

View details for Web of Science ID 000185165100007

View details for PubMedID 14513680
phi C31 integrase-mediated nonviral genetic correction of junctional epidermolysis bullosa HUMAN GENE THERAPY Ortiz-Urda, S., Thyagarajan, B., Keene, D. R., Lin, Q., Calos, M. P., Khavari, P. A. 2003; 14 (9): 923-928
Abstract

Patients afflicted with severe laminin 5-deficient junctional epidermolysis bullosa (JEB) often die in infancy with massive cutaneous blistering. Prior approaches to genetically correct this disorder have relied on stable integration of wild-type LAMB3 sequences, using retroviral vectors. To develop a nonviral approach to JEB gene therapy, we used the phiC31 integrase, which mediates unidirectional genomic integration of plasmids containing a specific attB site. An attB-containing laminin 5 beta3 expression plasmid was integrated into the genomes of primary keratinocytes from four unrelated, genetically characterized JEB patients. phiC31 integrase supported genomic integration into epidermal progenitor cells. Regeneration of human skin on immunedeficient mice, using these cells, produced human skin tissue with restored laminin 5 expression. Furthermore, corrected JEB tissue restored hemidesmosome formation and abolished histologic evidence of subepidermal blistering. These findings provide an approach to durable nonviral correction of JEB.

View details for PubMedID 12828862
Epstein-Barr virus vectors provide prolonged robust factor IX expression in mice BIOTECHNOLOGY PROGRESS Sclimenti, C. R., Neviaser, A. S., Baba, E. J., Meuse, L., Kay, M. A., Calos, M. P. 2003; 19 (1): 144-151
Abstract

We demonstrate that vectors incorporating components from Epstein-Barr virus (EBV) for retention and from human genomic DNA for replication greatly enhance the level and duration of marker gene expression in dividing cultured cells. The same types of vectors were tested in vivo by high-pressure tail vein injection of naked DNA in mice, resulting in liver delivery and expression. The therapeutic gene was a human factor IX (hFIX) minigene comprising genomically derived 5', 3', and intronic sequences that provided relatively good gene expression in vivo. We demonstrated that addition of the EBV EBNA1 gene and its family of repeats binding sites provided a 10- to 100-fold increase in prolonged hFIX expression in mouse liver. A single 25-microg dose of vector DNA generated normal (>5 microg/mL) levels of hFIX throughout the 8 month duration of the experiment. Vector DNA with or without the EBV sequences was retained in liver cells, and vector replication was not a factor in these nondividing liver cells. Instead, it appears that enhancement of stable hFIX expression by the EBV components was responsible for the increased level and duration of therapeutic gene expression. The EBV sequences also significantly enhanced stable expression of a vector carrying the full genomic hFIX gene delivered to mouse liver. These results underline the crucial importance of appropriate gene expression signals on gene therapy vectors and the utility of EBV sequences in particular for increasing stable gene expression.

View details for DOI 10.1021/bp0200907

View details for Web of Science ID 000180973100020

View details for PubMedID 12573017
Site-specific genomic integration produces therapeutic Factor IX levels in mice NATURE BIOTECHNOLOGY Olivares, E. C., Hollis, R. P., Chalberg, T. W., Meuse, L., Kay, M. A., Calos, M. P. 2002; 20 (11): 1124-1128
Abstract

We used the integrase from phage phiC31 to integrate the human Factor IX (hFIX) gene permanently into specific sites in the mouse genome. A plasmid containing attB and an expression cassette for hFIX was delivered to the livers of mice by using high-pressure tail vein injection. When an integrase expression plasmid was co-injected, hFIX serum levels increased more than tenfold to approximately 4 microg/ml, similar to normal FIX levels, and remained stable throughout the more than eight months of the experiment. hFIX levels persisted after partial hepatectomy, suggesting genomic integration of the vector. Site-specific integration was proven by characterizing and quantifying genomic integration in the liver at the DNA level. Integration was documented at two pseudo-attP sites, native sequences with partial identity to attP, with one site highly predominant. This study demonstrates in vivo gene transfer in an animal by site-specific genomic integration.

View details for DOI 10.1038/nbt753

View details for Web of Science ID 000179041500023

View details for PubMedID 12379870
Stable nonviral genetic correction of inherited human skin disease NATURE MEDICINE Ortiz-Urda, S., Thyagarajan, B., Keene, D. R., Lin, Q., Fang, M., Calos, M. P., Khavari, P. A. 2002; 8 (10): 1166-1170
Abstract

Current gene-transfer technologies display limitations in achieving effective gene delivery. Among these limitations are difficulties in stably integrating large corrective sequences into the genomes of long-lived progenitor-cell populations. Current larger-capacity viral vectors suffer from biosafety concerns, whereas plasmid-based approaches have poor efficiency of stable gene transfer. These barriers hinder genetic correction of many severe inherited human diseases, such as the blistering skin disorder recessive dystrophic epidermolysis bullosa (RDEB), caused by mutations in the large COL7A1 gene. To circumvent these barriers, we used the phi C31 bacteriophage integrase, which stably integrates large DNA sequences containing a specific 285-base-pair attB sequence into genomic 'pseudo-attP sites'. phi C31 integrase-based gene transfer stably integrated the COL7A1 cDNA into genomes of primary epidermal progenitor cells from four unrelated RDEB patients. Skin regenerated using these cells displayed stable correction of hallmark RDEB disease features, including Type VII collagen protein expression, anchoring fibril formation and dermal-epidermal cohesion. These findings establish a practical approach to nonviral genetic correction of severe human genetic disorders requiring stable genomic integration of large DNA sequences.

View details for DOI 10.1038/nm766

View details for PubMedID 12244305
Extrachromosomal plasmid vectors for gene therapy CURRENT OPINION IN MOLECULAR THERAPEUTICS Stoll, S. M., Calos, M. P. 2002; 4 (4): 299-305
Abstract

Extrachromosomal DNA is becoming widely utilized as a gene therapy vector. Plasmid DNA offers multiple advantages over viral gene therapy vectors, including large packaging capacity, stability without integration and reduced toxicity. Furthermore, plasmid DNA can be delivered to many different tissues, using a variety of delivery techniques currently being developed. This review will discuss the advantages of extrachromosomal DNA as a gene therapy vector, highlighting recent advances and successes in its use in vivo.

View details for Web of Science ID 000182260000003

View details for PubMedID 12222867
Phage TP901-1 site-specific integrase functions in human cells JOURNAL OF BACTERIOLOGY Stoll, S. M., Ginsburg, D. S., Calos, M. P. 2002; 184 (13): 3657-3663
Abstract

We demonstrate that the site-specific integrase encoded by phage TP901-1 of Lactococcus lactis subsp. cremoris has potential as a tool for engineering mammalian genomes. We constructed vectors that express this integrase in Escherichia coli and in mammalian cells and developed a simple plasmid assay to measure the frequency of intramolecular integration mediated by the integrase. We used the assay to document that the integrase functions efficiently in E. coli and determined that for complete reaction in E. coli, the minimal sizes of attB and attP are 31 and 50 bp, respectively. We carried out partial purification of TP901-1 integrase protein and demonstrated its functional activity in vitro in the absence of added cofactors, characterizing the time course and temperature optimum of the reaction. Finally, we showed that when expressed in human cells, the TP901-1 integrase carries out efficient intramolecular integration on a transfected plasmid substrate in the human cell environment. The TP901-1 phage integrase thus represents a new reagent for manipulating DNA in living mammalian cells.

View details for DOI 10.1128/JB.184.13.3657-3663.2002

View details for Web of Science ID 000176237400028

View details for PubMedID 12057961

View details for PubMedCentralID PMC135144
Directed evolution of a recombinase for improved genomic integration at a native human sequence NUCLEIC ACIDS RESEARCH Sclimenti, C. R., Thyagarajan, B., Calos, M. P. 2001; 29 (24): 5044-5051
Abstract

We previously established that a unidirectional site-specific recombinase, the phage phiC31 integrase, can mediate integration into mammalian chromosomes. The enzyme directs integration of plasmids bearing the phage attB recognition site into pseudo attP sites, a set of native sequences related to the phage attP recognition site. Here we use two cycles of DNA shuffling and screening in Escherichia coli to obtain evolved integrases that possess significant improvements in integration frequency and sequence specificity at a pseudo attP sequence located on human chromosome 8, when measured in the native genomic environment of living human cells. Such integrases represent custom integration tools that will be useful for modifying the genomes of higher eukaryotic cells.

View details for Web of Science ID 000172871800018

View details for PubMedID 11812835
Phage R4 integrase mediates site-specific integration in human cells GENE Olivares, E. C., Hollis, R. P., Calos, M. P. 2001; 278 (1-2): 167-176
Abstract

The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the phi C31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.

View details for Web of Science ID 000172537000017

View details for PubMedID 11707334
Epstein-Barr virus/human vector provides high-level, long-term expression of alpha(1)-antitrypsin in mice MOLECULAR THERAPY Stoll, S. M., Sclimenti, C. R., Baba, E. J., Meuse, L., Kay, M. A., Calos, M. P. 2001; 4 (2): 122-129
Abstract

We have constructed plasmid DNA vectors that contain Epstein-Barr virus (EBV) sequences and the human gene (SERPINA1) encoding alpha1-Antitrypsin (AAT). We demonstrate that a plasmid carrying the full SERPINA1 on a 19-kb genomic fragment and the EBV gene EBNA1 and its family of repeats binding sites undergoes efficient extrachromosomal replication in dividing mammalian tissue culture cells. Therefore, use of a whole genomic therapeutic gene to provide both replication and gene expression may be an effective gene therapy vector design, if the target cells are dividing. The efficacy of this same vector for expression of AAT in vivo in the nondividing cells of mouse liver was determined by hydrodynamic injection of naked plasmid DNA by means of the tail vein. A single injection of an EBV/genomic SERPINA1 vector provided >300 microg/ml of AAT, which approached normal plasma levels and persisted for the >9-month duration of the experiment. These data exceed most previously reported values, probably due to sequences in the genomic DNA that resist silencing of gene expression, possibly in combination with favorable effects on expression provided by the EBV sequences. These results demonstrate that plasmid DNA with the correct cis-acting sequences can provide in vivo long-term expression of protein at high levels that are therapeutically relevant for gene therapy.

View details for Web of Science ID 000170371100007

View details for PubMedID 11482983
Site-specific genomic integration in mammalian cells mediated by phage phi C31 integrase MOLECULAR AND CELLULAR BIOLOGY Thyagarajan, B., Olivares, E. C., Hollis, R. P., Ginsburg, D. S., Calos, M. P. 2001; 21 (12): 3926-3934
Abstract

We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.

View details for Web of Science ID 000168962200007

View details for PubMedID 11359900
An extrachromosomal tetracycline-regulatable system for mammalian cells. Nucleic acids research Sclimenti, C. R., Baba, E. J., Calos, M. P. 2000; 28 (17): E80-?
Abstract

We have modified the tetracycline-regulatable system so that all components are present on a stable extrachromosomal vector that can replicate in a wide variety of mammalian cells. An EBV/human ori vector is used to carry the system, overcoming the species specificity of conventional Epstein-Barr virus vectors. By placing the transcriptional transactivator gene under autoregulation, better induction characteristics are obtained. This system offers greater speed and sensitivity than previously reported methods. It can be applied within 3-4 weeks and produces an induction range of several hundred-fold with a low background.

View details for PubMedID 10954613

View details for PubMedCentralID PMC110717
A phage integrase directs efficient site-specific integration in human cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Groth, A. C., Olivares, E. C., Thyagarajan, B., Calos, M. P. 2000; 97 (11): 5995-6000
Abstract

The integrase from the Streptomyces phage phiC31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome. In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB. This assay was used to demonstrate that in the presence of the phiC31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes of attB and attP at 34 bp and 39 bp, respectively. Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attP into an established Epstein-Barr virus plasmid bearing attB was documented in human cells. This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.

View details for Web of Science ID 000087318700059

View details for PubMedID 10801973

View details for PubMedCentralID PMC18547
Mammalian genomes contain active recombinase recognition sites GENE Thyagarajan, B., Guimaraes, M. J., Groth, A. C., Calos, M. P. 2000; 244 (1-2): 47-54
Abstract

Recombinases derived from microorganisms mediate efficient site-specific recombination. For example, the Cre recombinase from bacteriophage P1 efficiently carries out recombination at its loxP target sites. While this enzyme can function in mammalian cells, the 34bp loxP site is expected to be absent from mammalian genomes. We have discovered that sequences from the human and mouse genomes surprisingly divergent from loxP can support Cre-mediated recombination at up to 100% of the efficiency of the native loxP site in bacterial assays. Transient assays in human cells demonstrate that such pseudo-lox sites also support Cre-mediated integration and excision in the human cell environment. Pseudo sites for Cre and other recombinases may be useful for site-specific insertion of exogenous genes into mammalian genomes during gene therapy and other genetic engineering processes.

View details for Web of Science ID 000085660700006

View details for PubMedID 10689186
Epstein-Barr virus plasmid model system for analyzing recombination in human cells PLASMID Phillips, J. E., Thyagarajan, B., Calos, M. P. 1999; 41 (3): 198-206
Abstract

Homologous recombination stimulated by a double-strand break at a desired target site offers a method to achieve site-specific integration useful for gene therapy and other genetic engineering. To test parameters needed for this strategy, we developed an Epstein-Barr virus shuttle vector model system as a genetic tool. This extrachromosomal plasmid assay system has several advantages over a chromosomal assay. The system detects all classes of recombination events without selection and allows rapid analysis of the frequency and nature of recombination events. We found that a double-strand break at the target site stimulated a large increase in recombination frequency. The resulting recombinants included one-sided insertion events, as well as two-sided or gene conversion events. A circular donor substrate was more effective in recombination than linearized donor DNA.

View details for Web of Science ID 000080916900003

View details for PubMedID 10366525
Effects of homology length and donor vector arrangement on the efficiency of double-strand break-mediated recombination in human cells SOMATIC CELL AND MOLECULAR GENETICS Phillips, J. E., Calos, M. P. 1999; 25 (2): 91-100
Abstract

We use an Epstein-Barr virus (EBV) plasmid model chromosome system to study how different donor plasmid constructs affect recombination stimulated by an I-SceI-induced double-strand break in the target sequence in human cells. The entire 3.5 kb lacZ gene was efficiently recombined into a target EBV vector lacking lacZ sequences, but having limited homology to the donor plasmid. A donor plasmid with lacZ flanked by sequence homologous to the target consistently generated gene conversion events and was more effective than a donor carrying lacZ outside the same sequence homology. Reducing the length of homology between the target and donor from 5.5 kb to 1 kb caused only a 3-fold drop in recombination frequency, contrasting with the exponential dependence on homology length seen when no DSB is present in the target. These results document a DSB-induced 175-fold increase in recombination of a heterologous gene into a target, requiring only limited flanking homology.

View details for Web of Science ID 000166887600004

View details for PubMedID 11225059
Epstein-Barr virus vectors for gene expression and transfer CURRENT OPINION IN BIOTECHNOLOGY Sclimenti, C. R., Calos, M. P. 1998; 9 (5): 476-479
Abstract

Vectors based on components of Epstein-Barr virus (EBV) have found increasingly wide applications in biotechnology. Three areas of recent advancement comprise the use of EBV vectors to improve the convenience of gene expression systems, the development of EBV vectors for gene transfer in gene therapy, and the use of EBV components to generate large vectors carrying sizable regions of genomic DNA.

View details for Web of Science ID 000076409800006

View details for PubMedID 9821275
Stability without a centromere PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Calos, M. P. 1998; 95 (8): 4084-4085
View details for Web of Science ID 000073120800003

View details for PubMedID 9539691
Assaying extrachromosomal gene therapy vectors that carry replication/persistence elements ADVANCED DRUG DELIVERY REVIEWS Sclimenti, C. R., Calos, M. P. 1998; 30 (1-3): 13-21
View details for Web of Science ID 000073361700003
EBV vectors for gene expression and delivery Current Opinion Biotechnology Calos, M.P., Sclimenti, C.R. 1998; 9
Telomeric sequences from human herpesvirus 6 do not mediate nuclear retention of episomal DNA in human cells ARCHIVES OF VIROLOGY Bulboaca, G. H., Deng, H., Dewhurst, S., Calos, M. P. 1998; 143 (3): 563-570
Abstract

Telomeric repeat sequences (TRS) have been identified close to, but not at, the genome termini of several lymphotropic herpesviruses, including human herpesviruses 6 and 7 (HHV-6, HHV-7). The functional significance of these motifs remains uncertain. Since telomeric sequences can mediate stable retention of episomal DNA in yeast, we have tested whether the TRS motifs from HHV-6 might mediate a similar function in human cells. Several candidate sequences were assessed for their ability to provide nuclear retention to an autonomously replicating vector in rapidly dividing human tissue culture cells, including HHV-6 TRS DNA, as well as telomeric DNA from human cells and sequences from Epstein-Barr virus (EBV). However, only a vector carrying the EBV-derived retention mechanism showed a significant level of nuclear retention. Neither the HHV-6 TRS motifs, nor human telomeric sequences, mediated nuclear retention of episomal DNA in human cells.

View details for Web of Science ID 000072832500013

View details for PubMedID 9572556
Assaying extrachromosomal gene therapy vectors that carry replication/persistence elements. Advanced drug delivery reviews Calos, M. P., Sclimenti, C. R. 1998; 30 (1-3): 13?21
Abstract

Persistence in the cell is a desirable property for most gene therapy vectors. For extrachromosomal vectors, persistence is limited in most cell types. To address this problem, we have developed vectors with the ability to replicate and be retained in the nucleus. These properties are conferred by specific elements present on the vectors and derived from genomic DNA and from Epstein-Barr virus. In order to begin evaluation of these vectors for use in gene therapy, we developed and present here two assays that measure the persistence of vector DNA in tissue culture cells under rapidly dividing and slowly dividing conditions. Our results indicate that inclusion of DNA replication and nuclear retention elements on a vector increases persistence of vector DNA in slowly dividing cells by at least 500%. Further improvement of the system is discussed.

View details for PubMedID 10837598
The potential of extrachromosomal replicating vectors for gene therapy TRENDS IN GENETICS Calos, M. P. 1996; 12 (11): 463-466
Abstract

Persistence of DNA vectors in target cells is advantageous in most applications of gene therapy. Particularly when target cells are undergoing proliferation, vector longevity will depend on either the integration of the vector into the chromosomes or the ability of the vector to replicate and be retained extrachromosomally. Vectors that efficiently integrate in a nonrandom fashion are currently unavailable, and those that can replicate extrachromosomally provide a major alternative strategy. Several classes of such vectors are under development, carrying mechanisms for prolonging DNA retention in mammalian nuclei that extend vector lifetime in non-proliferating cells as well. The vectors utilize either chromosomal or viral elements to mediate replication and retention, and have a large size capacity for insertion of genes of interest. I discuss the state of the art for these vectors, including the assets and limitations of their future use in gene therapy.

View details for Web of Science ID A1996VR55100017

View details for PubMedID 8973156
Long-term gene expression from autonomously replicating vectors in mammalian cells GENE THERAPY Wohlgemuth, J. G., Kang, S. H., Bulboaca, G. H., Nawotka, K. A., Calos, M. P. 1996; 3 (6): 503-512
Abstract

We tested the longevity of gene expression provided by autonomously replicating vectors. The vectors contain segments of human genomic DNA that provide efficient replication initiation and sequences derived from Epstein-Barr virus that provide nuclear retention. In order to monitor gene expression, the vectors also carry an expression cassette containing the chloramphenicol acetyl transferase gene. Replicating and control vectors were transfected into rapidly dividing human 293 cells, and gene expression and DNA retention were monitored. Gene expression decreased rapidly from a control vector lacking replication and retention functions, reaching near background levels by 10 days. Vectors having both replication and retention showed greatly prolonged gene expression, with vector DNA still detectable after 2 months. Autonomously replicating vectors also prolonged gene expression in rodent cells, in human lung epithelial cells, and in slowly dividing cell cultures. These results demonstrate the utility of this autonomous replication system for long-term retention and expression of introduced genes in mammalian cells. Such vectors could be useful for gene therapy, in combination with any of several methods for introduction of DNA.

View details for Web of Science ID A1996UR30200008

View details for PubMedID 8789800
REPLICATION OF CENTROMERE-II OF SCHIZOSACCHAROMYCES-POMBE MOLECULAR AND CELLULAR BIOLOGY Smith, J. G., CADDLE, M. S., Bulboaca, G. H., Wohlgemuth, J. G., Baum, M., Clarke, L., Calos, M. P. 1995; 15 (9): 5165-5172
Abstract

The centromeric DNAs of Schizosaccharomyces pombe chromosomes resemble those of higher eukaryotes in being large and composed predominantly of repeated sequences. To begin a detailed analysis of the mode of replication of a complex centromere, we examined whether any sequences within S. pombe centromere II (cen2) have the ability to mediate autonomous replication. We found a high density of segments with such activity, including at least eight different regions comprising most of the repeated and unique centromeric DNA elements. A physical mapping analysis using two-dimensional gels showed that autonomous replication initiated within the S. pombe sequences in each plasmid. A two-dimensional gel analysis of replication on the chromosomes revealed that the K and L repeat elements, which occur in multiple copies at all three centromeres and comprise approximately 70% of total centromeric DNA mass in S. pombe, are both sites of replication initiation. In contrast, the unique cen2 central core, which contains multiple segments that can support autonomous replication, appears to be repressed for initiation on the chromosome. We discuss the implications of these findings for our understanding of DNA replication and centromere function.

View details for Web of Science ID A1995RQ13400055

View details for PubMedID 7651433

View details for PubMedCentralID PMC230763
AUTONOMOUS REPLICATION IN DROSOPHILA-MELANOGASTER TISSUE-CULTURE CELLS CHROMOSOMA Smith, J. G., Calos, M. P. 1995; 103 (9): 597-605
Abstract

This study addresses the ability of DNA fragments from various sources to mediate autonomous DNA replication in cultured Drosophila melanogaster cells. We created a series of plasmids containing genomic DNA fragments from the Ultrabithorax gene of Drosophila and test ed them for autonomous replication after transfection into Schneider line 2 cells. We found that all plasmids containing Drosophila DNA were able to replicate autonomously, as were random human and Escherichia coli genomic DNA fragments. Most of the plasmids were detectable 18 days after transfection in the absence of selection, suggesting that transfected DNA is maintained in Drosophila cells without rapid loss or degradation. The finding that all plasmids containing Drosophila, human or bacterial DNA replicate autonomously in Drosophila cells suggests that the signals that direct autonomous replication in Drosophila contain a low degree of sequence specificity. A two-dimensional gel analysis of initiation on one of the plasmids was consistent with many dispersed initiation sites. Low sequence specificity and dispersed initiation sites also characterize autonomous replication in human cells and Xenopus eggs and may be general properties of autonomous replication in animal cells.

View details for Web of Science ID A1995QZ59100003

View details for PubMedID 7587582
STABLE AUTONOMOUSLY REPLICATING VECTORS FOR GENE-THERAPY Calos, M. P., Wohlgemuth, J. G., Kang, S. H., Bulboaca, G. H., Nawotka, K. A. WILEY-BLACKWELL. 1995: 389?389
View details for Web of Science ID A1995QT86401409
PHYSICAL MAPPING OF ORIGINS OF REPLICATION IN THE FISSION YEAST SCHIZOSACCHAROMYCES-POMBE MOLECULAR BIOLOGY OF THE CELL Wohlgemuth, J. G., Bulboaca, G. H., Moghadam, M., CADDLE, M. S., Calos, M. P. 1994; 5 (8): 839-849
Abstract

We isolated four fragments from the Schizosaccharomyces pombe genome that mediate autonomous replication. A two-dimensional gel analysis revealed that in each case initiation could be mapped to within the S. pombe sequences. In three of the fragments, initiation could be mapped to one discrete location. In the fourth fragment, subcloning and two-dimensional gel analysis suggested that two discrete origins of replication were located within 3 kb of each other. When in proximity, usually only one of these origins fired, suggesting origin interference. Two-dimensional gel analysis of the four origin fragments at their genomic locations demonstrated that each is used in the chromosomes, but in only a subset of cells or cell divisions. The S. pombe genome appears to contain many discrete origins, not all of which fire in any given cell and some of which are closely spaced. Not I/Sfi I mapping of the five origins from this and a previous study indicates that they are randomly distributed throughout the genome and appear to be representative of chromosomal origins of replication in this organism. We compare the features of S. pombe replication origins with those of S. cerevisiae and animal cells.

View details for Web of Science ID A1994PE82700002

View details for PubMedID 7803852
TRANSCRIPTION INHIBITS THE REPLICATION OF AUTONOMOUSLY REPLICATING PLASMIDS IN HUMAN-CELLS MOLECULAR AND CELLULAR BIOLOGY Haase, S. B., HEINZEL, S. S., Calos, M. P. 1994; 14 (4): 2516-2524
Abstract

This study addresses the effect of transcription on replication, using a system based on autonomously replicating plasmids in human cells. We added transcriptional elements from the human cytomegalovirus promoter/enhancer and the human beta-actin promoter to autonomously replicating plasmids based on human sequences and found that the transcriptional elements inhibited plasmid replication. Furthermore, conditional inhibition of plasmid replication was demonstrated by using a tetracycline-responsive promoter. We found that replication activity of plasmids carrying this promoter was inversely correlated with promoter activity. Replication activity was partially restored on plasmids when a transcriptional termination sequence was placed directly downstream of the promoter element. Transcriptional activity of the promoters and the efficacy of the terminator sequence were confirmed by using steady-state RNA analysis. These experiments suggest that transcription inhibits DNA replication on these plasmids and that the degree of inhibition is dependent on transcription strength. The possible significance of these results for chromosomal DNA replication are discussed.

View details for Web of Science ID A1994NC05700031

View details for PubMedID 8139554
SPECIFIC INITIATION AT AN ORIGIN OF REPLICATION FROM SCHIZOSACCHAROMYCES-POMBE MOLECULAR AND CELLULAR BIOLOGY CADDLE, M. S., Calos, M. P. 1994; 14 (3): 1796-1805
Abstract

Using a genetic assay for efficient autonomous replication, we have isolated from Schizosaccharomyces pombe a 6.2-kb fragment which shows the properties expected of an origin of DNA replication in S. pombe. A 2.8-kb subclone of the fragment has the same replication properties. Two-dimensional gel analysis of replication intermediates throughout plasmids carrying the 6.2- or 2.8-kb fragments shows that replication initiates only in a specific region, which can be localized to within several hundred base pairs, in the fragments. This region is also a site of replication initiation in the S. pombe chromosome where the fragments normally reside. These results provide strong evidence that initiation of replication in S. pombe is localized and mediated by specific DNA sequence signals.

View details for Web of Science ID A1994MX64000026

View details for PubMedID 8114712
EPSTEIN-BARR VIRUS-BASED VECTORS THAT REPLICATE IN RODENT CELLS GENE Krysan, P. J., Calos, M. P. 1993; 136 (1-2): 137-143
Abstract

Vectors based on Epstein-Barr virus (EBV) have been useful for cloning and gene expression studies in primate cells. However, these vectors do not replicate in rodent cells. We demonstrate here that the addition of large fragments of mammalian DNA to vectors containing the EBNA-1 gene (encoding the Epstein-Barr nuclear antigen-1) and the family of repeats from EBV generates autonomously replicating vectors which are stably maintained as extrachromosomal plasmids in hamster cells. By using a density-shift assay in Cs2SO4 density gradients, we demonstrate that the plasmids replicate once per cell cycle at high efficiency. These plasmids represent, for rodent cells, the only available class of stable, autonomous vectors replicating once per cell cycle. It is likely that this type of EBV-based autonomous replication system can also be extended to other organisms for which stable, extrachromosomal plasmid systems are not available.

View details for Web of Science ID A1993MU76700017

View details for PubMedID 8293997
AUTONOMOUS REPLICATION IN HUMAN-CELLS OF MULTIMERS OF SPECIFIC HUMAN AND BACTERIAL-DNA SEQUENCES MOLECULAR AND CELLULAR BIOLOGY Krysan, P. J., Smith, J. G., Calos, M. P. 1993; 13 (5): 2688-2696
Abstract

Using modules of a specific 2,712-bp human DNA sequence and a specific 2,557-bp Escherichia coli DNA sequence, we created plasmids containing between 1 and 12 modules of single or chimeric sequence composition and tested them in human cells for their autonomous replication ability. We found that replication efficiency per generation increased with successive addition of human modules, to essentially 100% by six copies. Although a single copy of the bacterial module had negligible replication ability, the replication efficiency per generation of 12 bacterial modules was 66%. Chimeras composed of human and bacterial modules displayed intermediate replication levels. We also used two-dimensional gel electrophoresis to physically map where replication initiated on a half human-half E. coli plasmid. Our results suggest that autonomous replication in human cells is stimulated by simple sequence features which occur frequently in human DNA but are more rare in bacterial DNA.

View details for Web of Science ID A1993KY63200006

View details for PubMedID 8386315

View details for PubMedCentralID PMC359641
THE REPLICATION BEHAVIOR OF SACCHAROMYCES-CEREVISIAE DNA IN HUMAN-CELLS CHROMOSOMA Tran, C. T., CADDLE, M. S., Calos, M. P. 1993; 102 (2): 129-136
Abstract

We studied the replication of random genomic DNA fragments from Saccharomyces cerevisiae in a long-term assay in human cells. Plasmids carrying large yeast DNA fragments were able to replicate autonomously in human cells. Efficiency of replication of yeast DNA fragments was comparable to that of similarly sized human DNA fragments and better than that of bacterial DNA. This result suggests that yeast genomic DNA contains sequence information needed for replication in human cells. To examine whether DNA replication in human cells would initiate specifically at a yeast origin of replication, we monitored initiation on a plasmid containing the yeast 2-micron autonomously replicating sequence (ARS) in yeast and human cells. We found that while replication initiates at the 2-micron ARS in yeast, it does not preferentially initiate at the ARS in human cells. This result suggests that the sequences that direct site specific replication initiation in yeast do not function in the same way in human cells, which initiate replication at a broader range of sequences.

View details for Web of Science ID A1993KH00700008

View details for PubMedID 8432194
ANALYSIS OF THE AUTONOMOUS REPLICATION BEHAVIOR IN HUMAN-CELLS OF THE DIHYDROFOLATE-REDUCTASE PUTATIVE CHROMOSOMAL ORIGIN OF REPLICATION NUCLEIC ACIDS RESEARCH CADDLE, M. S., Calos, M. P. 1992; 20 (22): 5971-5978
Abstract

Chinese hamster genomic DNA sequences from the region downstream of the dihydrofolate reductase (DHFR) gene reported to contain a chromosomal origin of bidirectional DNA replication (OBR-1) were tested for their ability to support autonomous DNA replication in human cells. A 13.3 kilobase fragment containing OBR-1 and surrounding sequences supported replication in short-term and long-term replication assays, while a 4.5 kb fragment containing OBR-1 did not support substantial replication in either assay. These results are consistent with our previous observations that large fragments of human DNA support replication, while smaller fragments are less efficient. The replication activities of plasmids containing OBR-1 were no greater than those of randomly chosen human fragments of similar size. Furthermore, two-dimensional gel analysis of plasmids containing OBR-1 indicated that initiation does not preferentially occur within the OBR-1 region. These results suggest that in the context of autonomous replication, the DHFR sequences tested do not contain genetic information specifying site-specific replication initiation. Possible implications of these results for chromosomal replication are discussed.

View details for Web of Science ID A1992KB04900012

View details for PubMedID 1461730
REPLICATION CONTROL OF AUTONOMOUSLY REPLICATING HUMAN SEQUENCES NUCLEIC ACIDS RESEARCH Haase, S. B., Calos, M. P. 1991; 19 (18): 5053-5058
Abstract

Three autonomously replicating plasmids carrying human genomic DNA and a vector derived from Epstein-Barr virus were studied by density labelling to determine the number of times per cell cycle these plasmids replicate in human cells. Each of the plasmids replicated semi-conservatively once per cell cycle. The results suggest that these human autonomously replicating sequences undergo replication following the same controls as chromosomal DNA and represent a good model system for studying chromosomal replication. We also determined the time within the S phase of the cell cycle that three of the plasmids replicate. Centromeric alpha sequences, which normally replicate late in S phase when in their chromosomal context, were found to replicate earlier when they mediate replication on an extrachromosomal vector. Reproducible patterns of replication within S phase were found for the plasmids, suggesting that the mechanism specifying time of replication may be subject to experimental analysis with this system.

View details for Web of Science ID A1991GH66000035

View details for PubMedID 1656387
AUTONOMOUS DNA-REPLICATION IN HUMAN-CELLS IS AFFECTED BY THE SIZE AND THE SOURCE OF THE DNA MOLECULAR AND CELLULAR BIOLOGY HEINZEL, S. S., Krysan, P. J., Tran, C. T., Calos, M. P. 1991; 11 (4): 2263-2272
Abstract

We previously developed short-term and long-term assays for autonomous replication of DNA in human cells. This study addresses the requirements for replication in these assays. Sixty-two random human genomic fragments ranging in size from 1 to 21 kb were cloned in a prokaryotic vector and tested for their replication ability in the short-term assay. We found a positive correlation between replication strength and fragment length, indicating that large size is favored for efficient autonomous replication in human cells. All large fragments replicated efficiently, suggesting that signals which can direct the initiation of DNA replication in human cells are either very abundant or have a low degree of sequence specificity. Similar results were obtained in the long-term assay. We also used the same assays to test in human cells a random series of fragments derived from Escherichia coli chromosomal DNA. The bacterial fragments supported replication less efficiently than the human fragments in the short-term and long-term assays. This result suggests that while the sequence signals involved in replication in human cells are found frequently in human DNA, they are uncommon in bacterial DNA.

View details for Web of Science ID A1991FC85200057

View details for PubMedID 1900922
REPLICATION INITIATES AT MULTIPLE LOCATIONS ON AN AUTONOMOUSLY REPLICATING PLASMID IN HUMAN-CELLS MOLECULAR AND CELLULAR BIOLOGY Krysan, P. J., Calos, M. P. 1991; 11 (3): 1464-1472
Abstract

We have used a two-dimensional gel electrophoresis mapping technique to determine where DNA replication initiates on a plasmid which utilizes a fragment of human DNA to replicate autonomously in human cells. Replication was found to initiate at multiple locations on the plasmid carrying the human sequence, in contrast to the pattern seen for an Epstein-Barr virus vector which served as a control with a fixed origin. The family of repeats, a portion of the Epstein-Barr virus origin of replication which is present our plasmid, was shown to function as a replication fork barrier. The nature of the stalled replicative intermediates on the human DNA-based plasmid further indicated that replication did not initiate at a single fixed position each time the plasmid replicated. The results suggest that the replication apparatus used to duplicate DNA in human cells may not have precise sequence requirements which target initiation to specific locations.

View details for Web of Science ID A1991EZ33100030

View details for PubMedID 1996103
The molecular biology of mutation in human cells: From shuttle vectors to artificial chromosomes The Cellular and Molecular Biology of Human Carcinogenesis Calos, M. P. 1990: 139?151
ISOLATION OF HUMAN SEQUENCES THAT REPLICATE AUTONOMOUSLY IN HUMAN-CELLS MOLECULAR AND CELLULAR BIOLOGY Krysan, P. J., Haase, S. B., Calos, M. P. 1989; 9 (3): 1026-1033
Abstract

We have isolated a heterogeneous collection of human genomic sequences which replicate autonomously when introduced into human cells. The novel strategy for the isolation of these sequences involved cloning random human DNA fragments into a defective Epstein-Barr virus vector. This vector alone was unable to replicate in human cells, but appeared to provide for the nuclear retention of linked DNA. The human sequences persist in a long-term replication assay (greater than 2 months) in the presence of the viral nuclear retention sequences. Using a short-term (4-day) assay, we showed that the human sequences are able to replicate in the absence of all viral sequences. The plasmids bearing human sequences were shown to replicate based on the persistence of MboI-sensitive plasmid DNA in the long-term assay and the appearance of DpnI-resistant DNA in the short-term assay. The human sequences were shown to be responsible for the replication activity and may represent authentic human origins of replication.

View details for Web of Science ID A1989T444300017

View details for PubMedID 2542763
IMPROVED EBV SHUTTLE VECTORS MUTATION RESEARCH Haase, S. B., HEINZEL, S. S., Krysan, P. J., Calos, M. P. 1989; 220 (2-3): 125-132
Abstract

Shuttle vectors based on Epstein-Barr virus (EBV) replicate autonomously in the nuclei of human cells. These vectors represent reasonable models for chromosomes, have low background mutation frequencies, and have been useful for studying induced mutation in human cells. Two improvements in the EBV vector system are discussed. Attempts are described to increase vector copy number per cell by using a limited period of replication driven by the simian virus 40 (SV40) origin of replication. Isolation of human sequences that can replace the viral origin of replication in providing for autonomous replication of the vectors is also described. These improvements are leading toward shuttle vectors that are more efficient and more closely resemble authentic chromosomes.

View details for Web of Science ID A1989T814800011

View details for PubMedID 2538736
COMPARISON OF ULTRAVIOLET IRRADIATION-INDUCED MUTAGENESIS OF THE LACI GENE IN ESCHERICHIA-COLI AND IN HUMAN-293 CELLS JOURNAL OF MOLECULAR BIOLOGY Hsia, H. C., Lebkowski, J. S., Leong, P. M., Calos, M. P., Miller, J. H. 1989; 205 (1): 103-113
Abstract

We report the sequence changes in the Escherichia coli lacI gene in 133 mutants detected after passage of an ultraviolet-irradiated shuttle vector human 293 cells. The results are compared with our previous studies of the lacI gene after ultraviolet light treatment in E. coli. In human cells, base substitutions predominate, and frameshifts are found much less frequently than in bacteria. The most frequent base change is the G.C to A.T transition. Overall, 110 to 112 transitions were G.C to A.T. Some of the hotspots seen in lacI in bacteria are prominent also in human 293 cells, suggesting that the same lesions are targeting mutations in both systems. Transitions are found almost exclusively at sequences at which pyrimidine-pyrimidine photoproducts can form. The data are consistent with the notion that a significant fraction of ultraviolet irradiation-induced mutagenesis in mammalian systems occurs by adding an A across from a photolesion. Double mutations are significantly more frequent in human cells than in bacteria. Reasons for this difference are discussed.

View details for Web of Science ID A1989R914200008

View details for PubMedID 2647996
USE OF SIMIAN VIRUS-40 REPLICATION TO AMPLIFY EPSTEIN-BARR VIRUS SHUTTLE VECTORS IN HUMAN-CELLS JOURNAL OF VIROLOGY HEINZEL, S. S., Krysan, P. J., Calos, M. P., DuBridge, R. B. 1988; 62 (10): 3738-3746
Abstract

We have increased the copy number of Epstein-Barr virus vectors that also carry the origin of replication of simian virus 40 (SV40) by providing a transient dose of SV40 T antigen. T antigen was supplied in trans by transfection of a nonreplicating plasmid which expresses T antigen into cells carrying Epstein-Barr virus-SV40 vectors. A significant increase in vector copy number occurred over the next few days. We also observed a high frequency of intramolecular recombination when the vector carried a repeat segment in direct orientation, but not when the repeat was in inverted orientation or absent. Furthermore, by following the mutation frequency for a marker on the vector after induction of SV40 replication, it was determined that SV40 replication generates a detectable increase in the deletion frequency but no measurable increase in the frequency of point mutations.

View details for Web of Science ID A1988Q064500026

View details for PubMedID 2843671
RECOMBINANT SHUTTLE VECTORS FOR THE STUDY OF MUTATION IN MAMMALIAN-CELLS MUTAGENESIS DuBridge, R. B., Calos, M. P. 1988; 3 (1): 1-9
View details for Web of Science ID A1988L773200001

View details for PubMedID 3282139
Use of lacI shuttle systems for analysis of mutation in human cells DNA Repair Calos, M. P. Marcel Dekker, New York. 1988: 277?294
MOLECULAR APPROACHES TO THE STUDY OF GENE MUTATION IN HUMAN-CELLS TRENDS IN GENETICS DuBridge, R. B., Calos, M. P. 1987; 3 (10): 293-297
View details for Web of Science ID A1987K746300006
ANALYSIS OF MUTATION IN HUMAN-CELLS BY USING AN EPSTEIN-BARR-VIRUS SHUTTLE SYSTEM MOLECULAR AND CELLULAR BIOLOGY DuBridge, R. B., Tang, P., Hsia, H. C., Leong, P. M., Miller, J. H., Calos, M. P. 1987; 7 (1): 379-387
Abstract

We developed highly sensitive shuttle vector systems for detection of mutations formed in human cells using autonomously replicating derivatives of Epstein-Barr virus (EBV). EBV vectors carrying the bacterial lacI gene as the target for mutation were established in human cells and later returned to Escherichia coli for rapid detection and analysis of lacI mutations. The majority of the clonal cell lines created by establishment of the lacI-EBV vector show spontaneous LacI- frequencies of less than 10(-5) and are suitable for studies of induced mutation. The ability to isolate clonal lines represents a major advantage of the EBV vectors over transiently replicating shuttle vectors (such as those derived from simian virus 40) for the study of mutation. The DNA sequence changes were determined for 61 lacI mutations induced by exposure of one of the cell lines to N-nitroso-N-methylurea. A total of 33 of 34 lacI nonsense mutations and 26 of 27 missense mutations involve G X C to A X T transitions. These data provide support for the mutational theory of cancer.

View details for Web of Science ID A1987F500500046

View details for PubMedID 3031469
EBV-lacI shuttle vectors: Addition of a selection for LacI mutants Mammalian Cell Mutagenesis Van Den Berg, D. J., DuBridge, R. B., Calos, M. P. Cold Spring Harbor Press, Cold Spring Harbor, New York. 1987: 307?314
Gene Transfer Vectors for Mammalian Cells Miller, J. H., Calos, M. P. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 1987
Determination of DNA sequence changes induced by ethyl methanesulfonate in human cells, using a shuttle vector system Molecular and Cellular Biology Lebkowski, J. S., Miller, J. H., Calos, M. P. 1986; 6: 1838-1842
Mutation of autonomously replicating plasmids Gene Transfer Calos, M. P. Plenum Press, New York. 1986: 243?261
THE LACI SHUTTLE - RAPID ANALYSIS OF THE MUTAGENIC SPECIFICITY OF ULTRAVIOLET-LIGHT IN HUMAN-CELLS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lebkowski, J. S., Clancy, S., Miller, J. H., Calos, M. P. 1985; 82 (24): 8606-8610
Abstract

A system has been devised that allows the effect of mutagens acting in human cells to be readily analyzed at the DNA sequence level. The bacterial gene lacI, carried on a shuttle vector, is introduced into human tissue culture cells by transfection and allowed to replicate in the cell nucleus. Twenty-four to 48 hr after transfection, the cells are exposed to a mutagen. After 1-2 days of further replication, vector DNA is purified and transfected back into Escherichia coli for scoring and analysis of mutations in lacI. The nucleotide sequence changes for 53 UV light-induced mutations have been deduced in this way. Most of the mutations are transitions and occur at pyrimidine-pyrimidine sequences. The mutagenic specificity observed closely resembles that of UV light in E. coli, suggesting that human and bacterial cells respond similarly to damage from UV light. Use of the lacI shuttle in this way should permit determination of the mutagenic specificity of a wide range of mutagens and carcinogens in human cells.

View details for Web of Science ID A1985AXL4200071

View details for PubMedID 3001711

View details for PubMedCentralID PMC390966
SIMIAN VIRUS-40 REPLICATION IN ADENOVIRUS-TRANSFORMED HUMAN-CELLS ANTAGONIZES GENE-EXPRESSION NATURE Lebkowski, J. S., Clancy, S., Calos, M. P. 1985; 317 (6033): 169-171
Abstract

Simian virus 40 (SV40) replicates efficiently in monkey kidney cells. However, we have now found that SV40-based vectors transfected into most human cells replicate poorly, if at all. In contrast, strong SV40 replication is observed in human embryonic kidney (HEK) cells transformed with the adenovirus early region, but not in untransformed HEK cells. Vector replication in adenovirus-transformed cells is dependent on the presence of the SV40 origin of replication and large-T antigen. However, vigorous replication occurs at levels of large-T antigen that are undetectable by immunofluorescence. These data suggest that the adenovirus oncogenes create a replication-permissive environment to which the SV40 replicon responds. Furthermore, replication and gene expression seem to be antagonistic on our vectors. High levels of large-T antigen are observed only when vector replication is blocked by mutations in the gene for large-T antigen or the origin of replication, or by direct inhibition of DNA polymerase with aphidicolin.

View details for Web of Science ID A1985AQK6900057

View details for PubMedID 2993921
Amplification of a bovine papilloma virus-simian virus 40 chimera Journal of Virology DuBridge, R. B., Lucky, M., Botchan, M. R., Calos, M. P. 1985; 56: 625-627
TRANSFECTED DNA IS MUTATED IN MONKEY, MOUSE, AND HUMAN-CELLS MOLECULAR AND CELLULAR BIOLOGY Lebkowski, J. S., DuBridge, R. B., ANTELL, E. A., GREISEN, K. S., Calos, M. P. 1984; 4 (10): 1951-1960
Abstract

Papovavirus-based shuttle vectors containing the bacterial lacI gene were used to show that a mutation frequency in the range of 1% occurs in lacI when such vectors are transfected into COS7 and CV-1 simian cells, NIH 3T3, 3T6, L, and C127 mouse cells, and human 293 and HeLa cells. This frequency is approximately four orders of magnitude higher than the spontaneous mutation frequency in either mammalian or bacterial cells. The mutations are predominantly base substitutions and deletions and also include insertions from the mammalian genome. Time course experiments argue that mutagenesis occurs soon after arrival of the DNA into the nucleus. However, replication of the vector is not required since mutations occur even when the vector lacks all viral sequences. The high mutation frequency appears to be the characteristic outcome of transfection of DNA into mammalian cells.

View details for Web of Science ID A1984TL59600002

View details for PubMedID 6095032
DELETION OF PLASMID SEQUENCES DURING SACCHAROMYCES-CEREVISIAE TRANSFORMATION JOURNAL OF BACTERIOLOGY Clancy, S., Mann, C., Davis, R. W., Calos, M. P. 1984; 159 (3): 1065-1067
Abstract

Saccharomyces cerevisiae was transformed with DNA by the lithium acetate method. Mutation of nonselected markers on the transforming vector was observed at a frequency several orders of magnitude higher than spontaneous mutation frequencies. These mutations were shown to be deletions. Linearization of the vector before transformation stimulated deletion formation.

View details for Web of Science ID A1984TG95800042

View details for PubMedID 6090387

View details for PubMedCentralID PMC215771
SPECIFICITY OF MUTATIONS INDUCED IN TRANSFECTED DNA BY MAMMALIAN-CELLS EMBO JOURNAL Miller, J. H., Lebkowski, J. S., GREISEN, K. S., Calos, M. P. 1984; 3 (13): 3117-3121
Abstract

DNA transfected into mammalian cells is subject to the high mutation frequency of approximately 1% per gene. We present data bearing on the derivation of the two main classes of mutations detected, base substitutions and deletions. The DNA sequence change is reported for nearly 100 independent base substitution mutations that occurred in shuttle vectors as a result of passage in simian cells. All of the mutations occur at G:C base pairs and involve either transition to A:T or transversion to T:A. To identify possible mutational intermediates, various topological forms of the vector DNA were introduced separately. Supercoiled and relaxed DNA are mutated at equal frequencies. However, linearized DNA leads to a greatly elevated frequency of deletions. Nicked and gapped templates stimulate both deletions and base substitutions. We discuss a model involving intracellular degradation of the transfected DNA which explains these observations.

View details for Web of Science ID A1984TX37100013

View details for PubMedID 6098464

View details for PubMedCentralID PMC557826
Genomic reorganization: mobile genetic elements. Science Calos, M. 1983; 221 (4618): 1366-1367
View details for PubMedID 17759003
HIGH MUTATION FREQUENCY IN DNA TRANSFECTED INTO MAMMALIAN-CELLS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Calos, M. P., Lebkowski, J. S., Botchan, M. R. 1983; 80 (10): 3015-3019
Abstract

The lacI gene of Escherichia coli was used to score mutation in mammalian cells of simian virus 40-based recombinant DNA vectors that provide for replication and selection in both bacterial and mammalian cells. Plasmid DNA was introduced into COS7 simian cells by DEAE-dextran transfection, allowed to replicate in the mammalian cells, and then returned to E. coli for analysis. Mutants in lacI were observed at frequencies of one to several percent, compared with a spontaneous mutation rate in E. coli of less than 10(-5). The lesions include a large number of base substitutions, in addition to deletions, duplications, and more complex rearrangements, including insertion into the plasmid of sequences originating in the host genome. We discuss possible sources of the high mutation frequency and its implications for experiments involving DNA transfer.

View details for Web of Science ID A1983QR49600044

View details for PubMedID 6574469
ON THE FORMATION OF SPONTANEOUS DELETIONS - THE IMPORTANCE OF SHORT SEQUENCE HOMOLOGIES IN THE GENERATION OF LARGE DELETIONS CELL Albertini, A. M., Hofer, M., Calos, M. P., Miller, J. H. 1982; 29 (2): 319-328
Abstract

Using lacl-Z fusion strains of Escherichia coli we have devised systems that detect deletions of varying lengths. We examined deletions 700-1000 base pairs long, and genetically characterized over 250 spontaneous deletions. Of these, we analyzed 24 by direct DNA sequencing and 18 by inspection of restriction fragment patterns. Deletions of this size occur almost exclusively at short repeated sequences in both (recA+ and recA- strain backgrounds, but are detected 25-fold more frequently in a recA+ background. The frequency of deletion formation correlates with the extent of homology between the short repeated sequences, although other factors may be involved. The largest hotspot, which accounts for 60% of the deletions detected, involves the largest homology in the system (14 of 17 base pairs). Altering a single base pair within this homology reduces deletion incidence by an order of magnitude. We discuss possible mechanisms of deletion formation and consider its relationship to the excision of transposable elements.

View details for Web of Science ID A1982NV49700005

View details for PubMedID 6288254
GENETIC AND SEQUENCE-ANALYSIS OF FRAMESHIFT MUTATIONS INDUCED BY ICR-191 JOURNAL OF MOLECULAR BIOLOGY Calos, M. P., Miller, J. H. 1981; 153 (1): 39-64
View details for Web of Science ID A1981MT04000003

View details for PubMedID 7040679
THE DNA-SEQUENCE CHANGE RESULTING FROM THE IQ1 MUTATION, WHICH GREATLY INCREASES PROMOTER STRENGTH MOLECULAR & GENERAL GENETICS Calos, M. P., Miller, J. H. 1981; 183 (3): 559-560
View details for Web of Science ID A1981MP91300026

View details for PubMedID 7038381
FURTHER CORRELATIONS OF THE LACI GENETIC-MAP WITH THE DNA-SEQUENCE JOURNAL OF MOLECULAR BIOLOGY Miller, J. H., Calos, M. P., Combepine, C., Hofer, M. 1981; 153 (1): 65-66
View details for Web of Science ID A1981MT04000004

View details for PubMedID 7040680
Genetic and sequencing studies of the specificity of transposition into the lac region of E. coli. Cold Spring Harbor symposia on quantitative biology Miller, J. H., Calos, M. P., Galas, D. J. 1981; 45: 243-257
View details for PubMedID 6271472
SEQUENCE-ANALYSIS OF TN9 INSERTIONS IN THE LACZ GENE JOURNAL OF MOLECULAR BIOLOGY Galas, D. J., Calos, M. P., Miller, J. H. 1980; 144 (1): 19-41
View details for Web of Science ID A1980KS38000002

View details for PubMedID 6260963
DNA-SEQUENCE ALTERATION RESULTING FROM A MUTATION IMPAIRING PROMOTER FUNCTION IN THE LAC REPRESSOR GENE MOLECULAR AND GENERAL GENETICS Calos, M. P., Miller, J. H. 1980; 178 (1): 225-227
View details for Web of Science ID A1980JR77700028

View details for PubMedID 6770231
MOLECULAR CONSEQUENCES OF DELETION FORMATION MEDIATED BY THE TRANSPOSON TN9 NATURE Calos, M. P., Miller, J. H. 1980; 285 (5759): 38-41
View details for Web of Science ID A1980JQ34300038

View details for PubMedID 6246435
TRANSPOSABLE ELEMENTS CELL Calos, M. P., Miller, J. H. 1980; 20 (3): 579-595
View details for Web of Science ID A1980JZ69100001

View details for PubMedID 6251969
GENETIC AND SEQUENCING STUDIES OF THE SPECIFICITY OF TRANSPOSITION INTO THE LAC REGION OF ESCHERICHIA-COLI COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Miller, J. H., Calos, M. P., Galas, D. J. 1980; 45: 243-257
View details for Web of Science ID A1980LV23700036
GENETIC-ANALYSIS OF TRANSPOSITIONS IN THE LAC REGION OF ESCHERICHIA-COLI JOURNAL OF MOLECULAR BIOLOGY Miller, J. H., Calos, M. P., Galas, D., Hofer, M., BUCHEL, D. E., MULLERHILL, B. 1980; 144 (1): 1-18
View details for Web of Science ID A1980KS38000001

View details for PubMedID 6260962
GENETIC AND SEQUENCING STUDY OF IS1 AND TN9 INSERTIONS IN THE ESCHERICHIA-COLI LAC OPERON COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Calos, M., Johnsrud, L., Miller, J. H. 1979; 43: 1263-1268
View details for Web of Science ID A1978HF86000071

View details for PubMedID 385226
DNA-SEQUENCE AT INTEGRATION SITES OF INSERTION ELEMENT IS1 CELL Calos, M. P., Johnsrud, L., Miller, J. H. 1978; 13 (3): 411-418
Abstract

We have detected two independent occurrences of insertion mutations in the lacl gene of E. Coli, and have used small plasmids carrying the l gene to purify large amounts of DNA containing these insertions. Analyses with restriction endonucleases and DNA sequencing techniques establish that both insertions involve the previously characterized element IS1. In each case, the integration of IS1 into the l gene DNA is associated with a directly repeated sequence of 9 nucleotides appearing at each end of the insertion element. Since one of these sequences was present in the wild-type gene, the second sequence either preexisted in the IS1 before integration, or else was generated by the process of insertion itself. The 9 base repeat is different in both cases. We discuss the relevance of these findings to the mechanism of integration of transposable elements.

View details for Web of Science ID A1978ES36300001

View details for PubMedID 350411
TRANSPOSON TN9 GENERATES A 9-BP REPEATED SEQUENCE DURING INTEGRATION CELL Johnsrud, L., Calos, M. P., Miller, J. H. 1978; 15 (4): 1209-1219
Abstract

We performed a genetic and sequencing analysis of insertions of the transposon Tn9 into the lac operon of E. coli. Genetic mapping of 70 insertions into lacl and Z shows that starting from the same point on the chromosome, Tn9 goes to at least 50 different points in these two genes. Although there are preferred regions for insertion, these consist of multiple integration points within a small area, as demonstrated by pairwise crosses and restriction mapping. Sequence analysis of three Tn9 insertions reveals that Tn9 integration is associated with a direct repeat of 9 base pairs (bp) of host sequence. We show that these extra 9 nucleotide pairs are generated upon insertion and not brought in with the element.

View details for Web of Science ID A1978GC55400009

View details for PubMedID 365355
DNA-SEQUENCE FOR A LOW-LEVEL PROMOTER OF LAC REPRESSOR GENE AND AN UP PROMOTER MUTATION NATURE Calos, M. P. 1978; 274 (5673): 762-765
Abstract

The promoter for a weakly expressed constitutive gene, the lactose repressor gene (lacI), has been sequenced, along with an 'up' promoter mutation Iq. The 10-fold enhancement in I expression found in Iq is the result of a single base change at position -35. To facilitate the sequencing, the lacI gene was cloned in a small plasmid.

View details for Web of Science ID A1978FL81400022

View details for PubMedID 355890

Michele Calos

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