Bio

Education & Certifications


  • BS, University of Wisconsin, Madison, Molecular Biology (2012)

Publications

All Publications


  • Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector. iScience Mizuno, N., Mizutani, E., Sato, H., Kasai, M., Ogawa, A., Suchy, F., Yamaguchi, T., Nakauchi, H. 2018; 9: 286–97

    Abstract

    Intra-embryo genome editing by CRISPR/Cas9 enables easy generation of gene-modified animals by non-homologous end joining (NHEJ)-mediated frameshift mutations or homology-directed repair (HDR)-mediated point mutations. However, large modifications, such as gene replacement or gene fusions, are still difficult to introduce in embryos without costly micromanipulators. Moreover, micromanipulation techniques for intra-embryo genome editing have been established in only a small set of animals. To overcome these issues, we developed a method of large-fragment DNA knockin without micromanipulation. In this study, we successfully delivered the knockin donor DNA into zygotes by adeno-associated virus (AAV) without removing the zona pellucida, and we succeeded in both large-DNA fragment knockin and whole exon exchange with electroporation of CRISPR/Cas9 ribonucleoprotein. By this method, we can exchange large DNA fragments conveniently in various animal species without micromanipulation.

    View details for PubMedID 30447647

  • An interspecies barrier to tetraploid complementation and chimera formation SCIENTIFIC REPORTS Yamaguchi, T., Sato, H., Kobayashi, T., Kato-itoh, M., Goto, T., Hara, H., Mizuno, N., Yanagida, A., Umino, A., Hamanaka, S., Suchy, F., Masaki, H., Ota, Y., Hirabayashi, M., Nakauchi, H. 2018; 8: 15289

    Abstract

    To study development of the conceptus in xenogeneic environments, we assessed interspecies chimera formation as well as tetraploid complementation between mouse and rat. Overall contribution of donor PSC-derived cells was lower in interspecies chimeras than in intraspecies chimeras, and high donor chimerism was associated with anomalies or embryonic death. Organ to organ variation in donor chimerism was greater in interspecies chimeras than in intraspecies chimeras, suggesting species-specific affinity differences among interacting molecules necessary for organogenesis. In interspecies tetraploid complementation, embryo development was near normal until the stage of placental formation, after which no embryos survived.

    View details for PubMedID 30327488

  • Generation of Vascular Endothelial Cells and Hematopoietic Cells by Blastocyst Complementation STEM CELL REPORTS Hamanaka, S., Umino, A., Sato, H., Hayama, T., Yanagida, A., Mizuno, N., Kobayashi, T., Kasai, M., Suchy, F., Yamazaki, S., Masaki, H., Yamaguchi, T., Nakauchi, H. 2018; 11 (4): 988–97

    Abstract

    In the case of organ transplantation accompanied by vascular anastomosis, major histocompatibility complex mismatched vascular endothelial cells become a target for graft rejection. Production of a rejection-free, transplantable organ, therefore, requires simultaneous generation of vascular endothelial cells within the organ. To generate pluripotent stem cell (PSC)-derived vascular endothelial cells, we performed blastocyst complementation with a vascular endothelial growth factor receptor-2 homozygous mutant blastocyst. This mutation is embryonic lethal at embryonic (E) day 8.5-9.5 due to an early defect in endothelial and hematopoietic cells. The Flk-1 homozygous knockout chimeric mice survived to adulthood for over 1 year without any abnormality, and all vascular endothelial cells and hematopoietic cells were derived from the injected PSCs. This approach could be used in conjunction with other gene knockouts which induce organ deficiency to produce a rejection-free, transplantable organ in which all the organ's cells and vasculature are PSC derived.

    View details for PubMedID 30245211

  • Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep. Transgenic research Vilarino, M., Suchy, F. P., Rashid, S. T., Lindsay, H., Reyes, J., McNabb, B. R., van der Meulen, T., Huising, M. O., Nakauchi, H., Ross, P. J. 2018

    Abstract

    The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR(ddPCR). Next, we determined the presence of mosaicism in~50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.

    View details for PubMedID 30284144

  • Interspecies chimeras CURRENT OPINION IN GENETICS & DEVELOPMENT Suchy, F., Nakauchi, H. 2018; 52: 36–41
  • Interspecies chimeras. Current opinion in genetics & development Suchy, F., Nakauchi, H. 2018; 52: 36–41

    Abstract

    By probing early embryogenesis and regeneration, interspecies chimeras provide a unique platform for discovery and clinical use. Although efficient generation of human:animal chimeric embryos remains elusive, recent advancements attempt to overcome incompatibilities in xenogeneic development and transplantation.

    View details for PubMedID 29859382

  • iPSC-Derived Organs In Vivo: Challenges and Promise CELL STEM CELL Suchy, F., Yamaguchi, T., Nakauchi, H. 2018; 22 (1): 21–24

    Abstract

    Transplanting iPSCs into the embryos of another species can generate functional organs for basic research and translational applications. We discuss forward-looking approaches and address key remaining challenges of generating iPSC-derived human organs in vivo.

    View details for PubMedID 29304339

  • CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep SCIENTIFIC REPORTS Vilarino, M., Rashid, S., Suchy, F., McNabb, B., van der Meulen, T., Fine, E. J., Ahsan, S., Mursaliyev, N., Sebastiano, V., Diab, S., Huising, M. O., Nakauchi, H., Ross, P. J. 2017; 7: 17472

    Abstract

    One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1-/- fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.

    View details for PubMedID 29234093

  • Rapid Chromatin Switch in the Direct Reprogramming of Fibroblasts to Neurons CELL REPORTS Wapinski, O. L., Lee, Q., Chen, A. C., Li, R., Corces, M., Ang, C., Treutlein, B., Xiang, C., Baubet, V., Suchy, F., Sankar, V., Sim, S., Quake, S. R., Dahmane, N., Wernig, M., Chang, H. Y. 2017; 20 (13): 3236–47

    Abstract

    How transcription factors (TFs) reprogram one cell lineage to another remains unclear. Here, we define chromatin accessibility changes induced by the proneural TF Ascl1 throughout conversion of fibroblasts into induced neuronal (iN) cells. Thousands of genomic loci are affected as early as 12 hr after Ascl1 induction. Surprisingly, over 80% of the accessibility changes occur between days 2 and 5 of the 3-week reprogramming process. This chromatin switch coincides with robust activation of endogenous neuronal TFs and nucleosome phasing of neuronal promoters and enhancers. Subsequent morphological and functional maturation of iN cells is accomplished with relatively little chromatin reconfiguration. By integrating chromatin accessibility and transcriptome changes, we built a network model of dynamic TF regulation during iN cell reprogramming and identified Zfp238, Sox8, and Dlx3 as key TFs downstream of Ascl1. These results reveal a singular, coordinated epigenomic switch during direct reprogramming, in contrast to stepwise cell fate transitions in development.

    View details for PubMedID 28954238

  • Lessons from Interspecies Mammalian Chimeras ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, VOL 33 Suchy, F., Nakauchi, H., Schekman, R. 2017; 33: 203–17

    Abstract

    As chimeras transform from beasts of Greek mythology into tools of contemporary bioscience, secrets of developmental biology and evolutionary divergence are being revealed. Recent advances in stem cell biology and interspecies chimerism have generated new models with extensive basic and translational applications, including generation of transplantable, patient-specific organs.

    View details for PubMedID 28806099

  • The Oligomeric States of the Purified Sigma-1 Receptor Are Stabilized by Ligands JOURNAL OF BIOLOGICAL CHEMISTRY Gromek, K. A., Suchy, F. P., Meddaugh, H. R., Wrobel, R. L., Lapointe, L. M., Chu, U. B., Primm, J. G., Ruoho, A. E., Senes, A., Fox, B. G. 2014; 289 (29): 20333-20344

    Abstract

    Sigma-1 receptor (S1R) is a mammalian member of the ERG2 and sigma-1 receptor-like protein family (pfam04622). It has been implicated in drug addiction and many human neurological disorders, including Alzheimer and Parkinson diseases and amyotrophic lateral sclerosis. A broad range of synthetic small molecules, including cocaine, (+)-pentazocine, haloperidol, and small endogenous molecules such as N,N-dimethyltryptamine, sphingosine, and steroids, have been identified as regulators of S1R. However, the mechanism of activation of S1R remains obscure. Here, we provide evidence in vitro that S1R has ligand binding activity only in an oligomeric state. The oligomeric state is prone to decay into an apparent monomeric form when exposed to elevated temperature, with loss of ligand binding activity. This decay is suppressed in the presence of the known S1R ligands such as haloperidol, BD-1047, and sphingosine. S1R has a GXXXG motif in its second transmembrane region, and these motifs are often involved in oligomerization of membrane proteins. Disrupting mutations within the GXXXG motif shifted the fraction of the higher oligomeric states toward smaller states and resulted in a significant decrease in specific (+)-[(3)H]pentazocine binding. Results presented here support the proposal that S1R function may be regulated by its oligomeric state. Possible mechanisms of molecular regulation of interacting protein partners by S1R in the presence of small molecule ligands are discussed.

    View details for DOI 10.1074/jbc.M113.537993

    View details for Web of Science ID 000339395200044

    View details for PubMedID 24847081

    View details for PubMedCentralID PMC4106346

  • Solution Structure of the 2A Protease from a Common Cold Agent, Human Rhinovirus C2, Strain W12 PLOS ONE Lee, W., Watters, K. E., Troupis, A. T., Reinen, N. M., Suchy, F. P., Moyer, K. L., Frederick, R. O., Tonelli, M., Aceti, D. J., Palmenberg, A. C., Markley, J. L. 2014; 9 (6)

    Abstract

    Human rhinovirus strains differ greatly in their virulence, and this has been correlated with the differing substrate specificity of the respective 2A protease (2Apro). Rhinoviruses use their 2Apro to cleave a spectrum of cellular proteins important to virus replication and anti-host activities. These enzymes share a chymotrypsin-like fold stabilized by a tetra-coordinated zinc ion. The catalytic triad consists of conserved Cys (C105), His (H34), and Asp (D18) residues. We used a semi-automated NMR protocol developed at NMRFAM to determine the solution structure of 2Apro (C105A variant) from an isolate of the clinically important rhinovirus C species (RV-C). The backbone of C2 2Apro superimposed closely (1.41-1.81 Å rmsd) with those of orthologs from RV-A2, coxsackie B4 (CB4), and enterovirus 71 (EV71) having sequence identities between 40% and 60%. Comparison of the structures suggest that the differential functional properties of C2 2Apro stem from its unique surface charge, high proportion of surface aromatics, and sequence surrounding the di-tyrosine flap.

    View details for DOI 10.1371/journal.pone.0097198

    View details for Web of Science ID 000338503400005

    View details for PubMedID 24937088

    View details for PubMedCentralID PMC4061012

  • Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence PROTEIN EXPRESSION AND PURIFICATION Gromek, K. A., Meddaugh, H. R., Wrobel, R. L., Suchy, F. P., Bingman, C. A., Primm, J. G., Fox, B. G. 2013; 89 (2): 203-209

    Abstract

    Sigma 1 receptor (S1R) is a eukaryotic membrane protein that functions as an inter-organelle signaling modulator and chaperone. Here we report an improved expression of S1R in Escherichia coli as a fusion to maltose binding protein (MBP) and a high-yield purification. Variants with linking amino acid sequences consisting of 0-5 alanine residues between MBP and S1R were created and tested in several E. coli expression strains in order to determine the best combination of construct and host for production of active MBP-S1R. Among the linker variations, the protein containing a 4-Ala linker exhibited superior expression characteristics (MBP-4A-S1R); this construct was most productively paired with E. coli B834-pRARE2 and a chemically defined growth and expression medium. A 3-step purification was developed, including extraction from the E. coli membrane fraction using a mixture of Triton X-100 and n-dodecyl-beta-D-maltopyranoside identified by screening constrainted by retention of binding function, and purification by amylose affinity and gel filtration chromatographies. This procedure yields ∼3.5mg of purified fusion protein per L of bacterial culture medium. Purified MBP-4A-S1R showed a 175-fold purification from the starting cellular lysate with respect to specific ligand binding activity, and is stable during concentration and freeze-thaw cycling.

    View details for DOI 10.1016/j.pep.2013.03.013

    View details for Web of Science ID 000319373300013

    View details for PubMedID 23562661

    View details for PubMedCentralID PMC3679933