Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing
GENES & DEVELOPMENT
2015; 29 (14): 1576-1585
Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.
View details for DOI 10.1101/gad.264861.115
View details for Web of Science ID 000358596300010
View details for PubMedCentralID PMC4526740
The evolution and function of the Pax/Six regulatory network in sponges
EVOLUTION & DEVELOPMENT
2013; 15 (3): 186-196
Examining the origins of highly conserved gene regulatory networks (GRNs) will inform our understanding of the evolution of animal body plans. Sponges are believed to be the most ancient extant metazoan lineage, and as such, hold clues about the evolution of genetic programs deployed in animal development. We used the emerging freshwater sponge model, Ephydatia muelleri, to study the evolutionary origins of the Pax/Six/Eya/Dac (PSED) GRN. Orthologs to Pax and Six family members are present in E. muelleri and are expressed in endothelial cells lining the canal system as well as cells in the choanoderm. Knockdown of EmPaxB and EmSix1/2 by RNAi resulted in defects to the canal systems. We further show that PaxB may be in a regulatory relationship with Six1/2 in E. muelleri, thus demonstrating that a component of the PSED network was present early in metazoan evolution.
View details for DOI 10.1111/ede.12032
View details for Web of Science ID 000318229300003
View details for PubMedID 23607302
RNA interference in marine and freshwater sponges: actin knockdown in Tethya wilhelma and Ephydatia muelleri by ingested dsRNA expressing bacteria
The marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available.We show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from 'knocking down' expression of the actin gene.This technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals.
View details for DOI 10.1186/1472-6750-11-67
View details for Web of Science ID 000293267400001
View details for PubMedID 21679422
View details for PubMedCentralID PMC3146823