RRC ID 77002
Author Ueki K, Nishida Y, Aoyama S, Uzawa H, Kanai A, Ito M, Ikeda K, Iida H, Miyatsuka T, Watada H.
Title Establishment of Pancreatic Beta Cell-specific Gene Knockout System Based on CRISPR-Cas9 Technology with AAV8-mediated gRNA Delivery.
Journal Diabetes
Abstract The Cre-loxP system provides valuable resources to analyze the importance of tissue-specific gene knockout, including pancreatic beta cells associated with the pathogenesis of diabetes mellitus. However, it is expensive and time-consuming to generate transgenic mice harboring floxed genes of interest and cross them with cell-specific Cre-expression mice. We establish a βCas9 system with mice expressing Cas9 in pancreatic beta cells and adeno-associated virus 8 (AAV8) -mediated gRNA delivery based on CRISPR-Cas9 technology to overcome those shortcomings. Interbreeding CAG-LoxP-Stop-LoxP (LSL)-Cas9 with Ins1-Cre mice generates normal glucosetolerant βCas9 mice expressing Cas9 with fluorescent reporter EGFP specifically in beta cells. We also show significant beta cell-specific gene knockout efficiency with AAV8-mediated delivery of gRNA for EGFP reporter by intraperitoneal injection in the mice. As a proof of concept, we administer AAV8 to βCas9 mice for expressing gRNA for Pdx1, a culprit gene of maturity-onset diabetes of the young 4 (MODY4). As reported previously, we demonstrate that those mice show glucose intolerance with trans-differentiation of Pdx1 knockout beta cells into glucagonexpressing cells. We successfully generate a convenient beta cell-specific gene knockout system with βCas9 mice and AAV8-mediated gRNA delivery.
Published 2023-8-25
DOI 10.2337/db23-0445
PII 153551
PMID 37625131
IF 7.72
Mice RBRC03934