Establishment of Pancreatic ß-Cell-Specific Gene Knockout System Based on CRISPR-Cas9 Technology With AAV8-Mediated gRNA Delivery.
Diabetes
; 72(11): 1609-1620, 2023 Nov 01.
Article
en En
| MEDLINE
| ID: mdl-37625131
ABSTRACT
The Cre-loxP system provides valuable resources to analyze the importance of tissue-specific gene knockout (KO), including pancreatic ß-cells associated with the pathogenesis of diabetes. 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 ßCas9 system with mice expressing Cas9 in pancreatic ß-cells and adeno-associated virus 8 (AAV8)-mediated guide RNA (gRNA) delivery based on CRISPR-Cas9 technology to overcome those shortcomings. Interbreeding CAG-loxP-STOP-loxP (LSL)-Cas9 with Ins1-Cre mice generates normal glucose-tolerant ßCas9 mice expressing Cas9 with fluorescent reporter EGFP specifically in ß-cells. We also show significant ß-cell-specific gene KO efficiency with AAV8-mediated delivery of gRNA for EGFP reporter by intraperitoneal injection in the mice. As a proof of concept, we administered AAV8 to ßCas9 mice for expressing gRNA for Pdx1, a culprit gene of maturity-onset diabetes of the young 4. As reported previously, we demonstrate that those mice show glucose intolerance with transdifferentiation of Pdx1 KO ß-cells into glucagon-expressing cells. We successfully generated a convenient ß-cell-specific gene KO system with ßCas9 mice and AAV8-mediated gRNA delivery.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Idioma:
En
Revista:
Diabetes
Año:
2023
Tipo del documento:
Article
País de afiliación:
Japón