High-fidelity mouse line generated by CRISPR-Cas9 assisted gene targeting.

Objective: α-cells are the second most prominent cell type in pancreatic islets and are responsible for producing glucagon to increase plasma glucose levels in times of fasting. α-cell dysfunction and inappropriate glucagon secretion occur in both type 1 and type 2 diabetes. Thus, there is growing interest in studying both normal function and pathophysiology of α-cells. However, tools to target gene ablation or activation specifically of α-cells have been limited, compared to those available for β-cells. Previous and transgenic mouse lines have suffered from transgene silencing, and the only available "knock-in" mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop a mouse line that would maintain normal glucagon expression and would be less susceptible to transgene silencing.

Methods: We utilized CRISPR-Cas9 technology to insert an sequence into the 3' UTR of the () locus in mouse embryonic stem cells (ESCs). Targeted ESC clones were then injected into mouse blastocysts to obtain mice. Recombination efficiency in GCG pancreatic α-cells and glucagon-like peptide 1 positive (GLP1) enteroendocrine L-cells was measured in ; mice injected with tamoxifen during fetal development and adulthood.

Results: Tamoxifen injection of ; mice induced high recombination efficiency of the locus in perinatal and adult α-cells (88% and 95%, respectively), as well as in first-wave fetal α-cells (36%) and adult enteroendocrine L-cells (33%). Mice homozygous for the allele were phenotypically normal.

Conclusions: We successfully derived a mouse line that expresses CreER in pancreatic α-cells and enteroendocrine L-cells without disrupting gene expression. These mice will be a useful tool for performing temporally controlled genetic manipulation specifically in these cell types.