High-fidelity Glucagon-CreER 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 Glucagon-Cre and Glucagon-CreER transgenic mouse lines have suffered from transgene silencing, and the only available Glucagon-CreER "knock-in" mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop a Glucagon-CreER^T2 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 IRES-CreER^T2 sequence into the 3' UTR of the Glucagon (Gcg) locus in mouse embryonic stem cells (ESCs). Targeted ESC clones were then injected into mouse blastocysts to obtain Gcg-CreER^T2 mice. Recombination efficiency in GCG⁺ pancreatic α-cells and glucagon-like peptide 1 positive (GLP1⁺) enteroendocrine L-cells was measured in Gcg-CreER^T2 ;Rosa26-LSL-YFP mice injected with tamoxifen during fetal development and adulthood.

Results: Tamoxifen injection of Gcg-CreER^T2 ;Rosa26-LSL-YFP mice induced high recombination efficiency of the Rosa26-LSL-YFP 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 Gcg-CreER^T2 allele were phenotypically normal.

Conclusions: We successfully derived a Gcg-CreER^T2 mouse line that expresses CreER^T2 in pancreatic α-cells and enteroendocrine L-cells without disrupting preproglucagon gene expression. These mice will be a useful tool for performing temporally controlled genetic manipulation specifically in these cell types.