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.