[HTML][HTML] High-fidelity Glucagon-CreER mouse line generated by CRISPR-Cas9 assisted gene targeting

AM Ackermann, J Zhang, A Heller, A Briker… - Molecular …, 2017 - Elsevier
AM Ackermann, J Zhang, A Heller, A Briker, KH Kaestner
Molecular metabolism, 2017Elsevier
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 …
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-CreERT2 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-CreERT2 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-CreERT2 mice. Recombination efficiency in GCG+ pancreatic α-cells and glucagon-like peptide 1 positive (GLP1+) enteroendocrine L-cells was measured in Gcg-CreERT2;Rosa26-LSL-YFP mice injected with tamoxifen during fetal development and adulthood.
Results
Tamoxifen injection of Gcg-CreERT2;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-CreERT2 allele were phenotypically normal.
Conclusions
We successfully derived a Gcg-CreERT2 mouse line that expresses CreERT2 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.
Elsevier