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

Abstract：

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 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.

journal_name

Mol Metab

journal_title

Molecular metabolism

authors

Ackermann AM,Zhang J,Heller A,Briker A,Kaestner KH

doi

10.1016/j.molmet.2017.01.003

subject

Has Abstract

pub_date

2017-01-12 00:00:00

pages

236-244

issue

issn

2212-8778

pii

S2212-8778(16)30381-7

journal_volume

pub_type

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