Elsevier

Molecular Metabolism

Volume 6, Issue 3, March 2017, Pages 236-244
Molecular Metabolism

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

https://doi.org/10.1016/j.molmet.2017.01.003Get rights and content
Under a Creative Commons license
open access

Highlights

  • Developed a new Gcg-CreERT2 knock-in mouse line using CRISPR-Cas9 gene targeting.

  • Gcg-CreERT2 mice exhibit high recombination in α-cells and L-cells.

  • There is no evidence of preproglucagon haploinsufficiency in Gcg-CreERT2 mice.

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-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.

Keywords

Islet
α-cell
Enteroendocrine L-cell
Glucagon
GLP1
CRISPR

Abbreviations

Cre
Cre recombinase
CreERT2
tamoxifen-inducible Cre recombinase-estrogen receptor fusion protein
CRISPR
clustered regularly interspaced short palindromic repeat
DAPI
4′,6-diamidino-2-phenylindole
ESC
embryonic stem cell
FACS
fluorescence-activated cell sorting
GCG
glucagon
GLP1
glucagon-like peptide 1
IRES
internal ribosomal entry site
LSL
loxP-stop-loxP
gRNA
guide RNA
UTR
untranslated region
YFP
yellow fluorescent protein

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