title: High-fidelityGlucagon-CreERmouse line generated by CRISPR-Cas9 assisted gene targeting.
abstract: 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. PreviousGlucagon-CreandGlucagon-CreERtransgenic mouse lines have suffered from transgene silencing, and the only availableGlucagon-CreER"knock-in" mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop aGlucagon-CreERT2mouse line that would maintain normal glucagon expression and would be less susceptible to transgene silencing.
  
  
  
  We utilized CRISPR-Cas9 technology to insert anIRES-CreERT2sequence into the 3' UTR of theGlucagon(Gcg) locus in mouse embryonic stem cells (ESCs). Targeted ESC clones were then injected into mouse blastocysts to obtainGcg-CreERT2mice. Recombination efficiency in GCG+pancreatic α-cells and glucagon-like peptide 1 positive (GLP1+) enteroendocrine L-cells was measured inGcg-CreERT2;Rosa26-LSL-YFPmice injected with tamoxifen during fetal development and adulthood.
  
  
  
  Tamoxifen injection ofGcg-CreERT2;Rosa26-LSL-YFPmice induced high recombination efficiency of theRosa26-LSL-YFPlocus 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 theGcg-CreERT2allele were phenotypically normal.
  
  
  
  We successfully derived aGcg-CreERT2mouse line that expresses CreERT2in pancreatic α-cells and enteroendocrine L-cells without disruptingpreproglucagongene expression. These mice will be a useful tool for performing temporally controlled genetic manipulation specifically in these cell types.
authors.affiliation: Division of Endocrinology and Diabetes, The Children's Hospital of Philadelphia, 3400 Civic Center Boulevard, Philadelphia, PA, 19104, USA; Institute of Diabetes, Obesity, and Metabolism, Perelman School of Medicine, The University of Pennsylvania, 3400 Civic Center Boulevard, Philadelphia, PA, 19104, USA. Electronic address: ackermanna@email.chop.edu.
authors.forename: Amanda M
authors.initials: AM
authors.lastname: Ackermann
authors.affiliation: Institute of Diabetes, Obesity, and Metabolism, Perelman School of Medicine, The University of Pennsylvania, 3400 Civic Center Boulevard, Philadelphia, PA, 19104, USA; Department of Genetics, Perelman School of Medicine, The University of Pennsylvania, 3400 Civic Center Boulevard, Philadelphia, PA, 19104, USA. Electronic address: zhangjia@upenn.edu.
authors.forename: Jia
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Institute of Diabetes, Obesity, and Metabolism, Perelman School of Medicine, The University of Pennsylvania, 3400 Civic Center Boulevard, Philadelphia, PA, 19104, USA; Department of Genetics, Perelman School of Medicine, The University of Pennsylvania, 3400 Civic Center Boulevard, Philadelphia, PA, 19104, USA. Electronic address: aryelh@mail.med.upenn.edu.
authors.forename: Aryel
authors.initials: A
authors.lastname: Heller
authors.affiliation: Department of Genetics, Perelman School of Medicine, The University of Pennsylvania, 3400 Civic Center Boulevard, Philadelphia, PA, 19104, USA. Electronic address: abriker@villanova.edu.
authors.forename: Anna
authors.initials: A
authors.lastname: Briker
authors.affiliation: Institute of Diabetes, Obesity, and Metabolism, Perelman School of Medicine, The University of Pennsylvania, 3400 Civic Center Boulevard, Philadelphia, PA, 19104, USA; Department of Genetics, Perelman School of Medicine, The University of Pennsylvania, 3400 Civic Center Boulevard, Philadelphia, PA, 19104, USA. Electronic address: kaestner@mail.med.upenn.edu.
authors.forename: Klaus H
authors.initials: KH
authors.lastname: Kaestner
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D064113:CRISPR-Cas Systems / Q000187:drug effects; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics / Q000378:metabolism; D055879:Gene Knock-In Techniques; D018390:Gene Targeting; D005818:Genetic Engineering / Q000379:methods*; D005821:Genetic Techniques; D005934:Glucagon / Q000235:genetics* / Q000378:metabolism; D050416:Glucagon-Secreting Cells / Q000378:metabolism / Q000502:physiology; D007515:Islets of Langerhans / Q000187:drug effects; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D013629:Tamoxifen / Q000494:pharmacology; D019076:Transgenes / Q000187:drug effects
chemical_list: D020413:3' Untranslated Regions; D013629:Tamoxifen; D005934:Glucagon
keywords: CRISPR; CRISPR, clustered regularly interspaced short palindromic repeat; Cre, Cre recombinase; CreERT2, tamoxifen-inducible Cre recombinase-estrogen receptor fusion protein; DAPI, 4′,6-diamidino-2-phenylindole; ESC, embryonic stem cell; Enteroendocrine L-cell; FACS, fluorescence-activated cell sorting; GCG, glucagon; GLP1; GLP1, glucagon-like peptide 1; Glucagon; IRES, internal ribosomal entry site; Islet; LSL, loxP-stop-loxP; UTR, untranslated region; YFP, yellow fluorescent protein; gRNA, guide RNA; α-cell