Chiyo Shiota1,2, Krishna Prasadan3, Ping Guo3, Joseph Fusco3, Xiangwei Xiao3, George K Gittes4. 1. Division of Pediatric Surgery, Department of Surgery, Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine, 4401 Penn Ave, Pittsburgh, PA, 15224, USA. chiyo.shiota@vanderbilt.edu. 2. Department of Molecular Physiology and Biophysics, Vanderbilt University, 2215 Garland Ave, Nashville, TN, 37232, USA. chiyo.shiota@vanderbilt.edu. 3. Division of Pediatric Surgery, Department of Surgery, Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine, 4401 Penn Ave, Pittsburgh, PA, 15224, USA. 4. Division of Pediatric Surgery, Department of Surgery, Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine, 4401 Penn Ave, Pittsburgh, PA, 15224, USA. gittesgk@upmc.edu.
Abstract
AIMS/HYPOTHESIS: The Cre/loxP system, which enables tissue-specific manipulation of genes, is widely used in mice for diabetes research. Our aim was to develop a new Cre-driver mouse line for the specific and efficient manipulation of genes in pancreatic alpha cells. METHODS: A Gcg CreERT2 knockin mouse, which expresses a tamoxifen-inducible form of Cre from the endogenous preproglucagon (Gcg) gene locus, was generated by homologous recombination. The new Gcg CreERT2 mouse line was crossed to the Rosa26 tdTomato (R26 tdTomato ) Cre reporter mouse line in order to evaluate the tissue specificity, efficiency and tamoxifen dependency of Gcg CreERT2 -mediated recombination. Cell types of pancreatic islets were identified using immunohistochemistry. Biochemical and physiological data, including blood glucose levels, plasma glucagon and glucagon-like peptide (GLP)-1 levels, and pancreatic glucagon content, were collected and used to assess the overall effect of Gcg gene targeting on Gcg CreERT2/w heterozygous mice. RESULTS: Tamoxifen-treated Gcg CreERT2/w ;R26 tdTomato/w mice displayed Cre reporter activity, i.e. expression of tdTomato red fluorescent protein (RFP) in all known cells that produce proglucagon-derived peptides. In the adult pancreas, RFP was detected in 94-97% of alpha cells, whereas it was detected in a negligible (~ 0.2%) proportion of beta cells. While more than 98% of cells labelled with tamoxifen-induced RFP were glucagon-positive cells, 14-25% of pancreatic polypeptide (PP)-positive cells were also positive for RFP, indicating the presence of glucagon/PP bihormonal cell population. Tamoxifen-independent expression of RFP occurred in approximately 6% of alpha cells. In contrast to alpha cells and GLP-1-producing neurons, in which RFP expression persisted for at least 5 months after tamoxifen administration (presumably due to rare neogenesis in these cell types in adulthood), nearly half of RFP-positive intestinal L cells were replaced with RFP-negative L cells over the first 2 weeks after tamoxifen administration. Heterozygous Gcg CreERT2/w mice showed reduced Gcg mRNA levels in islets, but maintained normal levels of pancreatic and plasma glucagon. The mice did not exhibit any detectable baseline physiological abnormalities, at least in young adulthood. CONCLUSIONS/ INTERPRETATION: The newly developed Gcg CreERT2 knockin mouse shows faithful expression of CreERT2 in pancreatic alpha cells, intestinal L cells and GLP-1-producing neurons. This mouse line will be particularly useful for manipulating genes in alpha cells, due to highly specific and efficient CreERT2-mediated recombination in this cell type in the pancreas.
AIMS/HYPOTHESIS: The Cre/loxP system, which enables tissue-specific manipulation of genes, is widely used in mice for diabetes research. Our aim was to develop a new Cre-driver mouse line for the specific and efficient manipulation of genes in pancreatic alpha cells. METHODS: A Gcg CreERT2 knockin mouse, which expresses a tamoxifen-inducible form of Cre from the endogenous preproglucagon (Gcg) gene locus, was generated by homologous recombination. The new Gcg CreERT2 mouse line was crossed to the Rosa26 tdTomato (R26 tdTomato ) Cre reporter mouse line in order to evaluate the tissue specificity, efficiency and tamoxifen dependency of Gcg CreERT2 -mediated recombination. Cell types of pancreatic islets were identified using immunohistochemistry. Biochemical and physiological data, including blood glucose levels, plasma glucagon and glucagon-like peptide (GLP)-1 levels, and pancreaticglucagon content, were collected and used to assess the overall effect of Gcg gene targeting on Gcg CreERT2/w heterozygous mice. RESULTS:Tamoxifen-treated Gcg CreERT2/w ;R26 tdTomato/w mice displayed Cre reporter activity, i.e. expression of tdTomato red fluorescent protein (RFP) in all known cells that produce proglucagon-derived peptides. In the adult pancreas, RFP was detected in 94-97% of alpha cells, whereas it was detected in a negligible (~ 0.2%) proportion of beta cells. While more than 98% of cells labelled with tamoxifen-induced RFP were glucagon-positive cells, 14-25% of pancreatic polypeptide (PP)-positive cells were also positive for RFP, indicating the presence of glucagon/PP bihormonal cell population. Tamoxifen-independent expression of RFP occurred in approximately 6% of alpha cells. In contrast to alpha cells and GLP-1-producing neurons, in which RFP expression persisted for at least 5 months after tamoxifen administration (presumably due to rare neogenesis in these cell types in adulthood), nearly half of RFP-positive intestinal L cells were replaced with RFP-negative L cells over the first 2 weeks after tamoxifen administration. Heterozygous Gcg CreERT2/w mice showed reduced Gcg mRNA levels in islets, but maintained normal levels of pancreatic and plasma glucagon. The mice did not exhibit any detectable baseline physiological abnormalities, at least in young adulthood. CONCLUSIONS/ INTERPRETATION: The newly developed Gcg CreERT2 knockin mouse shows faithful expression of CreERT2 in pancreatic alpha cells, intestinal L cells and GLP-1-producing neurons. This mouse line will be particularly useful for manipulating genes in alpha cells, due to highly specific and efficient CreERT2-mediated recombination in this cell type in the pancreas.
Authors: Harini Chakravarthy; Xueying Gu; Martin Enge; Xiaoqing Dai; Yong Wang; Nicolas Damond; Carolina Downie; Kathy Liu; Jing Wang; Yuan Xing; Simona Chera; Fabrizio Thorel; Stephen Quake; Jose Oberholzer; Patrick E MacDonald; Pedro L Herrera; Seung K Kim Journal: Cell Metab Date: 2017-02-16 Impact factor: 27.287
Authors: H Katsuta; T Akashi; R Katsuta; M Nagaya; D Kim; Y Arinobu; M Hara; S Bonner-Weir; A J Sharma; K Akashi; G C Weir Journal: Diabetologia Date: 2009-10-23 Impact factor: 10.122
Authors: Eva Tudurí; Maria M Glavas; Ali Asadi; Robert K Baker; Cara E Ellis; Galina Soukhatcheva; Marjolaine Philit; Frank K Huynh; James D Johnson; C Bruce Verchere; Timothy J Kieffer Journal: Sci Rep Date: 2019-07-25 Impact factor: 4.379