GcgCreERT2 knockin mice as a tool for genetic manipulation in pancreatic alpha cells
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.
A GcgCreERT2 knockin mouse, which expresses a tamoxifen-inducible form of Cre from the endogenous preproglucagon (Gcg) gene locus, was generated by homologous recombination. The new GcgCreERT2 mouse line was crossed to the Rosa26tdTomato (R26tdTomato) Cre reporter mouse line in order to evaluate the tissue specificity, efficiency and tamoxifen dependency of GcgCreERT2-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 GcgCreERT2/w heterozygous mice.
Tamoxifen-treated GcgCreERT2/w;R26tdTomato/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 GcgCreERT2/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.
The newly developed GcgCreERT2 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.
KeywordsCre/loxP CreERT2 Pancreatic alpha cells Proglucagon
Bacterial artificial chromosome
Flippase recognition target
Nucleus of the solitary tract
- PPG neuron
Red fluorescent protein
Pancreatic islet alpha cells are specialised to produce the glucagon that counteracts insulin for glucose homeostasis. Defects in insulin production and release by islet beta cells, insulin signalling to target organs or both are primary causes of diabetes mellitus. However, it has become evident that dysregulation of glucagon secretion by alpha cells also contributes to diabetes development and severity, indicating that alpha cells could also be a therapeutic target for better management of the disease [1, 2]. In addition, recent studies have suggested that alpha cells might be a potential source for the generation of new beta cells to cure diabetes [3, 4, 5].
The Gcg gene encodes preproglucagon, which consists of an N-terminal signal peptide and proglucagon. In the pancreas, the Gcg gene is only expressed in alpha cells. Outside the pancreas, the Gcg gene is expressed in intestinal L cells  and in a subset of neurons in the lower brain stem , most of which are in the nucleus of the solitary tract (NST) and some in the intermediate reticular nucleus [8, 9]. Tissue-specific differential processing of proglucagon yields glucagon in alpha cells, but yields glucagon-like peptide (GLP)-1 and GLP-2 in L cells and neurons. Intestinal GLP-1 is one of the incretins that are released after food intake and augment insulin secretion from beta cells, thereby lowering the blood glucose level . GLP-1-producing NST neurons, so-called preproglucagon (PPG) neurons, project to multiple brain regions where GLP-1 receptors are expressed. This central GLP-1 controls neurological and cognitive functions, including appetite regulation and glucose homeostasis , and activation of PPG neurons reduces food intake and body weight in mice .
Mouse models have been extensively used in islet studies. The use of Cre/lox site-specific recombination systems, which allow cell-type-specific deletion or activation of genes by expressing Cre recombinase in distinct cell populations, has greatly enhanced our knowledge of islet biology, both in normal conditions as well as in the pathogenesis of diabetes. For genetic manipulation of alpha cells, the transgenic mouse line in which the Cre gene is expressed under the control of the 1.6 kb fragment of the rat Gcg gene promoter has been widely used over the years . Previously, we have also generated Gcg-Cre transgenic mice using an 8 kb mouse Gcg promoter and codon-optimised Cre (improved Cre; iCre) , and other groups have developed Gcg-Cre mice using a construct based on the bacterial artificial chromosome (BAC) [9, 14]. The transgenic approach is unable to control the insertion site and copy number of transgenes, leading to some unpredictability. A relatively low recombination rate in alpha cells has been reported in some studies with the rat Gcg-Cre mouse [15, 16], perhaps due to silencing of the transgene , while we noted off-target recombination in a large percentage of beta cells in our mouse Gcg-Cre mouse despite the use of a large 8 kb promoter . Although not conclusive, it has been suggested that the Gcg gene is expressed at low levels in beta cells or their progenitors, and amplified Gcg promoter activity due to multiple copies of transgene produced enough Cre to cause recombination, even though endogenous Gcg promoter activity was low. In fact, gene expression analysis of single mouse beta cells has consistently demonstrated that beta cells express genes for other islet hormones at very low levels [18, 19].
Given the need for more precise manipulations of alpha cells, we developed an alternative Cre-driver mouse line that enables specific and efficient Cre-mediated recombination in alpha cells. To this end, we designed a new Cre-driver mouse with the following features: (1) use of the Gcg promoter to drive Cre expression in alpha cells to take advantage of its strong and specific activity in alpha cells within the pancreas, even though there will also be activity in GLP-1-producing cells; (2) use of a knockin strategy to express Cre under the control of endogenous regulatory elements for Gcg gene transcription; and (3) use of CreERT2, a tamoxifen-inducible form of Cre, to reduce off-target recombination, which would more likely occur during embryogenesis and early postnatal development. Although we did not incorporate a bicistronic expression system into our knockin strategy due to concerns about CreERT2 expression levels, Ackermann et al recently developed GcgIRES-CreERT2 knockin mice that express both glucagon and CreERT2 from a targeted Gcg allele and demonstrated specific and efficient Cre-mediated recombination in alpha cells . Here, we describe our new GcgCreERT2 knockin mouse, with additional information regarding a GcgiCre knockin mouse that we also generated. Given the similarity of our GcgCreERT2 knockin mice to those reported by Ackermann et al , the results from the extended characterisation of CreERT2 expression in our mice may be applicable to their mice, and thus would supplement their report.
Vector construct and gene targeting
Plasmid and E. coli necessary for the BAC recombineering were a gift from Neal G. Copeland (National Cancer Institute, Frederick, MD, USA). CreERT2 fragment was obtained from pCAG-CreERT2 (a gift from C. Cepko, Harvard Medical School, Boston, MA, USA: Addgene plasmid no. 14797) .
Chimeric mice were bred with Rosa26FLPo knockin mice (The Jackson Laboratory, Bar Harbor, ME, USA, stock no. 012930) to remove the neomycin resistance gene cassette (Fig. 1c), and resulting GcgCreERT2/w mice were backcrossed to C57BL/6J mice (The Jackson Laboratory, stock no. 000664) for seven generations while collecting data for this study. The Rosa26tdTomato Cre reporter (R26tdTomato) strain was obtained from The Jackson Laboratory (stock no. 007909) and bred with GcgCreERT2 mice to obtain double-mutant mice. Mouse genotypes were determined by PCR using DNA from tail biopsies. To induce Cre-mediated recombination, GcgCreERT2 mice were treated with s.c. injection of tamoxifen dissolved in corn oil (10 mg/ml), using regimens indicated in the Results section.
Mouse colonies were maintained in a specific pathogen-free barrier facility in the Children’s Hospital of Pittsburgh of UPMC (Pittsburgh, PA, USA). All mouse handling and experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh, and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Primary antibodies used for immunohistochemistry
Rabbit anti-human E-cadherin
Cell Signaling, Danvers, MA, USA
Rat anti-mouse E-cadherin
Life Technologies/Invitrogen, Grand Island, NY, USA
Millipore/Novagen, Billerica, MA, USA
Phoenix, Burlingame, CA, USA
Millipore/Linco, Billerica, MA, USA
Guinea pig anti-human insulin
Millipore/Linco, Billerica, MA, USA
Rabbit anti-mouse MafB
Bethyl, Montgomery, TX, USA
Guinea pig anti-rat PP
Millipore/Linco, Billerica, MA, USA
Rabbit anti-human somatostatin
Agilent/Dako, Santa Clara, CA, USA
Blood glucose measurement and hormone assay
Blood glucose levels were measured using an Accu-Chek glucose meter (Roche, Indianapolis, IN, USA). Plasma glucagon and total GLP-1 levels were determined with glucagon RIA and a total GLP-1 ELISA kit (Millipore, Billerica, MA, USA), respectively. Pancreatic glucagon was measured as previously described . All assays were performed in duplicate.
Islet isolation, cell sorting and gene expression analyses
For islet isolation, pancreas was digested by intraductal injection of CIzyme RI enzyme solution (VitaCyte, Indianapolis, IN, USA), followed by 15 min incubation at 37°C . Islets were hand-picked after a density-gradient purification using Histopaque (Sigma-Aldrich, St Louis, MO, USA)  and dispersed into single cells in Accumax solution (Stemcell, Cambridge, MA, USA). Resulting single-cell populations were sorted on a FACSAria cell sorter (BD Biosciences, San Jose, CA, USA) to isolate tdTomato red fluorescent protein (RFP)-positive and -negative cells. RNA was extracted from the isolated islets or the sorted cells using an RNeasy Mini kit (Qiagen, Hilden, Germany) or an Arcturus PicoPure RNA isolation kit (Applied Biosystems, Waltham, MA, USA), and cDNA was synthesised using a QuantiTect Reverse Transcription Kit (Qiagen). Gene expression was measured by real-time PCR using QuantiTect primers (Qiagen) and iTaq SYBR Green mix (Bio-Rad, Hercules, CA, USA). cDNA samples were run in duplicate.
Data are expressed as means ± SD. Statistical significance was assessed with an unpaired two-tailed Student t test or two-way ANOVA, and p < 0.05 was considered statistically significant. The experimenters were not blind to group assignment and no data were omitted.
Alpha cell-specific expression of CreERT2 in GcgCreERT2 mouse islets
Tamoxifen-induced recombination in GcgCreERT2 mouse alpha cells
RFP labelling in GcgCreERT2/w;R26tdTomato/w mouse islet cells
Alpha cells (%)
64.9 ± 5.4
81.8 ± 6.8
6.1 ± 0.2
93.7 ± 6.0
5.8 ± 1.5
96.6 ± 1.5
Beta cells (%)
10.1 ± 0.8
0.9 ± 0.4
0.2 ± 0.1
0.1 ± 0.1
0.1 ± 0.2
0.2 ± 0.1
PP cells (%)
2.6 ± 0.2
13.8 ± 6.3
2.2 ± 1.5
24.9 ± 4.4*
Delta cells (%)
0.4 ± 0.3
2.2 ± 1.5
1.6 ± 0.2
2.3 ± 2.2
We next examined CreERT2-mediated recombination in adult mice. At 5 weeks of age, GcgCreERT2/w;R26tdTomato/w mice were randomised into groups receiving either three doses of 1 mg tamoxifen or vehicle over a week, and pancreases were fixed at 2 or 5–6 months of age. Pancreatic sections were double-stained for glucagon and insulin or for pancreatic polypeptide (PP) and somatostatin. Although some RFP-positive cells were seen in the islets of vehicle-injected mice at both ages examined, a dramatic increase in RFP expression was observed among alpha cells in tamoxifen-injected mice at 2 months of age, and the pattern of RFP expression was maintained to 5–6 months of age (Fig. 3b). Quantitative analysis further demonstrated an efficient induction of RFP expression by tamoxifen in alpha cells (> 90%), with low frequency of tamoxifen-independent (‘leaky’) RFP expression (< 6%) in this cell type (Table 2). Importantly, more than 98% of RFP-positive cells were alpha cells, indicating that CreERT2-mediated recombination occurred almost exclusively in alpha cells. Consistently, RFP expression in beta and delta cells was negligible. However, a substantial proportion of PP cells (10–25%) were labelled with RFP (Table 2). Since only a few RFP-positive cells were negative for glucagon staining (1.2 ± 0.7% and 1.1 ± 0.5% of total RFP-positive cells at 2 and 5–6 months of age, respectively), this indicates that some alpha cells stained positively with anti-PP antibody. Indeed, double staining for glucagon and PP demonstrated the co-existence of both hormones in some cells (Fig. 3c), which is consistent with previous immunohistochemical studies on rat  or mouse  islets.
In addition to morphological assessments, expression of Gcg and Ins2 genes was measured by quantitative RT-PCR (qRT-PCR) in flow-sorted RFP-positive and -negative islet cells (Fig. 3d). Gcg gene expression was detected nearly exclusively in RFP-positive cells (Fig. 3e), whereas Ins2 gene expression was detected solely in RFP-negative cells, as expected (Fig. 3f). Collectively, the data confirmed that recombination of the floxed target sequence is tamoxifen-dependent and highly specific to alpha cells in GcgCreERT2 mouse pancreases.
Recombination by GcgCreERT2 in extrapancreatic proglucagon-producing cells
In the brain, RFP expression was detected in neurons of the NST, but not in other areas (Fig. 4c). As expected, RFP-positive neurons were still seen 5 months after tamoxifen injection.
Normal physiology in GcgCreERT2/w mice
GcgiCre knockin mice with an unexpected duplication mutation of the Gcg locus
We generated GcgiCre knockin mice using the same strategy as the GcgCreER2 mice, with the only difference being insertion of an iCre coding sequence instead of a CreERT2 sequence. While the iCre sequence was knocked into the Gcg gene as we designed, subsequent analysis of GcgiCre/CreERT2 hemizygous mice, in which we observed glucagon expression, indicated that the targeted allele still carried an intact Gcg gene, and that a duplication mutation had occurred in the GcgiCre mouse line, most likely during homologous recombination for gene targeting in ES cells. To determine the structure of the duplication, we performed a long-range PCR analysis of GcgiCre/CreERT2 mouse genomic DNA using primers specific to iCre or the 3′ portion of exon 2, which does not exist in the GcgiCre or GcgCreERT gene, in combination with various primers for upstream and downstream of the Gcg gene. The results revealed that the duplicated region included all exons of the Gcg gene, which spans 9 kb, and extended upstream for at least 17 kb and downstream for at least 7.8 kb. The 3′ portion of the adjacent Fap gene, which is located upstream of Gcg and encodes fibroblast activation protein, was involved in the duplication. Due to such a large size of the duplication, we could not determine duplication junction points or the relative position of the GcgiCre gene to the Gcg gene. Despite the lack of full information, we thought it was worthwhile to characterise this mouse line because the GcgiCre gene has at least 17 kb upstream sequence of the Gcg gene.
RFP labelling in GcgiCre/w;R26tdTomato/w mouse islet cells
Cell type (%)
95.2 ± 3.2
93.9 ± 1.2
0.6 ± 0.1
0.92 ± 0.3
37.9 ± 5.8
24.5 ± 2.3*
3.3 ± 1.1
4.5 ± 4.2
In the intestine, RFP expression was observed in L cells that were marked with GLP-1 staining (Fig. 6b). RFP-negative L cells were often seen in crypts, indicating a time lag between GLP-1 expression and RFP expression in newly formed L cells, with the latter requiring iCre expression and recombination of the R26TdTomato gene prior to RFP expression. In contrast to GcgCreERT2 mice, non-specific RFP expression was widely seen in the brain in GcgiCre/w;R26TdTomato/w mice (Fig. 6c, d). RFP-labelled cells often formed large clusters, suggesting that the recombination of the R26TdTomato gene occurred in early developmental stages with subsequent clonal expansion (Fig. 6d).
While we and Ackermann et al  used a knockin strategy to generate new GcgCreERT2 or GcgiCre mouse lines, two other groups have established new Gcg-Cre mouse lines by means of BAC transgenesis and successfully applied them to gene manipulation in L cells  or PGP neurons . BAC transgenes include long flanking sequences on both sides of the Cre inserted into the Gcg gene, and thereby confer better control of Gcg-Cre gene expression by cis elements similar to the endogenous Gcg gene. Nevertheless, it was reported that approximately 20% of beta cells were labelled with Cre reporter in one of the BAC transgenic lines . This is in contrast to our two new mouse models, in which activation of Cre reporter in beta cells was negligible (< 1%), even in the GcgiCre mice in which the targeted Gcg gene locus was disarranged by a duplication mutation.
Upon tamoxifen injection, the GcgCreERT2 mice displayed Cre reporter activation specifically in cells that express preproglucagon in adult mice. However, labelling NST neurons by the reporter seemed less effective compared with a recent study using BAC Gcg-Cre mice . A further study is required to determine the efficiency of Cre-mediated recombination in the GcgCreERT2 mouse brain, but it is possible that induction of recombination by tamoxifen is weaker in the brain than in other organs since the penetration of 4-hydroxytamoxifen, the primary active tamoxifen metabolite, into the brain is partially restricted by the P-glycoprotein (ABCB1) transporter . In contrast, the efficiency of Cre reporter activation in GcgCreERT2 mouse L cells was similar to that in the BAC Gcg-iCre mice . Consistent with previous estimates of the turnover time of enteroendocrine cells using 3H-thymidine labelling , half of the Cre reporter-positive L cells disappeared during the first 2 weeks after tamoxifen treatment, indicating that the effect of genetic manipulation on L cells does not last longer in this inducible system. Taken together, our GcgCreERT2 mouse favours studies on alpha cells, although each study must give careful consideration to the possible effects of Cre-mediated gene modifications in extrapancreatic sites.
The GcgIRES-CreERT2 mice reported by Ackermann et al express both preproglucagon and CreERT2 as two independent proteins from a single Gcg-IRES-CreERT2 mRNA . The GcgiCre mice described here express preproglucagon in addition to Cre from the targeted allele due to a duplication mutation. In contrast, our GcgCreERT2 mice are heterozygous null mutants for the Gcg gene. Although we did not detect any physiological abnormalities in the current analysis, Gcg heterozygosity may affect the functionality of preproglucagon-producing cells in extreme conditions or during ageing. Therefore, including GcgCreERT2 mice as a control is crucial.
The lineage labelling analysis of the GcgCreERT2 and GcgiCre mouse islets showed that 14–25% of PP-positive cells co-expressed glucagon in adult animals. This result is comparable with findings from a recent study that identified glucagon/PP double-positive cells (16% of PP-positive cells) by RNA fluorescence in situ hybridisation analysis of mouse islet cells . Our data showed that the size of this bihormonal cell population was relatively stable over the experimental period, suggesting the presence of a subpopulation of alpha cells. Lineage analysis using PP cell-specific Cre might determine if this subpopulation is an independent lineage from the majority of alpha cells that are negative for PP.
In conclusion, we describe a basic characterisation of a new GcgCreERT2 knockin mouse. The data demonstrate that this mouse line is a useful tool, particularly for studies on pancreatic alpha cells. In addition to GcgCreERT2 knockin mice, we report a new GcgiCre knockin mouse that also displayed specific and efficient recombination in alpha cells. However, it should be noted that the GcgiCre knockin mouse line is not adequate for some studies due to widespread Cre lineage labelling in the brain.
The mouse strains reported here are available at The Jackson Laboratory as stock no. 030681 for GcgCreERT2 mice and no. 030663 for GcgiCre mice.
We thank A. J. Styche (Children’s Hospital of Pittsburgh) for technical assistance in performing flow cytometry.
The data are available on request from the corresponding authors.
This work was supported in part by the Children’s Hospital of Pittsburgh Foundation to CS and by National Institutes of Health grants to GKG (DK098196, DK111460, DK112836).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
CS, XX and GKG contributed to the conception and design of the research. CS, KP, PG and JF performed the experiments. CS prepared the figures and drafted the manuscript. GKG edited the manuscript. All authors contributed to the interpretation of the data and to the critical revision of the manuscript, and approved the final manuscript. CS and GKG are responsible for the integrity of the work as a whole.
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