Ins1 Cre knock-in mice for beta cell-specific gene recombination
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Pancreatic beta cells play a central role in the control of glucose homeostasis by secreting insulin to stimulate glucose uptake by peripheral tissues. Understanding the molecular mechanisms that control beta cell function and plasticity has critical implications for the pathophysiology and therapy of major forms of diabetes. Selective gene inactivation in pancreatic beta cells, using the Cre-lox system, is a powerful approach to assess the role of particular genes in beta cells and their impact on whole body glucose homeostasis. Several Cre recombinase (Cre) deleter mice have been established to allow inactivation of genes in beta cells, but many show non-specific recombination in other cell types, often in the brain.
We describe the generation of Ins1 Cre and Ins1 CreERT2 mice in which the Cre or Cre-oestrogen receptor fusion protein (CreERT2) recombinases have been introduced at the initiation codon of the Ins1 gene.
We show that Ins1 Cre mice induce efficient and selective recombination of floxed genes in beta cells from the time of birth, with no recombination in the central nervous system. These mice have normal body weight and glucose homeostasis. Furthermore, we show that tamoxifen treatment of adult Ins1 CreERT2 mice crossed with Rosa26-tdTomato mice induces efficient recombination in beta cells.
These two strains of deleter mice are useful new resources to investigate the molecular physiology of pancreatic beta cells.
KeywordsBeta cells Cre recombinase Glucose homeostasis Hypothalamus Insulin Pancreatic islets Transgenic mice
Cre-oestrogen receptor fusion protein
Enhanced yellow fluorescent protein
Green fluorescent protein
Rat insulin promoter
Pancreatic beta cells participate in glucose homeostasis by secreting insulin in response to rises in glycaemia to stimulate glucose uptake by peripheral tissues. To maintain this capacity over a lifetime, the insulin secretion capacity of individual beta cells as well as their number may be modulated to compensate for the development of insulin resistance in target tissues. Failure of this beta cell adaptive capacity is a major cause of the development of diabetic hyperglycaemia. On the other hand, the enhancement of natural mechanisms of beta cell adaptation might prove useful for regenerative therapies in autoimmune type 1 diabetes. Understanding the molecular basis of beta cell plasticity is therefore a central focus in diabetes research .
Genetically modified mice are being used extensively to study the role of specific genes in diabetes pathophysiology [2, 3, 4, 5], and several genetic systems have been developed to overexpress or inactivate genes selectively in beta cells [6, 7, 8, 9, 10, 11, 12]. In particular, recent recombinant technologies have provided the possibility to selectively inactivate genes of interest in a tissue- and time-dependent manner, as critically reviewed recently . A particularly useful approach consists in flanking the DNA sequence to be deleted by tandemly oriented 34 nucleotide-long lox sequences, which are recognised by Cre recombinase (Cre), a recombination enzyme that leads to the elimination of the sequence present between the two lox sites. Modification of the target DNA sequence with lox sites is performed by homologous recombination in embryonic stem cells, and the mice that are ultimately generated are crossed with mice that express Cre under a tissue-specific promoter to allow tissue-specific gene deletion. The use of Cre-oestrogen receptor fusion protein (CreERT2) , which is Cre fused to a modified form of the ligand-binding domain of the human oestrogen receptor that can be activated by tamoxifen, allows precise time-controlled genetic recombination.
One key aspect of the Cre-lox system is that transgenic expression of the Cre gene must be specific to the desired target tissue. Deletion in beta cells has been performed using mice expressing Cre under the control of various promoters such as that of the rat Ins2 gene [12, 15], the mouse Ins1 promoter  or the human insulin promoter . In contrast to the mouse Ins2 gene, which is also expressed in the embryonic and adult brain, the Ins1 gene is selectively expressed by the beta cells [18, 19], thereby providing a potential specificity advantage to drive Cre reporter expression only in beta cells. Existing transgenics that use insulin gene regulatory regions typically allow for beta cell gene inactivation; however, they often also lead to expression in brain regions, mostly the hypothalamus for the Ins2 promoter, leading to phenotypes that are complicated by the non-specific site of expression of the recombinase. Other approaches have used the promoters of transcription factor genes expressed at different stages of beta cell development, such as Pdx-1, Neurog3, Nkx2.2, Pax4, Pax6 or Isl-1 (for detailed review of these lines, see ). Although all of these lead to Cre expression in beta cells, they also lead to gene deletion either in islet non-beta cells or diverse non-pancreatic cellular lineages, including widely used Pdx1-Cre mouse lines that induce recombination in the exocrine pancreas. There is thus a need for more selective Cre lines for gene inactivation in beta cells.
The design of a mouse model that transcribes Cre exclusively in beta cells faces several obstacles. One is that gene regulatory sequences are not exclusively encoded in the 5′ regions of genes but are often determined or modulated by long-range cis-regulatory interactions, most of which have not been annotated. This obstacle is best addressed by inserting the recombinase in the endogenous genomic locus of interest, rather than using predefined promoter fragments. Another complexity is that most genes that are enriched in beta cells are also expressed in other cell types, some of which are central to glucose homeostasis. Notably, of the two non-allelic mouse insulin genes, Ins2 is the most abundantly expressed in beta cells, yet it is also expressed in hypothalamus and brain . Thus, even genetic models that succeed in recapitulating Ins2 expression will unavoidably lead to expression outside the beta cells, while recapitulation of Ins1 expression is expected to lead to selective beta cell expression
Here, we generated mice in which Cre was introduced at the initiator codon of the Ins1 locus by homologous recombination technology so as to ensure faithful expression of the recombinase in beta cells. We show that this approach allowed for highly efficient expression of Cre or CreERT2 recombinase in beta cells and that the specificity of expression was guaranteed with no expression in hypothalamus or other brain areas and with recombination as early as the time of birth.
Mouse genotyping was performed by PCR analysis using primers listed in electronic supplementary material (ESM) Table 1.
Histological and immunodetection procedures
For immunodetection of enhanced yellow fluorescent protein (eYFP), tdTomato, insulin, glucagon, pancreatic polypeptide and somatostatin in the pancreas or in the brain, mice were fixed by perfusion with cold 4% paraformaldehyde in sodium phosphate buffer (0.1 mol/l, pH 7.4). The organs were dissected and kept for 4 h in the same paraformaldehyde solution before overnight incubation in 30% sucrose, then frozen in isopentane and stored at −80°C until used.
For immunofluorescence detection studies in eYFP reporter mice 20 μm cryosections were preincubated for 30 min in a permeabilisation blocking buffer (0.1 mol/l PBS, pH 7.4, 3% BSA A8022, 0.3% Triton X-100, 9002-93-1; Sigma-Aldrich), then incubated overnight at 4°C with a rabbit polyclonal antibody to green fluorescent protein (GFP) (ab290, diluted 1:2,000; Abcam, Cambridge, UK), a polyclonal guinea pig anti-insulin (AO564, diluted 1:400; Dako, Baar, Switzerland) or anti-glucagon (4031-01F, diluted 1:500; Linco Research, St Charles, MO, USA), a polyclonal anti-pancreatic polypeptide (diluted 1:1,000; Linco Research) or a polyclonal rabbit anti-somatostatin (A0566, diluted 1:200; Dako). After washing, the sections were then incubated for 90 min with Alexa Fluor 488 goat anti-guinea pig IgG (H+L) (A11073, diluted 1:200; Invitrogen, LuBioScience, Lucerne, Switzerland) or with a goat anti-rabbit immunoglobulin-Cy3 conjugate (111-165-144, diluted 1/100; Jackson ImmunoResearch, West Grove, PA, USA), or for the Rosa26-tdTomato mice with Alexa Fluor 488 goat anti-rabbit IgG (H+L) (A-11008, diluted 1:200; Invitrogen). Immunofluorescence analysis of tdTomato was carried out in 3 μm paraffin sections, essentially as described [22, 23], using a rabbit polyclonal anti-red fluorescent protein (600-401-379; Rockland Immunochemical, Gilbertsville, PA, USA).
For enzymatic detection of eYFP, after overnight incubation with primary antibody, sections were washed and incubated for 90 min with a biotinylated goat anti-rabbit antibody (BA-1000, diluted 1:750; Vector Laboratories, Burlingame, CA, USA), incubated for 30 min with a biotin–avidin complex (Vector) and stained using 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich).
Sections were observed using an Axio Imager D1 microscope (Carl Zeiss, Oberkochen, Germany) interfaced with Axiovision software (Carl Zeiss) or a 510 Meta inverted confocal laser scanning microscope (LSM) with LSM software (release 3.5; Zeiss).
Glucose tolerance tests
For glucose tolerance tests, 16 h fasted male and female mice of indicated ages were injected i.p. with glucose (2 g/kg), and blood glucose levels were determined from tail–tip bleedings at the indicated times relative to glucose injections. Blood glucose levels were measured using a glucometer (Ascensia Breeze 2; Bayer HealthCare, Switzerland).
Data are expressed as means±SD or SEM. Statistical analysis was performed using an unpaired two-tailed Student’s t test. Values of p < 0.05 were considered significant.
All breeding and cohort maintenance performed in our animal facility and all experiments were approved by the Service Vétérinaire du Canton de Vaud and the Comitè Ètic d’Experimentació Animal of the University of Barcelona.
Generation of the Ins1 Cre and Ins1 CreERT2 mice followed the strategy described in Fig. 1a–c and the Methods section, allowing expression of Cre or CreERT2 from the translation initiation codon of the Ins1 gene. Embryonic stem cells with homologous recombination were used to generate chimeric mice. These were crossed with Flp deleter mice to remove the neo cassette. The resulting Ins1 Cre mice were crossed with Rosa26-eYFP mice to determine the anatomical sites of Cre-dependent lox site recombination. PCR analysis of the recombined Rosa26-eYFP locus (Fig. 1c) indicated recombination in whole pancreas DNA preparations but not in DNA extracted from liver, hypothalamus, cortex or cerebellum (Fig. 1d).
Here we describe the generation of two new mouse models for efficient and selective induction of lox-dependent recombination in pancreatic beta cells. The Ins1 Cre mice induce complete recombination by the time of birth in all beta cells, whereas the Ins1 CreERT2 mice can be used for tamoxifen-dependent gene recombination in adult mice.
Several Cre transgenic mice have already been established for beta cell-specific gene recombination. The present models use a knock-in strategy to express Cre from the endogenous Ins1 locus. This locus is known to have a more restricted beta cell expression than the Ins2 locus, which displays transient expression in the hypothalamus and other brain structures . The use of the Ins2 promoter to drive Cre expression led to mouse models with compound genetic recombination in the islets and brain, leading to complicated interpretation of the phenotype of mice used . Ins2 knock-in mouse lines have also been generated , but are similarly expected to recapitulate endogenous expression and result in ectopic expression in brain. On the other hand, two recent studies have reported transgenic models expressing Cre or CreER driven by Ins1 promoter sequences or by an Ins1 locus bacterial artificial chromosome [16, 26]. In keeping with our model, these mice lack Cre expression in the brain [16, 26]. Our model provides further advantages in that Cre is inserted in the native Ins1 locus and is thus theoretically controlled by all known and unknown native cis-regulatory sequences, providing greater ability to recapitulate the endogenous Ins1 expression pattern. We have indeed verified that recombination is efficiently induced in pancreatic islet beta cells at birth, whereas no recombination could be observed in the brain. Furthermore, we report an inducible Ins1 CreERT2 mouse line that is capable of recombination in 60–70% of the beta cells. Importantly, there was no leakiness of CreERT2 expression as judged by the absence of tdTomato expression in non-treated or vehicle-treated mice.
An important aspect of the Ins1 Cre mouse model is that its glucose homeostasis and body weight control are indistinguishable from those of control littermates. It has been reported that one of the rat insulin promoter (RIP)-driven Cre mouse lines  has a defect in glucose homeostasis , casting some doubt on the results of reports showing an impact of beta cell gene deletion using this recombinase line. We have not tested body weight and glucose homeostasis in cohorts of Ins1 CreERT2 mice. However, because both the Ins1 Cre and Ins1 CreERT2 mice were generated by the same knock-in strategy we anticipate similar effects. In the different transgenic mouse lines, such as RIP-Cre, functional differences may result from insertional mutagenesis caused by the random insertion of the transgene, usually as large concatemers. This can potentially lead to unwanted effects such as the formation of loss-of-function alleles, abnormalities in the activity of nearby genes, and unexpected expression pattern and strength of the transgene. The knock-in approach used here results in a well-defined targeted modification that is therefore more precise and less prone to induction of artefacts. Another theoretical consideration is that the Ins2 promoter is stronger than Ins1  and could thus result in higher concentrations of Cre that are potentially deleterious. Importantly, our knock-in approach does not affect glucose homeostasis despite suppressing one Ins1 allele, which is also in agreement with previous studies reporting perfect glycaemic control in mice with deletion of single alleles of the Ins1 or Ins2 locus .
In summary, we have generated two new Cre deleter mice that can be used for beta cell-specific constitutive (Ins1 Cre) or inducible (Ins1 CreERT2) deletion of genes. The constitutive deleter mice induce deletion in nearly all beta cells from the immediate postnatal period to adulthood and they display normal control of body weight and glycaemia. The inducible deleter mice will be useful to test the impact of gene deletion in adult mice or at a defined time during development, although deletion does not occur in all beta cells. These two mouse strains are therefore very valuable new tools for beta cell research.
We thank V. Grau (IDIBAPS, Barcelona, Spain) for expert technical assistance. The Ins1 Cre and Ins1 CreERT2 mice were generated by Genoway and we thank K. Thiam (Genoway, Lyon, France) for discussing the targeting strategy.
This project was funded by a framework programme 6 project of the European Union (EuroDia; LSHM-CT-2006-518153) and by grants from the Swiss National Science Foundation (no. 3100A0B-128657) to BT and Ministerio de Economía y Competitividad (SAF2011-27086) to JF. JF is a Wellcome Trust Senior Investigator (WT101033).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
BT and JF conceived the project, supervised its realisation, analysed the data and wrote the manuscript. DT, MAM, MR and EH performed the experiments, analysed the data and contributed to writing of the manuscript. BT and JF are the guarantors of this work. All authors have approved the final version.
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