Characterization and Validation of Cre‐Driver Mouse Lines
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Abstract
Conditional gene manipulations in mice are increasingly popular strategies in biomedical research. These approaches rely on the production of conditional genetically engineered mutant mouse (GEMM) lines with mutations in protein?encoding genes. These conditional GEMMs are then bred with one or several transgenic mouse lines expressing a site?specific recombinase, most often the Cre recombinase, in a tissue?specific manner. Conditional GEMMs can only be exploited if Cre transgenic mouse lines are available to generate somatic mutations, and thus the number of Cre transgenic lines has significantly increased over the last 15 years. Once produced, these transgenic lines must be validated for reliable, efficient, and specific Cre expression and Cre?mediated recombination. In this overview, the minimum level of information that is ideally required to validate a Cre?driver transgenic line is first discussed. The vagaries associated with validation procedures are considered next, and some solutions are proposed to assess the expression and activity of constitutive or inducible Cre recombinase before undertaking extensive breeding experiments and exhaustive phenotyping. Curr. Protoc. Mouse Biol. 1:1?15. © 2011 by John Wiley & Sons, Inc.
Keywords: site?specific recombination; conditional mutagenesis; inducible Cre; functional genomic
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Specificity and Efficiency of Cre Expression
- Specificity and Efficiency of Cre‐Mediated Deletion
- Anatomical Pattern of Cre‐Mediated Deletion
- Additional or Alternative Procedures to Detect Cre Expression
- Phenotypic Characterization of Cre Lines
- Summary
- Acknowledgments
- Literature Cited
- Figures
- Tables
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Figure 1. Flow chart combining simple assays allowing one to characterize Cre recombinase expression and activity in transgenic Cre lines from the genomic to the cellular level. Each step can also be applied to complete or confirm available data on Cre‐driver lines. G, generation; tg, transgenic; β–gal, β‐galactosidase; AP, alkaline phosphatase; RT‐qPCR, quantitative real‐time reverse transcriptase PCR; qPCR, quantitative PCR. View Image -
Figure 2. (A ) Schematic representation of the CreERT2 transgene used for pronuculei injection (Feil et al., ; Indra et al., ). The pair of primers used for RT‐qPCR amplification of Cre transcripts is located in the β‐globin region between a tissue‐specific promoter X (pX) and the CreERT2 gene. (B ) RXRαΔAF2(LNL) mouse line. This line is used as a floxed reporter line for determination of Cre recombinase activity by qPCR. A floxed Neo cassette is inserted between exon 9 and 10 of the RXRα gene, and a mutation ~undefined) is present in exon 10 (Mascrez et al., ). Three pairs of primers have been designed that allow specific amplification of the wild‐type (WT, green), floxed (LNL, blue), and excised (L, pink) alleles. Ex, exon; In, Intron; pA, polyadenylation site; pX, promoter of the gene X. View Image -
Figure 3. Characterization of Cre expression in the ubiquitous Ppm1a‐CreERT2 (A, B ) and digestive tract‐specific Vil1‐CreERT2 (C ) mouse lines. (A, C) Comparison of relative Cre expression between different transgenic lines ( N is at least 2 for each line) determined by RT‐qPCR in 25 tissue samples. (B) Comparison of relative expression of Cre versus the endogenous Ppm1a mRNAs ( N = 2). View Image -
Figure 4. Determination of Cre‐mediated excision by qPCR in the ubiquitous Ppm1a‐CreERT2 (A ) and digestive tract‐specific Vil1‐CreERT2 (B ) mouse lines. Comparison of the percentage of excised allele (L) versus floxed (LNL) and wild type (WT) allele in double transgenic animals Ppm1a‐CreERT2 /RXRαΔAF2(LNL) and Vil1‐CreERT2 /RXRαΔAF2(LNL) mice injected with vehicle (top) or with tamoxifen (bottom). For tamoxifen injections, tamoxifen (Sigma, cat. no. T56648) was prepared at 10 mg/ml in sunflower seed oil (Sigma, cat. no. S5007). Intraperitoneal injection of 100 µl of this solution was performed for 5 consecutive days (1 mg/mouse/day) with mice aged 10 weeks old, and whose weight was >20 g. Identical amounts of sunflower seed oil (vehicle) were administered following the same protocol to control mice. View Image -
Figure 5. Schematic representation of the construction and activity of the three most popular colorimetric reporter lines used to test for Cre activity in Cre‐driver transgenic lines. β‐gal, β‐galactosidase; AP, alkaline phosphatase; CMV, cytomegalovirus; PGK, phosphoglycerate kinase; CAT, chloramphenicol acetyltransferase; pA, polyadenylation site. View Image -
Figure 6. Characterization of Cre activity at histological level in the Ppm1a‐CreERT2 mice line. (A ) Cre‐mediated expression of the reporter gene β‐galactosidase in nine organs dissected from double‐transgenic Ppm1a‐CreERT2 /ROSA26 mice injected with tamoxifen. (B ) XGal (ROSA, ACZL) and hAP (Z/AP) staining of muscle sections revealing (i) the reporter expression pattern in three different colorimetric reporter lines crossed with a CMV‐Cre deleter mice (bottom row) (Dupe et al., ), and (ii) the localization of Cre activity in Ppm1a‐CreERT2 mice crossed with each of these reporter lines (top row). View Image -
Figure 7. Characterization of Cre activity at histological level in the Tph2‐CreERT2 and Ins1‐CreERT2 mouse lines. (A ) Sections through the pons, midbrain, and medulla revealing Cre activity in the raphe nuclei through Cre‐mediated expression of the reporter gene β‐galactosidase (Tph2‐CreERT2 /ROSA26 mice injected with tamoxifen, top row) and Cre expression through ISH with a specific Cre probe (Tph2‐CreERT2 mice, bottom row). (B ) IHC detection of Cre protein and Xgal staining of pancreas sections of tamoxifen or vehicle‐injected Ins1‐CreERT2 /ROSA26 mice revealed specific Cre protein content and Cre activity restricted to the islets of Langerhans. ISH procedures have been described elsewhere (Chotteau‐Lelievre et al., ; Gofflot et al., ). For IHC, primary rabbit anti‐Cre (1:8000 dilution, VWR, cat. no. 69050‐3) was used with goat anti‐rabbit antibody coupled to horseradish peroxidase (1:100 dilution; Invitrogen, cat. no. G‐21234) as secondary antibody. After washing, Cre was visualized by FITC‐tyramide amplification (1/50, 30‐min incubation) (PerkinElmer, cat. no. SAT701B). View Image -
Figure 8. Phenotypic analysis of the Vil1‐CreERT2 transgenic line. (A ) Schematic representation of the mouse BAC used for the construction of the Vil1‐CreERT2 transgene. (B ) Expression of mRNA levels of the GPCR Tgr5 in the ileum of control and Vil1‐CreERT2 transgenic mice. (C ) Mean ± SD of the area under the curve during an oral glucose tolerance test in control ( N = 8) and Vil1‐CreERT2 ( N = 8) mice fed a high‐fat, high‐sucrose diet for 12 weeks. (D ‐E ) Insulin and GLP‐1 levels measured in the serum 15 min after the administration of the oral glucose load in the control and Vil1‐CreERT2 transgenic mice of panel (B). View Image
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