Abstract
Conditional gene knockout technology is a powerful tool to study the function of a gene in a specific tissue, organ or cell lineage. The most commonly used procedure applies the Cre-LoxP strategy, where the choice of the Cre driver promoter is critical to determine the efficiency and specificity of the system. However, a considered choice of an appropriate promoter does not always protect against the risk of unwanted recombination and the consequent deletion of the gene in other tissues than the desired one(s), due to phenomena of non-specific activation of the Cre transgene. Furthermore, the causes of these phenomena are not completely understood and this can potentially affect every strain of Cre-mice. In our study on the deletion of a same gene in two different tissues, we show that the incidence rate of non-specific recombination in unwanted tissues depends on the Cre driver strain, ranging from 100 %, rendering it useless (aP2-Cre strain), to ~5 %, which is still compatible with their use (RIP-Cre strain). The use of a simple PCR strategy conceived to detect this occurrence is indispensable when producing a tissue-specific knockout mouse. Therefore, when choosing the Cre-driver promoter, researchers not only have to be careful about its tissue-specificity and timing of activation, but should also include a systematical screening in order to exclude mice in which atypical recombination has occurred and to limit the unnecessary use of laboratory animals in uninterpretable experiments.
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Acknowledgments
We thank Dr. Pedro Herrera for kindly providing us with RIP-Cre mice. V.S. was supported by a grant from the Fondation pour la Recherche Médicale (FRM) Grant FDT20140930804. This work was supported by Grants from Région Nord-Pas de Calais, FEDER, INSERM, A.N.R. (FXREn), Société Francophone du Diabète (SFD), Université Lille 2, Université Lille Nord de France and European Genomic Institute for Diabetes (EGID, ANR-10-LABX-46) and European Commission. B.S. is a member of the Institut Universitaire de France.
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Fig. S1
Breeding scheme used to obtain AT-POI-KO mice and their corresponding controls. POIfl/fl aP2-Cre−/− mice were crossed with POIwt/wt aP2-Cre+/? mice to generate POIfl/wt aP2-Cre−/− and POIfl/wt aP2-Cre+/− offspring. Next, POIfl/fl aP2-Cre−/− and POIfl/wt aP2-Cre+/− mice were bred together to generate POIfl/fl aP2-Cre+/− (AT-POI-KO) and POIfl/fl aP2-Cre−/− littermate mice (control mice) (TIFF 10960 kb)
Fig. S2
The null allele is present in all aP2-Cre+/− obtained from 1st cross. a, Percentages of mice from the 1st cross distributed by tail genotype for AT-POI-KO breeding. b, Representative gel showing PCR products for POI-amplified locus on genomic DNA extracted from tail biopsy of AT-POI-KO mice (TIFF 13998 kb)
Fig. S3
The null allele is present in all aP2-Cre+/− mice obtained from 2nd cross. a, Percentages of mice from the 2nd cross distributed by tail genotype from AT-POI-KO breeding. b, Representative gel showing PCR products for POI-amplified locus on genomic DNA extracted from tail biopsies of AT-POI-KO mice (TIFF 14170 kb)
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Spinelli, V., Martin, C., Dorchies, E. et al. Screening strategy to generate cell specific recombination: a case report with the RIP-Cre mice. Transgenic Res 24, 803–812 (2015). https://doi.org/10.1007/s11248-015-9889-1
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DOI: https://doi.org/10.1007/s11248-015-9889-1