Application Guide - QIAxcel Advanced

34 QIAxcel Advanced Application Guide 10/2016 Rapid and effective genotyping of Cre transgenic mice Norman Moullan Laboratory of Integrative Systems Physiology (LISP), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Sizes of Cre gene-specific DNA fragments from the Cre gene were unambiguously identified using the QIAxcel® system. The accuracy of the system allowed rapid and reliable identification of Cre transgenic mice. Introduction The Cre-lox system, which is not naturally present in the mouse genome, is a molecular tool for genome manipulation and has been successfully used to generate mouse mutants (1, 2). Initially, transgenic mouse lines are produced: one expressing the Cre recombinase and one carrying 2 loxP sites (34 bp sequences). Upon crossing Cre and loxP strains, the Cre recombinase cuts specifically at the loxP sites in tissues where Cre transgene is expressed (3, 4). Subsequent recombination of loxP sequences leads to rearrangements of the genome. Depending on the orientation of the loxP sites, deletions, inversions, or chromosomal translocations are generated. By targeting Cre recombinase to tissues of interest, conditional knockout mutants can be generated (2). To test the successful insertion of the Cre transgene into the genome of transgenic mice, PCR- amplified Cre gene sequences were identified using QIAxcel system. Materials and methods Rapid genomic DNA extraction from mouse ear tissue was performed by incubating samples in 0.1N NaOH at 70–95°C for up to 10 minutes. Samples were placed on ice for 5 minutes and 30 µl 170 mM Tris·HCl (pH 8.0) was added. Samples were vortexed and centrifuged for 4 minutes at maximum speed, and 3 µl of the supernatant was used for PCR amplification. Primers specific for the Cre transgene (sense: 5’- GAACC TGATG GACAT GTTCA GG -3’; anti-sense: 5’- AGTGC GTTCG AACGC TAGAG CCTGT -3’) were used to amplify a 320 bp fragment. Primers specific for the myogenin gene (sense 5’- TTACG TCCAT CGTGG ACAGC -3’ and anti-sense 5’- TGGGC TGGGT GTTAG CCTTA -3’) were included in the reaction to amplify a 250 bp control fragment.

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