title: A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot.
abstract: The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific genomic locus, but limits the detection of its correct genomic integration by standard PCR methods. Genomic Long Range PCR (LR-PCR), using primers adjacent to the homology arms, has been used as an alternative to radioactive-based Southern blot screenings. However, LR-PCRs are often difficult and render many false positive and false negative results. Here, we propose an alternative screening method based on the detection of a genetic modification at the mRNA level, which we successfully optimized in two mouse models. This screening method consists of a reverse-transcription PCR (RT-PCR) using primers that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting constructs which can also be useful to screen KO and KI mutant mice or cell lines including those generated by CRISPR/Cas9.
authors.affiliation: Department of Cellular and Molecular Medicine, Center for Chromosome Stability and Center for Healthy Aging, Panum Institute, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Eliene
authors.initials: E
authors.lastname: Albers
authors.affiliation: Department of Cellular and Molecular Medicine, Center for Chromosome Stability and Center for Healthy Aging, Panum Institute, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Mauro
authors.initials: M
authors.lastname: Sbroggiò
authors.affiliation: Transgenic Core Facility, Department of Experimental Medicine, Panum Institute, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Javier
authors.initials: J
authors.lastname: Martin-Gonzalez
authors.affiliation: Department of Cellular and Molecular Medicine, Center for Chromosome Stability and Center for Healthy Aging, Panum Institute, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Avram
authors.affiliation: Department of Cellular and Molecular Medicine, Center for Chromosome Stability and Center for Healthy Aging, Panum Institute, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Munk
authors.affiliation: Department of Cellular and Molecular Medicine, Center for Chromosome Stability and Center for Healthy Aging, Panum Institute, University of Copenhagen, Copenhagen, Denmark. ajlopez@sund.ku.dk.
authors.forename: Andres J
authors.initials: AJ
authors.lastname: Lopez-Contreras
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D053595:Embryonic Stem Cells / Q000502:physiology*; D019398:Genes, BRCA1; D008822:Mice, Transgenic; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
keywords: RT-PCR; Southern blot; Transgenic mouse; mESC screening