TaqMan® Real Time PCR Based Assays for Screening and Validating Transgenic and Knockout Constructs

We have developed TaqMan® real-time PCR based copy number assays to screen and validate transgenic and knockout mice for the presence and absence of reporter genes and target genes. The method offers high specificity and accuracy, and simplified workflow over traditional screening techniques including PCR, southern blot and cytogenetic analysis, and can be applied to validate and quantify gene constructs in cells and other animal and plant species. We designed TaqMan® copy number assays for several reporter genes including beta-galactosidase (LacZ), green fluorescent protein (GFP), neomycin resistance gene, ampicillin, hygromycin and Cre recombinase that are commonly used as markers in targeted mutagenesis and gene manipulation.  The reporter gene assays along with the TaqMan® mouse copy number assays were tested in 40 transgenic and knockout mouse samples. In knockout samples, we detected the copy numbers of the reporter genes introduced to disrupt targeted knockout regions and the knockout mouse gene regions. In transgenic samples, we measured the copy number of the reporter genes and the transgenes. The copy number results were subsequently compared with the expected genotypes of mouse samples. Our studies demonstrate that the assays accurately and specifically detect transgenic and knockout mice with high reproducibility. We also developed TaqMan® gene expression assays for several commonly used reporters including LacZ, GFP, yellow fluorescent protein and luciferase. The assays can be used to verify and quantify reporter gene products from expression vectors that have been introduced from cells or animals.