Floxed mini-gene targeting strategy for the generation of conditional “knock-in” of Gα
allele. A Exon 5 of the genomic locus of the gene Gnai2 was replaced by homologous recombination using a vector containing a G184S mutant Exon 5 (E5*) with a 3’ extension containing Flp Recombinase Target (FRT)-sites (red chevrons) flanking a neo selection cassette and a 5’ extension with Lox P sites (green triangles) flanking a minigene containing wild-type E5-9. B After introduction of the targeting vector, the neo marker is removed by breeding the Gαi2
fl-neo-G184S positive mice with transgenic Flp recombinase expressing animals. C To generate whole-body Gαi2
*G184S conditional knock-in mice, transgenic mice carrying tamoxifen-sensitive, Cre-ERT2 gene driven by the Gt(ROSA)26Sortm1 (ROSA) promoter were bred with Gαi2
fl/fl-G184S mice. After tamoxifen administration there is recombination of genomic Lox P sites which leads to removal of the E5-9 minigene and the mutant E5* is positioned in the normal Gnai2 gene to express the Gαi2*G184S mutant allele. D Southern blot analysis of ES cell genomic DNA following digestion with EcoNI and hybridization with the 5’-Neo probe to screen for 5’ homologous recombination event. E Southern blot verification analysis of ES cell genomic DNA following digestion with AflII and hybridization with an external 3’ probe. In figures d and e lanes 1 to 4 are correctly targeted ES cell clones, lane 5 is a non-targeted (WT) ES cell clone.