Fig. 7: Generation of a novel MIAT floxed mouse line.

A Targeting strategy of MIAT conditional knockout mouse model. Flox region is hypothetical exon 1–3. B Genotyping strategies to screen embryonic stem (ES) cells. 5’ homologous arm: 9.1 kb fragment should be amplified in the homologous recombinant ES cell clones, and none of fragment should be amplified in the negative ES cell clones. 3’ homologous arm: 5.7 kb fragment should be amplified in the homologous recombinant ES cell clones, and 12.4 kb fragment should be amplified in the negative ES cell clones. As shown in agarose gel electrophoresis of PCR products, six positive homologous recombinant ES clones were identified. C–D Genotyping strategies to screen F1 MIAT^fl/+ mice. The chimeric male mice were crossed with Flp mice to generate F1 mice. 5’ homologous arm: 8.6 kb fragment should be amplified in the homologous recombinant F1 mice, and 11.7 kb fragment should be amplified in the negative F1 mice. 3’ homologous arm: 3.9 kb fragment should be amplified in the homologous recombinant F1 mice, and 12.4 kb fragment should be amplified in the negative F1 mice. By long-PCR identification, seven heterozygous F1 mice were identified. All positive PCR products were confirmed by sequencing. Regions 1 and 2 were for identifying the 5’ homologous recombination, and regions 3 and 4 were for identifying the 3’ homologous recombination (C). For genotyping the offspring, short-PCR was used to identify heterozygous (HE) and wild-type (WT) mice. M: DNA size marker shown in the right (D). Genotyping PCR images show germline transmission of the targeted MIAT floxed allele. Agarose gel electrophoresis of PCR products is shown in the bottom (C–D).