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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: Circ Cardiovasc Genet. 2014 Jul 15;7(4):423–433. doi: 10.1161/CIRCGENETICS.113.000281

Figure 2. Generation of a knock-in mouse having the human NKX2-5 disease-causing missense mutation in the homeodomain at the position of 52Arg→Gly (Arg52Gly, R52G).

Figure 2

(A) Structure of wild-type Nkx2-5 locus, and the targeting construct. Southern blotting hybridized with the 5′ probe showed 8.3 kb EcoRI-digested genomic DNA in wild-type alleles and 6.5 kb fragments in the targeted alleles. PCR primers used for genotyping are shown. (B) Identification of wild-type (lane 1) and mutant alleles (lane 2) in ES cells using Southern blotting. (C) Sequence analyses of genomic DNA demonstrate CGT in wild-type vs. (C/G)GT in +/R52G. (D) Western blotting of total Nkx2-5 proteins and GAPDH in P1 hearts dissected from Nkx2-5+/+ (lane 1) vs. Nkx2-5+/R52G (lane 2) mice. (E) Growth retardation in E10.5 homozygous Nkx2-5R52G/R52G mouse in comparison to wild-type littermate. Nuclear localized wild-type or mutant Nkx2-5 proteins were visualized by immunohistochemistry (brown). (F) Generation of germline Nkx2-5 knockout mice with elimination of floxed-exon 2 by Cre-deleter mice. (G) Western blotting of Nkx2-5 proteins and GAPDH in P1 hearts dissected from Nkx2-5+/+ (lane 1) vs. Nkx2-5+/− (lane 2) mice.