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. 2004 Jun;24(12):5281–5289. doi: 10.1128/MCB.24.12.5281-5289.2004

FIG. 1.

FIG. 1.

Generation of floxed SRF transgenic mice by homologous recombination. (A) Targeting strategy. Homologous recombination introduced LoxP sites at the two ends of exon 2 and a floxed neomycin resistance into intron 2. Maps of the wild-type Srf locus, the targeting vector, the recombined allele, and the excised allele are shown. Exons are shown as boxes. The position of the 5′ and 3′ probes used for Southern blot analysis in panel B are indicated. Arrows show primer positions for PCR. Arrowheads indicate the LoxP sites. (B) Southern blot analysis. Genomic DNA from ES cells was digested with NdeI and EcoRV. The 5′ and 3′ probes revealed a 20-kb fragment in the wild-type ES cells (+/+), and two bands (20 and 8 kb for 5′ probe and 20 and 12 kb for 3′ probe) in recombined ES cells (Sf/+). (C) Genotyping of offspring by PCR. Primers SF1 and SF2 amplified a 448-bp fragment for wild-type mice and a 492-bp fragment for homozygous floxed SRF (Sf/Sf) mice due to the presence of the LoxP sequence. Both bands were amplified in heterozygous mice (Sf/+). (D) Western blot analysis. Equal amounts of proteins from the hearts of adult Sf/Sf and wild-type (wt) mice were loaded onto the gel, and two bands corresponding to the SRF protein were revealed with anti-SRF antibody (Santa Cruz Biochemicals). (E) Characterization of exon 2 excision. DNA extracted from E9.5 embryonic hearts was amplified by using SF1, SF2, and SF3 oligonucleotides. A 310-bp fragment was detected after the excision of exon 2 in both homozygous (Sf/Sf) and heterozygous (Sf/+), Cre-expressing embryos. A sample containing DNA extracted from mice containing an allele lacking exon 2 of SRF (SΔ/+) was used as a positive control. (F) Absence of normal SRF protein in E10.5 embryo hearts. Western blot analysis revealed two bands of approximately 67 and 54 kDa in homozygous (Sf/Sf) and heterozygous (Sf/+) control embryos. No bands corresponding to normal SRF was detected in Cre-expressing homozygous (Sf/Sf) hearts. A smaller band of ∼46 kDa corresponding to the exon 2 truncated protein was observed in these samples.