Figure 2

Targeted disruption of the mouse Csrp2 gene. (A) A neomycin resistance cassette (neo) was incorporated into Csrp2 (exon 4) as outlined in the Method section. The different Csrp2 alleles (WT vs k.o.) from progeny of mating heterozygotes were discriminated by PCR analysis of tail biopsy DNAs. The positions of the oligonucleotide primers used for amplification of the wild-type (218 bp) and mutant (422 bp) PCR fragments are indicated. In the genotyping experiment shown, the different DNAs were genotyped as homozygous null (-/-), heterozygous (+/-) or wild-type (+/+). (B) Southern hybridisation of littermate offspring from heterozygous intercrosses genotyped as heterozygote (WT), wild-type (WT), or homozygote null (Csrp2^-/-). The DNA was digested with BamHI, fragments were separated in a 1% agarose gel and transferred to a nylon membrane. The blot was hybridized with an external 1.5 kbp XhoI probe (see Methods), resulting in fragments of ~7.3 (knock out) or ~12.6 kb (wild-type) in size. (C) Northern blot of total kidney RNA isolated from wild-type (+/+), heterozygote (+/-), and homozygote (-/-) Csrp2 null mice. The RNAs were hybridized with a Csrp2 specific cDNA probe. The autoradiograph showed the typical 1.2. kb Csrp2 signal in Csrp2^+/+, a weaker band with Csrp2^+/-, and a faint band with the Csrp2^-/- mice. To verify the integrity of RNAs, the blot was subsequently hybridized with a GAPDH-specific cDNA probe. (D) Western blot of kidney homogenates extracted from wild-type (WT) and mutant mice (Csrp2^-/-). As a positive control and to demonstrate the specificity of the CRP2 specific antibody, cell extracts taken from COS-7 cells that were transfected with myc-epitope tagged version of murine CRP1, CRP2 and CRP3 were taken. The expression of these proteins was demonstrated by subsequent probing with a myc-epitope specific antibody. In the Csrp2 nulls, no CRP2 band at any size was detected.