Figure 3. “Knockin” Mouse Model.

(A) Targeting construct. As we have described elsewhere (Kos et al. 2003), targeting initially resulted in a “knockout” allele, due to disruption of normal transcription, presumably by the intronic loxP-flanked neomycin resistance cassette. After breeding to Cre transgenic mice, this neomycin cassette was excised, as illustrated.

(B) Northern blot analysis using kidney total RNA illustrates that the expression of the Actn4 transcript in K228E/K228E is similar to the expression in wild-type mice.

(C) RT-PCR and sequencing of the relevant portion of Actn4 exon 8 confirms that the transcript in mice homozygous for the targeted allele contains the desired point mutation (top, wild-type; bottom, targeted).

(D) Western blot showing markedly decreased expression of α-actinin-4 protein in K228E/K228E homozygous mice and moderately decreased expression in heterozygotes. Shown are blots using protein from cultured fibroblasts. Results were similar using protein extracted from lung, brain, liver, and kidney (data not shown). β-actin control is shown below.

(E) Western blot showing expression of α-actinin-4 in lymphocytes from a human K228E/+ heterozygote (Kaplan et al. 2000) and three wild-type controls (two related, one unrelated). β-actin control is shown below.