Generation and characterization of <i>Limk2-</i>mutant mice.

<p>(A) Retroviral gene trap vector VICTR48 (EU676804) was used to produce OmniBank clone OST80053, which contains an insertion within intron 12 of the <i>Limk2</i> gene. The <i>Limk2</i> transcript variants 1–3 (v1–3) represent protein isoforms a-c, respectively, according to current NCBI Reference Sequence annotation for accessions NM_010718.3, NM_173053.1, and NM_001034030.1 (intron/exon numbering based on transcript v1). This mutation would be expected to truncate the <i>Limk2</i> gene product within the kinase domain following coding exon 12, disrupting all reported transcript variants of the <i>Limk2</i> gene. Open boxes denote untranslated exons, filled boxed denote coding exons. Exons that code for the LIM domains are shown in orange, those that code for the PDZ domain are shown in blue, and those that code for the kinase domain are shown in red. LTR, viral long terminal repeat; SA, splice acceptor sequence; <i>neo</i>, neomycin phosphotransferase gene; pA, polyadenylation sequence; <i>Pgk</i>, phosphoglycerate kinase-1 promoter; <i>Btk</i>-SD, Bruton's tyrosine kinase splice donor sequence. *The Limk2 v3 transcript is elsewhere referred to as Limk2t <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047168#pone.0047168-Ikebe1" target="_blank">[45]</a> or tLimk2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047168#pone.0047168-Takahashi2" target="_blank">[46]</a>. (B) RT-PCR expression analysis of <i>Limk2</i> transcripts. Endogenous <i>Limk2</i> transcription was detected in the brain, eye and testis of wild type (+/+) and heterozygous (+/−) mice. No endogenous <i>Limk2</i> expression was detected in homozygous (−/−) tissues. Primers 10F and 14R are complementary to <i>Limk2</i> exons 10 and 14/15, respectively, and amplify a product of 489 nucleotides. (C) Immunoblotting analysis with a rat monoclonal antibody that recognizes mouse LIMK2 residues 145–260, which includes the PDZ domain, revealed LIMK2 in several tissues from wild type mice (+/+) such as spleen, brain, lung and retina. LIMK2 was undetectable in these same tissues isolated from homozygous <i>Limk2</i> (−/−) mutant mice. (D) Mouse primary keratinocytes were cultured from neonatal animals and probed with antibodies that recognize phospho-cofilin, total cofilin or GAPDH. Cell cultures were obtained from several litters to quantify the ratio of p-cofilin to cofilin. There was a statistically significant reduction in p-cofilin levels in the knockout compared to wild type. The difference between the means was 66%±7 (<i>n</i> = 4 per genotype; P<0.0001; student's T test).