Figure 1: Generation and validation of Coro7gt/gt mice.
From: Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology
(a) The knockout vector consists of the lacZ gene as a reporter and the neomycin phosphotransferase gene. Genomic locus of the Coro7 gene depicting exons 1, 2, 3, 4, 5 and 6 is illustrated. CRN7 reporter insertion allele: FRT sites, splice acceptor, IRES, lacZ reporter cassette, neomycin selection cassette, 3′ lox P sites flanking the selection cassette and exon 4. The 5′ and 3′ homology arms are derived from the C57BL/6N genetic background. Schematic showing positions of the Southern blot neo probe, AseI restriction enzyme sites, PCR genotyping primers (P1: Ex4F (F-forward), P2: GR7 (R-reverse), arrowheads show direction) and Reverse transcription (RT)-PCR primers (Ex1F-Ex3R and Ex6F, arrowheads show direction; Ex16R is not highlighted here but is further downstream). (b) PCR genotyping was performed using tail genomic DNA from wild-type (+/+), heterozygous (+/gt) and homozygous (gt/gt) mice using primers P1 and P2. (c) Southern blot analysis of AseI-digested genomic DNA from wild-type, heterozygous and homozygous mice. A radiolabelled probe specific for the neomycin cassette was used for hybridization. (d) RT-PCR analysis was done using primer pairs from exon 1 and exon 3 (Ex1F-Ex3R) as well as exon 6 and exon 16 (Ex6F-Ex16R) at the total RNA levels using fibroblasts from wild-type and knockout mice. Control RT-PCR products were generated by a GAPDH-specific primer pair. (e) For Northern blot analysis mRNA was isolated from wild-type and knock-out mice fibroblasts and hybridized with a cDNA probe amplified from exon 6 to exon 16. *indicates non-specific binding to 28S and 18S rRNA. (f) Immunoblot analysis using lysates from various tissue from wild-type (WT), heterozygous (HET) and homozygous (HOM) mice. Lysates were probed with CRN7-specific mAb K37-142-1.
