Figure 1
Generation of the Tmem30a-knockout-first allele. (A) Scheme showing the targeting strategy for the disruption of the Tmem30a gene. The knockout-first allele design is shown with the LacZ reporter. Exon 3 is flanked by two loxP sites. PCR primers used to genotype the target allele are shown beneath the diagram. Primer pair F1-R1 was designed to genotype the loxP site upstream of exon 3. Primer pair F2-R2 was designed to genotype the loxP site downstream of exon 3. Primer pair F3-R3 was designed to genotype the loxP site upstream of the human beta actin promoter (hBactP). After crossing with the Flper deletion line, the FRT-flanked reporter cassette was removed, resulting in a floxed allele. The critical exon (exon 3) is flanked by two loxP sequences. When the floxed allele was crossed to a Cre-expressing line, exon 3 was deleted, resulting in a frame-shifting deleted allele. (B) Genotyping of Tmem30a-knockout first mice. Using primer pair F1-R1, PCR amplification of genomic DNA extracted from mouse tails produced products of 220 bp in knockout mice. Using primer pair F2-R2, PCR amplification of genomic DNA extracted from mouse tails produced products of 214 bp in knockout mice and 179 bp in wild-type mice. Gel picture was cropped to save space. Full gel picture was listed as Figure S9. (C) Genotyping of Tmem30a conditional knockout mice. Using primer pair F2-R2, PCR amplification of genomic DNA from mouse tails produced products of 214 bp in homozygous conditional knockout mice and 179 bp in wild-type mice. Two products were amplified in heterozygous mice. Gel picture was cropped to save space. Full gel picture was listed as Figure S9. (D) Colocalization of TMEM30A with ATP8A2 in the mouse retina. Immunofluorescence staining of cryosections of retinas from wild-type mice at 30 days of age using a polyclonal antibody to ATP8A2 (green) and a monoclonal antibody to TMEM30A (red). Nuclei were counter stained with DAPI (blue). Both proteins mainly localized to the outer segment of the photoreceptor cells. Weak expression in the inner segment was observed. Scale bar: 25 μm.
