Figure 1. Clrn1 RT–PCR analysis in mouse retina and KO mouse generation. (A) Schematic diagram illustrating the intron/exon structure of the murine Clrn1 gene, and the structure of the spliced Clarin 1 Is Necessary for Hearing but Not Vision transcripts showing primer locations. (B) Two transcripts are readily amplified from mouse retina. A common forward primer (F2) was paired with exon-specific reverse primers (R2-R6; see Figure S1) in lanes 1–5. (C) Quantitative RT–PCR analysis comparing major splice variant expression levels in 6 adult C57BL/6J mouse retinas (3 male, 3 female), using F3/R1 primers and fluorescent FAM-BHQ probes (see Figure S1) specific for exon 1/3 or exon 1/4 splice junctions. The inset gel image shows end-point RT-PCR analysis of the same samples using F2/R2 primers. (D) Postnatal developmental and adult expression of Clrn1 using F2/R2 primers. (E) Prenatal developmental expression of Clrn1. Whole embryos, heads, and brain tissue RNAs were amplified using F1/ R1 primers. (F) RT–PCR analysis of isolated prenatal eyes, a P21 retina, an adult control retina, and a No RT (no reverse transcription) negative control using F1/R1 primers. (G) Regional expression in the adult mouse brain is demonstrated (F1/R1 primers). All brain regions appear to transcribe the Clrn1 gene. (H) Clrn1 expression in adult mouse organs (F1/R1 primers). A more restricted expression profile is evident in mouse tissues outside of the nervous system, similar to expression in human [12]. (I) The 59 untranslated region (59 UTR), exon 1 (containing the transcriptional start site (TSS) and the ATG start codon), and the proximal 269 bp of the first intron were removed in embryonic stem cells by homologous recombination. The uppermost construct repre- sents a 50,000 bp genomic fragment on chromosome 3q21, while the two lower constructs represent approximately 20,000 bp centered on exon 1. The genomic region (*) and neomycin resistance gene (NeoR) were spliced out by Cre recombinase activity at loxP (P) sites. (J) RT–PCR analysis of Clrn1 total KO (2/2), littermate heterozygous controls (+/2), and WT (+/+) animals using F2/R2 primers. No RT = no reverse transcription. (+) = Plasmid DNA containing cloned Clrn1 cDNA (exons 1, 3, and 4). doi:10.1371/journal.pgen.1000607.g001 Generation of the Clrn1 Knockout Mouse A schematic diagram of the genetic recombination used to generate the Clrn1 KO mouse is shown in Figure 1I. We eliminated the production of all known coding transcripts in the eye and ear by utilizing Cre recombinase and loxP sites to recombinatorily remove part of the gene promoter, the transcrip- tional start site (TSS), the entire first coding exon including the 59 UTR and starting ATG, and a small portion of the 1st intron (collectively labeled with an asterisk; Figure 1I). Heterozygous animals on a C57BL/6 background were mated and provided viable WT, heterozygous Clrn1 KO (+/2), and homozygous Clrn1 KO (2/2) progeny. Figure 1J shows retinal RT-PCR analysis of littermate WT and Clrn1 KO (homozygous and heterozygous) animals. Using primers (F2/R2) that span from exon 1 to exon 4, both primary transcripts were observed in both WT (+/+) and Clrn1 heterozygous (+/2) animals, whereas no amplification was observed in the homozygous (2/2) Clrn1 KO animals. Furthermore, although this was not a quantitative assay, it is apparent that the WT mouse expressed more than either heterozygous animal. Moreover, no amplification was apparent when the RT enzyme was omitted from the cDNA synthesis reaction, ensuring that positive amplification was due to Clrn1 mRNA expression and not genomic DNA contamination. Inner Retinal Expression of Clrn1 In contrast to all other known disease-causing mutations resulting in retinitis pigmentosa (RP), Clrn1 mRNA expression was restricted to the inner nuclear layer (INL) of the retina, and was undetectable in photoreceptor or retinal pigmented epithelial (RPE) cells. This was an unexpected and unusual result, and to the authors’ knowledge, this is the first time that a mutant protein produced by the inner retina results in an RP phenotype. In situ hybridization was performed with a Clrn1 riboprobe on developmental retinal tissue from both WT and Clrn1 KO mice (Figure 2). In WT animals, Clrn1 mRNA was not detected at E18.5 PLoS Genetics | www.plosgenetics.org 3 August 2009 | Volume 5 | Issue 8 | e1000607