Screen And Identification Of The Causative Gene Of The Retinal Degeneration Fast Mouse Model

Retinitis pigmentosa is one of the main diseases that cause loss of vision, and it has a prevalence of 1/4000-1/3500. Our laboratory found and has been establishing an inbred mouse model of retinitis pigmentosa. ERG recordings of this kind of mouse reveal that adult mice lack the a and b wave. The inheritance pattern is autosomal recessive. The occurrence and progress of the retina degeneration is relatively early and fast in this mouse model. It is potentially a good animal model for retinitis pigmentosa. This research aimed to screen and identify the causative gene of the retinal degeneration fast (rdf) mouse model. We took the following methods to carry out this research:1. Chose some candidate genes and compared their expression level in the retina of rdf mice with wild type Kunming mice.2. Cloned and sequenced the selected candidate gene and analyzed the structure of the suspected causative gene, and then checked the inheritance of this suspected causative gene in several generations of rdf mouse.3. Constructed two hybrid mice family by crossing the affected mice with wild type Kungming and C57BL/6J mouse, respectively. Genotyped the family members and identified the phenotype by ERG recording, and then analyzed the segregation type of the genotype with the phenotype.4. Checked the inheritance of the mutant gene in cogenic mice.5. Profiled the expression of the mRNA in the retina of affected mice. Analyzed the mRNA coding region sequence.Results1. RT-PCR results revealed that, compared with the wild type Kunming mice, expression of the pde6b gene in the retina was barely detectable in adult rdf mice and was significantly decreased in postnatal 7 days rdf mice.2. Genomic analysis of the pde6b gene revealed that there was a long terminal repeat (LTR) sequence inserted in the first intron of the pde6b gene. RT-PCR using specific primers for retrovirus revealed that retrovirus env gene was expressed in the rdf mice retina, and sequence analysis of the amplified fragment showed 100% homology to xmv retrovirus env sequence. The insertion point was confirmed by sequence analysis of sequences amplified using primers specific for both ends of the retrovirus to 1505bp in the first intron of the pde6b gene in the rdf mice.3. Cross family members were collected and 10 mice in F1 generation, 61 mice in F2 generation, 60 mice in N2 generation were obtained. Genotypes of these mice showed cosegregation with the phenotypes. Cross of heterozygous F2 generations produced 51 mice and the genotypes of these F3 generation offspring also cosegregated with the phenotypes.4. The mutant pde6b gene was constantly inherited in congenic mice and it had direct relationship with the retina degeneration phenotype.5. Cloned and sequenced the pde6b coding region sequence of both the wild type Kunming mice and the rdf mice. The sequence of the Kunming mice was different from the sequence submitted in the GeneBank database (NM₀08806) in some points: a transition of A to G at 706, and a transversion of T to C at 1049 in the latter sequence. Similar results were obtained in rdf mice pde6b sequence.ConclusionThis research identified the causative gene of retinal degeneration fast mouse. A retrovirus insertion in the 1505bp of the first intron of the pde6b gene causes a defect in this gene, and homologous presentation of the mutant allele in the genome induces retinal degeneration phenotype.