Abstract
Abstract: :
Purpose: The Rd4 mouse inherits an autosomal dominant retinal degeneration that cosegregates with a large inversion encompassing nearly all of chromosome 4. The aim of this study is to identify the gene(s) responsible for the retinal degeneration in the Rd4 mouse. Methods: We employed fluorescence in situ hybridization (FISH) experiments to analyze the inversion breakpoints. Metaphase chromosomes were prepared from primary cultures of mouse ear–clips using standard cytogenetics procedures. Bacterial artificial chromosome (BAC) DNAs were labeled with digoxigenin–11–dUTP or Biotin. Single color or dual–color FISH were done and the signals were detected with anti–digoxigenin fluorescein or Texas Red Avidin. Results: We previously reported that the proximal breakpoint of the Rd4 inversion is in the centromere itself and the distal breakpoint lies below the marker D4Mit59. According to the Mouse Genome Informatics genetic map, D4Mit59 maps 78 cM from the centromere of chromosome 4. To date there are more than 20 known genes placed on this region (Human–Mouse Homology Map, NCBI). To narrow down the region further, 5 BAC clones covering the critical region were used for FISH experiments. We found that one BAC clone is disrupted by the breakpoint. There are 3 known genes, 5 unknown genes, and 3 predicted genes mapped on this BAC clone. We used several genomic DNA fragments prepared from the BAC clone as probes for FISH experiments to define the position of the breakpoint. Two of these fragments were mapped proximal and distal to the breakpoint, respectively, indicating that the breakpoint localizes to the interval between them. According to the complete genomic sequence of the BAC clone from NCBI, the size of the interval is about 50 kb. Conclusions: We narrowed down the telomeric breakpoint region of the Rd4 inversion to 50 kb. Analysis of genomic sequence and Northern blots with probes corresponding to genes in the 50 kb region will allow us to identify the candidate gene for Rd4.
Keywords: genetics • positional cloning • retinal degenerations: hereditary