Abstract
Abstract: :
Purpose: Lens opacity 4 (Lop4) is a mutation in a novel gene that leads to the development of nuclear cataract in mice. The Lop4 locus has been previously mapped to 96 cM on mouse chromosome 2 in the region of synteny with HSA20p12–q13 and cryopreserved. The current Lop4 critical region encompasses >300 genes. In order to further map and study the Lop4 locus, we reconstituted the Lop4 strain into live animals and evaluated their genetic background. In addition, we identified Eya2 and Zfp64 as potential candidate genes. Methods: A male and a female Lop4 were recovered, determined to have nuclear cataracts, and allowed to breed. Lenses and a spleen were collected from a single F1 female. For analysis of the genetic background 60 microsatellite markers with an average separation of 30 cM, were used to scan the Lop4 genome. For exon analysis of Eya2 and Zfp64, primers were designed to anneal at least 50 bp away from intron/exon boundaries. Results: We completed evaluation of 25 polymorphic microsatellite markers on a DNA sample from reconstituted Lop4 animal. Initial findings reviled that 19 out of 25 polymorphic markers have a C3H allele. The candidate gene analysis revealed that seven out of nine Zfp64 exons did not show any sequence differences between Lop4 and wild type mice. The sequence analysis of 15 out of 16 Eya2 exons did not reveal any differences between Lop4 and wild type mice. Conclusions: The initial findings suggest that the Lop4 locus is not a C3H congenic background and that establishing of the Lop4 congenic line will be necessary in order to pursue fine resolution linkage mapping. Evaluation of Eya2 and Zfp64 did not identify any mutations, but two more exons for Zfp64 and one more exon for Eya2 are currently being evaluated. In addition, expression studies of Eya2 and Zfp64 by RT–PCR and northern blotting in wild type and Lop4 lens tissues are also currently in progress.
Keywords: gene screening • genetics • gene mapping