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D.M. Maddox, W.L. Hicks, S. Byers, R. Taft, B. Chang, J.K. Naggert, P.M. Nishina; IVF–Assisted Mapping of the NMF12 and Nob3 Mutations . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3553.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Often the limiting factor in completing a positional cloning project is the rate at which F2 mice can be generated. In order to streamline the mapping process, we have adopted an in vitro fertilization (IVF) assisted approach to positionally clone two recessive retinal mutations, NMF12 and Nob3. Methods: IVF was used to generate F1 (NMF12 x DBA/2J; Nob3 x BALB/cJ) and the F2 intercross mice for each mutation. Fine mapping was performed using MIT markers and single nucleotide polymorphisms (SNPs). Results: Four hundred and thirty–four NMF12 X DBA/2J F2 mice were generated simultaneously for fine structure mapping. The critical region for the NMF12 mutation has been reduced to a 1.7 megabase (Mb) region on mouse Chromosome 2, containing 37 annotated transcripts. One of these transcripts encodes the gene for solute carrier family 4, sodium bicarbonate transporter–like, member 11 (Slc4a11). As a targeted mutation in a member of the sodium bicarbonate transporter family, Slc4a7 has recently been shown to result in a retinal degenerative phenotype similar to that of NMF12 (Bok et al., 2003). Slc4a11 was selected as the best candidate gene and cDNA is being sequenced for coding region alterations. In order to map the Nob3 mutation, 401 Nob3 X BALB/cJ F2 mice were generated. The critical region for the Nob3 mutation has been reduced to a 7.2 Mb region, containing a very strong candidate gene: metabotropic glutamate receptor 6 (Grm6 or mGluR6). A null mutation of this gene in mice has been reported to result in a phenotype similar to that observed in Nob3 mutant mice. Real–time PCR experiments and Northern blotting experiments indicate a 3–fold reduction in the level of mGluR6 mRNA in the Nob3 mutants, suggesting that nonsense–mediated degradation is occurring. cDNA from the Nob3 mice is being sequenced to determine whether mutations exist in the coding sequence of mGluR6. Conclusions: By utilizing an IVF–assisted approach, within one year, we have been able to identify strong candidate genes for both the NMF12 and Nob3 mutations. This demonstrates that IVF–assisted mapping is a powerful technique for simultaneously generating large numbers of mice that can be used to narrow the critical region containing the mutation. In the future, the efficacy of this exciting technique will continue to grow exponentially as the number of spontaneous and chemically mutagenized mouse models increases.
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