Mapping and manipulation of the murine ryanodine receptor gene (Ryr1)

• Main Author: Kathirvel, Paramasivam
• Published: University of Edinburgh 2000
• Subjects: 591.35
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.653223

In this present study, experiments were carried out a) to characterise the mouse skeletal muscle ryanodine receptor (<i>Ryr1)</i> gene cDNA, b) to construct a contiguous map of the murine Ryr1 gene and c) to evaluate the phenotype of the <i>Ryr1</i> knockout transgenic mice and to develop a mouse model, which carries a homologue of the C1843T mutation, associated with MH in pigs and some humans. The murine <i>Ryrl</i> cDNA has been characterised by cDNA cloning and sequence analysis. Murine <i>Ryrl</i> exon-specific RT-PCR primers were designed and used to generate <i>Ryrl</i> cDNA fragments from skeletal muscle total RNA of the mouse strain 129. The cloned RT-PCR products are overlapping and yield about 15 kb cDNA sequence. The cDNA sequence has higher sequence similarity with mammalian sequences than other vertebrate and invertebrate ryanodine receptor gene(s). A contiguous map of the <i>Ryr1</i> gene has been constructed by DNA fingerprint analysis and STS mapping. Large fragment genomic clones containing murine <i>Ryr1</i> sequences have been isolated from two libraries - a Bacterial Artificial Chromosome (BAC) library and P1-derived artificial chromosome (PAC) library. Of the 33 clones isolated, 4 were from the BAC and 29 were from the PAC library. The BACs form tow contiguous fragments separated by a gap. The relationships among the BACs were confirmed by DNA fingerprinting using human <i>RYR1 </i>cDNA fragments. This experiment also confirms the isolated clones are skeletal muscle ryanodine receptor isoform <i>(Ryr1)</i> specific. Sequence information from these BAC genomic clones and cDNA sequence information from this study has been used to develop STS markers. A contiguous map of the clones and flanking sequences has been established by STS analysis in BAC/PAC clones, which together span about 495 kb. The presence of all tested STS markers in two single PACs indicates that the murine <i>Ryr1</i> gene is about 150 kb.