| Popis: |
In this present study, experiments were carried out a) to characterise the mouse skeletal muscle ryanodine receptor (Ryr1) gene cDNA, b) to construct a contiguous map of the murine Ryr1 gene and c) to evaluate the phenotype of the Ryr1 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 Ryrl cDNA has been characterised by cDNA cloning and sequence analysis. Murine Ryrl exon-specific RT-PCR primers were designed and used to generate Ryrl 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 Ryr1 gene has been constructed by DNA fingerprint analysis and STS mapping. Large fragment genomic clones containing murine Ryr1 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 RYR1 cDNA fragments. This experiment also confirms the isolated clones are skeletal muscle ryanodine receptor isoform (Ryr1) 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 Ryr1 gene is about 150 kb. |
