Objective To determine the function of, in vivo, renin and its role in the pathogenesis of hypertension. Methods A renin 2 gene restriction map was constructed by endonuclease digest ion and Southern blotting hy...Objective To determine the function of, in vivo, renin and its role in the pathogenesis of hypertension. Methods A renin 2 gene restriction map was constructed by endonuclease digest ion and Southern blotting hybridization. Transgenic rats were produced via micro injection method.Results The 24 kb fragments containing mouse full length ren 2 and it s flanking sequence were cleaved by single enzymes (EcoRⅠ, KpnⅠ and BamHⅠ) an d combined enzymes (EcoRⅠ/KpnⅠ, KpnⅠ/BamHⅠ and BamHⅠ/EcoRⅠ), respectively. The digests were electrophoresed in 0.8% agarose plates and transferred onto NC membranes. Radioactive 735 bp and 1400 bp probes obtained from half and full l ength renin 1 cDNA were used in southern blotting hybridization. According to t he electrophoresis and hybridization patterns, a ren 2 restriction map was cons tructed. 1603 fertilized rat ova after injection with purified 24 kb renin 2 ge ne were implanted into the oviducts of 81 pseudopregnant recipients in about 2 0 ova per female rat. 306 progenies were obtained from 50 foster mothers (averag e of pregnancies was 56.6%). 248 survived pups were identified by PCR analys is and Southern hybridization, and eight positive rats were found to be the transgenic rats (founder, F). All of them carried long fragments (24 kb) of renin 2 gene with normal blood pressure. Preliminary breeding and screening were carried out in the founder. Total survival pups (17.8%) and overall efficiencies (1%) were h arvested as the same as those reported in the literatures. A systemic observatio n and the problems occurred during production of transgenic rats were also descr ibed besides the technique procedure used in this study.Conclusions Mapping of full length murine ren 2 can be used in invest igation of the structure and function of the gene. The results denoted that the ren 2 tran sgenic rats were successfully established in this study and the technique used i n the production of transgenic rats was proved to be valid in leading to wide s pread application of transgenic technique to many other related researches.展开更多
文摘Objective To determine the function of, in vivo, renin and its role in the pathogenesis of hypertension. Methods A renin 2 gene restriction map was constructed by endonuclease digest ion and Southern blotting hybridization. Transgenic rats were produced via micro injection method.Results The 24 kb fragments containing mouse full length ren 2 and it s flanking sequence were cleaved by single enzymes (EcoRⅠ, KpnⅠ and BamHⅠ) an d combined enzymes (EcoRⅠ/KpnⅠ, KpnⅠ/BamHⅠ and BamHⅠ/EcoRⅠ), respectively. The digests were electrophoresed in 0.8% agarose plates and transferred onto NC membranes. Radioactive 735 bp and 1400 bp probes obtained from half and full l ength renin 1 cDNA were used in southern blotting hybridization. According to t he electrophoresis and hybridization patterns, a ren 2 restriction map was cons tructed. 1603 fertilized rat ova after injection with purified 24 kb renin 2 ge ne were implanted into the oviducts of 81 pseudopregnant recipients in about 2 0 ova per female rat. 306 progenies were obtained from 50 foster mothers (averag e of pregnancies was 56.6%). 248 survived pups were identified by PCR analys is and Southern hybridization, and eight positive rats were found to be the transgenic rats (founder, F). All of them carried long fragments (24 kb) of renin 2 gene with normal blood pressure. Preliminary breeding and screening were carried out in the founder. Total survival pups (17.8%) and overall efficiencies (1%) were h arvested as the same as those reported in the literatures. A systemic observatio n and the problems occurred during production of transgenic rats were also descr ibed besides the technique procedure used in this study.Conclusions Mapping of full length murine ren 2 can be used in invest igation of the structure and function of the gene. The results denoted that the ren 2 tran sgenic rats were successfully established in this study and the technique used i n the production of transgenic rats was proved to be valid in leading to wide s pread application of transgenic technique to many other related researches.
