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Published on: December 5, 2017
Studies on renin-2 gene in transgenic rats
1Cardiovascular Institute & Fu Wai Hospital, CAMS and PUMC, Beijing 100037, China.
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 digestion and Southern blotting hybridization. Transgenic rats were produced via microinjection method.
Results:
The 24 kb fragments containing mouse full-length ren-2 and its flanking sequence were cleaved by single enzymes (EcoR I, Kpn I and BamH I) and combined enzymes (EcoR I/Kpn I, Kpn I/BamH I and BamH I/EcoR I), 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-length renin-1 cDNA were used in southern blotting hybridization. According to the electrophoresis and hybridization patterns, a ren-2 restriction map was constructed. 1603 fertilized rat ova after injection with purified 24 kb renin-2 gene were implanted into the oviducts of 81 pseudopregnant recipients in about 20 ova per female rat. 306 progenies were obtained from 50 foster mothers (average of pregnancies was 56.6%). 248 survived pups were identified by PCR analysis 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 harvested as the same as those reported in the literatures. A systemic observation and the problems occurred during production of transgenic rats were also described besides the technique procedure used in this study.
Conclusions:
Mapping of full-length murine ren-2 can be used in investigation of the structure and function of the gene. The results denoted that the ren-2 transgenic rats were successfully established in this study and the technique used in the production of transgenic rats was proved to be valid in leading to wide-spread application of transgenic technique to many other related researches.
