Cardiac-specific overexpression of angiotensin II type 1 receptor in transgenic rats.
This chapter describes the methods required for overexpression of the angiotensin II type I receptor (AT1) in cardiomyocytes of transgenic rats. This includes cloning of the transgenic construct consisting of the alpha-myosin heavy chain (MHC) promoter, the human AT1 cDNA and SV40 T-antigen splicing and polyadenylation sites, and purification of the transgenic DNA for microinjection by electroelution. The individual steps for the introduction of the transgene into the germline of rats by pronuclear microinjection are described, with special emphasis on the adaptation made to the standard procedure in mice. The identification of transgenic rats by PCR and Southern blot and the principles of establishing transgenic lines as well as characterizing transgene expression by Northern blot and RT-PCR are outlined.
Hoffmann
Sigrid
S
Medical Research Center, University of Heidelberg, Mannheim, Germany.
D000818:Animals; D030801:Animals, Genetically Modified; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006321:Heart; D008845:Microinjections; D018995:Myosin Heavy Chains / Q000235:genetics; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; D018076:DNA, Complementary; D044140:Receptor, Angiotensin, Type 1; D018995:Myosin Heavy Chains