Core C (Vector Core) will provide access to a wide array of gene transfer technology. The Core has assembled a comprehensive inventory of DNA plasmids, cell lines, and viruses useful for the development of vectors. The Core will provide services in terms of vector creation, amplification, purification, and analysis for Projects 1, 2, and 4. This dedicated facility will be run by Dr. Qianhong Li, who has extensive experience with recombinant viruses and has studied virus-mediated gene therapy for myocardial ischemia/reperfusion injury for eight years. We have developed an rAAV vector system that is helper adenovirus free, thereby eliminating the problem of adenovirus contamination in the final preparation. We have already created and successfully used rAAV/LacZ, rAAV/iNOS, and rAAV/ecSOD for in vivo cardiac gene transfer in mice. rAAV vectors currently available in the Vector Core include rAAV/LacZ, rAAV/hr GFP, """"""""empty"""""""" rAAV, rAAV/ecSOD, and rAAV/iNOS. The primary responsibility of the Vector Core will be to design, create, produce, and analyze rAAV vectors for long-term in vivo gene transfer. Specific responsibilities include: (i) cloning the gene of interest into an AAV DNA plasmid containing ITR sequences, (ii) cloning the mitochondrial leader sequence into an AAV DNA plasmid containing the ITR and the therapeutic gene, (iii) amplifying and maintaining purified stocks of AAV DNA plasmid containing the therapeutic gene, (iv) amplifying and maintaining purified stocks of an AAV DNA plasmid containing the rep and cap genes as well as a helper DNA plasmid encoding the Ad5 E2a and E4 gene regions, (v) constructing rAAV vectors in 293 cells, (vi) purifying, titrating, and analyzing rAAV vectors, (vii) long-term storage of rAAV vectors, and (viii) developing mitochondrially-targeted rAAV vectors (mt-rAAV) which will be used in Project 4 to study the effects of targeting iNOS and HO-1 specifically to the mitochondria.
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