title: Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts.
abstract: Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.
authors.affiliation: Department of Cardiology, Boston Children's Hospital; Department of Pediatrics, Harvard Medical School; jding@enders.tch.harvard.edu.
authors.forename: Jian
authors.initials: J
authors.lastname: Ding
authors.affiliation: Department of Cardiology, Boston Children's Hospital; Department of Pediatrics, Harvard Medical School.
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Lin
authors.affiliation: Department of Genetics, Harvard Medical School; Howard Hughes Medical Institute.
authors.forename: Jian-Ming
authors.initials: JM
authors.lastname: Jiang
authors.affiliation: Department of Genetics, Harvard Medical School; Howard Hughes Medical Institute.
authors.forename: Christine E
authors.initials: CE
authors.lastname: Seidman
authors.affiliation: Department of Genetics, Harvard Medical School; Howard Hughes Medical Institute.
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Seidman
authors.affiliation: Department of Cardiology, Boston Children's Hospital; Department of Pediatrics, Harvard Medical School.
authors.forename: William T
authors.initials: WT
authors.lastname: Pu
authors.affiliation: Department of Cardiology, Boston Children's Hospital; Department of Pediatrics, Harvard Medical School; dwang@enders.tch.harvard.edu.
authors.forename: Da-Zhi
authors.initials: DZ
authors.lastname: Wang
mesh_terms: D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D009206:Myocardium / Q000378:metabolism*; D034741:RNA, Small Interfering
chemical_list: D034741:RNA, Small Interfering