Homologous recombination is required for AAV-mediated gene targeting.
High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) Nature Genet., 18, 325-330; D. W. Russell and R. K. Hirata (2000) J. Virol., 74, 4612-4620; R. Hirata et al. (2002) Nat. Biotechnol., 20, 735-738], but the mechanism of targeting is unclear and random integration often occurs in parallel. We assessed the role of specific DNA repair and recombination pathways in rAAV gene targeting by measuring correction of a mutated enhanced green fluorescent protein (EGFP) gene in cells where homologous recombination (HR) or non-homologous end-joining (NHEJ) had been suppressed by RNAi. EGFP-negative cells were transduced with rAAV vectors carrying a different inactivating deletion in the EGFP, and in parallel with rAAV vectors carrying red fluorescent protein (RFP). Expression of RFP accounted for viral transduction efficiency and long-term random integration. Approximately 0.02% of the infected GFP-negative cells were stably converted to GFP positive cells. Silencing of the essential NHEJ component DNA-PK had no significant effect on the frequency of targeting at any time point examined. Silencing of the SNF2/SWI2 family members RAD54L or RAD54B, which are important for HR, reduced the rate of stable rAAV gene targeting approximately 5-fold. Further, partial silencing of the Rad51 paralogue XRCC3 completely abolished stable long-term EGFP expression. These results show that rAAV gene targeting requires the Rad51/Rad54 pathway of HR.
Vasileva
Ana
A
Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.
Linden
R Michael
RM
Jessberger
Rolf
R
D045744:Cell Line, Tumor; D004265:DNA Helicases / Q000378:metabolism; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000502:physiology; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors; D000229:Dependovirus / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D034622:RNA Interference; D011995:Recombination, Genetic
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C491221:RAD54B protein, human; C110901:X-ray repair cross complementing protein 3; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D051747:DNA-Activated Protein Kinase; D004265:DNA Helicases; C103532:RAD54L protein, human