Transient depletion of Ku70 and Xrcc4 by RNAi as a means to manipulate the non-homologous end-joining pathway.
Non-homologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells and is likely responsible for the non-homologous integration of transgenes. In higher eukaryotes, this pathway predominates over the homologous recombination (HR) pathway and therefore may account for the low level of HR events that occur in mammalian cells. We evaluated the effects of transient RNAi-induced down-regulation of key components of the NHEJ pathway in human HCT116 cells. Treatment with siRNA targeting Ku70 and Xrcc4 reduced corresponding protein levels by 80-90% 48h after transfection, with a return to normal levels by 96h. Additionally, down-regulation of Ku70 and Xrcc4 resulted in a concomitant depletion of both Ku70 and Ku86 proteins. Biological consequences of transient RNAi-mediated depletion of Ku70 and Xrcc4 included sensitization to gamma radiation and a significant decrease in the expression of a linear GFP reporter gene. The results highlight the possibility of a successful means to manipulate the NHEJ pathway by RNAi.
Bertolini
Luciana R
LR
Department of Animal Science, University of California-Davis, Davis, CA 95616, USA.
Bertolini
Marcelo
M
Anderson
Gary B
GB
Maga
Elizabeth A
EA
Madden
Knut R
KR
Murray
James D
JD
D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics / Q000528:radiation effects*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics*; D049452:Green Fluorescent Proteins; D045325:HCT116 Cells; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000528:radiation effects; D014162:Transfection
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; C101467:XRCC4 protein, human; D049452:Green Fluorescent Proteins; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen