Increased Gene Targeting in Ku70 and Xrcc4 Transiently Deficient Human Somatic Cells

• Published in: Molecular biotechnology Vol. 41; no. 2; pp. 106 - 114
• Main Authors: Bertolini, Luciana R, Bertolini, Marcelo, Maga, Elizabeth A, Madden, Knut R, Murray, James D
• Format: Journal Article
• Language: English
• Published: New York New York : Humana Press Inc 01-02-2009; Humana Press Inc; Springer; Springer Nature B.V
• Subjects: Analysis of Variance; Antigens, Nuclear - genetics; Antigens, Nuclear - metabolism; Biochemistry; Biological and medical sciences; Biological Techniques; Biotechnology; Cell Biology; Cellular biology; Chemistry; Chemistry and Materials Science; Chi-Square Distribution; Deoxyribonucleic acid; DNA; DNA Repair - genetics; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Gene Targeting - methods; Genomics; HCT116 Cells; Human Genetics; Humans; Hypoxanthine Phosphoribosyltransferase - metabolism; Ku Autoantigen; Mammals; Molecular biology; Polymerase Chain Reaction; Protein Science; Proteins; Recombination, Genetic; RNA Interference; Thioguanine - metabolism; RNAi; RNA interference; Gene targeting; Homologous recombination; Non-homologous end joining; Xrcc4; DNA integration; Ku; Human; Gene silencing; Targeting; Gene; DNA; Xrcc4 . Gene targeting . Homologous recombination; Somatic cell; RNA interference . RNAi; DNA integration . Ku
• ISSN: 1073-6085; 1559-0305
• DOI: 10.1007/s12033-008-9098-8

The insertion of foreign DNA at a specific genomic locus directed by homologous DNA sequences, or gene targeting, is an inefficient process in mammalian somatic cells. Given the key role of non-homologous end joining (NHEJ) pathway in DNA double-strand break (DSB) repair in mammalian cells, we investigated the effects of decreasing NHEJ protein levels on gene targeting. Here we demonstrate that the transient knockdown of integral NHEJ proteins, Ku70 and Xrcc4, by RNAi in human HCT116 cells has a remarkable effect on gene targeting/random insertions ratios. A timely transfection of an HPRT-based targeting vector after RNAi treatment led to a 70% reduction in random integration events and a 33-fold increase in gene targeting at the HPRT locus. These findings bolster the role of NHEJ proteins in foreign DNA integration in vivo, and demonstrate that their transient depletion by RNAi is a viable approach to increase the frequency of gene targeting events. Understanding how foreign DNA integrates into a cell's genome is important to advance strategies for biotechnology and genetic medicine.