RESEARCH Increased Gene Targeting in Ku70 and Xrcc4 Transiently Deficient Human Somatic Cells Luciana R. Bertolini Æ Marcelo Bertolini Æ Elizabeth A. Maga Æ Knut R. Madden Æ James D. Murray Published online: 30 August 2008 Ó Humana Press 2008 Abstract 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 target- ing. 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 tran- sient 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. Keywords Non-homologous end joining Á RNA interference Á RNAi Á DNA integration Á Ku Á Xrcc4 Á Gene targeting Á Homologous recombination Introduction Foreign DNA can be integrated into host cell chromosomes at defined loci by homologous recombination (HR) or at random sites by non-homologous insertion (RI). Targeted DNA integration in mammalian cells is difficult to control because RI is 1,000 to 10,000 times more frequent than targeted HR, making it difficult to inactivate a gene or correct a mutation [1]. The integration of foreign DNA is likely to occur as the DNA ends become substrates for cellular double-strand break (DSB) repair mechanisms, which occur through either non-homologous end joining (NHEJ) or HR mechanisms [2–4]. NHEJ is the main form of DNA repair in mammalian cells, a process also involved in V(D)J recombination [5]. In this process, two DNA ends with no obvious sequence homology are joined in a non- template-specific manner. In contrast, HR occurs through the use of a homologous template to repair a DNA lesion. Although our understanding of these two pathways is currently limited and the outcome is significantly different, they appear to share functional similarities as well as temporal differences in cell cycle or developmental state that may dictate which pathway predominates at a specific time. Proteins in the NHEJ pathway include the Ku complex proteins Ku70 and Ku86, DNA-PKcs, Artemis nuclease, DNA ligase IV with its co-factor Xrcc4, and the factor Cernunnos-XLF that interacts with the Xrcc4-DNA ligase IV complex [6–8]. In the NHEJ-mediated DNA repair model, DSB ends are first bound by the Ku complex that protects the DNA ends from degradation and maintains their close juxtaposition [9, 10]. The NHEJ-specific DNA ligase IV, along with its cofactor Xrcc4, is then recruited to the DSB-Ku complex to catalyze the final step of the pathway [11, 12]. Mammalian cells deficient for NHEJ L. R. Bertolini Á M. Bertolini Á E. A. Maga Á J. D. Murray (&) Department of Animal Science, University of California, Davis, CA, USA e-mail: jdmurray@ucdavis.edu K. R. Madden Biogrammatics, Inc., Carlsbad, CA, USA J. D. Murray Department of Population Health and Reproduction, University of California, Davis, CA, USA Mol Biotechnol (2009) 41:106–114 DOI 10.1007/s12033-008-9098-8