Manipulation of gene targeting frequency in mammalian cells

• Main Author: Dominguez-Bendala, Juan
• Published: University of Edinburgh 2000
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.649616

With the development of nuclear transfer from somatic cells in several species, gene targeting can now be utilised for the design of more accurate animal models for human diseases and the generation of genetically modified livestock. However, its use is limited by the low frequency of homologous recombination in somatic cells. Future applications of gene targeting, such as the development of human gene therapies, will also require dramatic improvements in the efficiency of homologus recombination. The aim of this work has been devise strategies for the stimulation of gene targeting efficiency <i>in vitro.</i> Using a very sensitive test system based on the directly selectable knockout of the <i>HPRT</i> gene in ES cells <i>in vitro,</i> a variety of experimental approaches were assessed for their ability to enhance effective targeting frequency - measured as the ratio of homologous to total integrants. These can be grouped into three main subcategories: (1) Modifications of the targeting vector (nuclear localisation signals, dsRNA vectors); (2) Alteration of the target conditions (methylation status, chromatin configuration); and (3) Manipulation of the expression of recombination-related genes (down-regulation of homologous recombination repressors and overexpression of recombinases). Loss of p53, Ku80 or DNA-PK_cs function did not result in enhanced targeting efficiency in ES cells. In contrast, constitutive overexpression of the eukaryotic recombinase Rad51 yielded a 4-fold increase in effective targeting frequency compared to wild-type control cells. Significant increases were also observed in <i>Dnmt1-/-</i> and poly(ADP-rybosyl)polymerase (PARP) -defective cells, as well as in cells treated with chemical inhibitors of PARP activity. These results contribute to the knowledge of the mechanisms underlying homologous recombination in mammalian cells, and suggest possible avenues of research to overcome the practical limitations of gene targeting.