Comparison of several protocols for the increase in homologous recombination in normal porcine fetal fibroblasts and the application to an actual locus



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Texas A&M University


Together with the advancements in animal cloning, the ability to efficiently target specific genes in somatic cells would greatly enhance several areas of research. While it has been possible for quite some time to target specific genes in the germ cells of mice, the advancements in somatic cell gene targeting has been slowed for two main reasons. First, the finite lifespan of somatic cells, due mainly to the inability of the somatic cells to regenerate or maintain their telomeres, poses a major problem given the lengthy selection process needed to identify a targeting event. The second problem is the overall inefficiency of homologous recombination. A double strand break or introduction of foreign DNA into a cell can be processed either through the homologous recombination or non-homologous end joining pathways. Of these two, non-homologous end joining is dominant in somatic cells. A two plasmid recombination system was used to study the effects of the manipulation of several non-homologous end joining and homologous recombination pathway molecules on the rates of homologous recombination in porcine fetal fibroblasts. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. Results indicate a strong positive relationship between inactivation of p53, cell synchronization, and efficient DNA nuclear delivery in enhancing the rate of homologous recombination. These findings were then applied to an actual locus in the pig, the ?1,3 galactosyltransferase gene. Results from these transfections are compared to published accounts of successful targeting at this locus and possibilities for the differences found are discussed.