International Society for Animal Genetics Conference, 2004, Tokyo, Japan. Abstract A 013.
Bacterial artificial chromosomes (BACs) provide valuable resources for the analysis and mani-pulation of large genomic fragments. Using recombineering to make directed genetic modifica-tions of genomic DNA cloned into BACs, we are developing approaches to assist in the resol-ution of complex human diseases with the pig biomedical model. To overcome difficulties ass-ociated with the introduction of BACs with large inserts into mammalian cells, we carried out experiments to determine the effects of various parameters regarding the efficiency of DNA transfection into porcine fibroblast cells. The BAC clone 403O9 from the RPCI-44 library was recombineered by introduction of a cassette encoding enhanced green fluorescent protein (EGFP) and used to monitor the effectiveness of BAC DNA uptake into the fibroblast cells. Since the rate of successful BAC transfection depends on factors including the purity of BAC DNA, the transfecting agent, and the target cell line, we compared the effects of varying these parameters on transfection efficiency. The efficiencies were determined by flow cytometry analysis of transient EGFP expression in the transfected cells. We determined that the time course of EGFP expression varied depending on the method of transfection (non-liposomal lipid reagent with DNA-condensing enhancer, lipid-based transfection reagent, or electroporation), observed the transfected cells expressed EGFP for up to two weeks, and optimized the conditions for transfection. Co-transfecting an EGFP BAC with another BAC of interest may provide a rapid method of selecting successful transfectants. (Supported by USDA grant AG2002-35205-12712).