197 INFLUENCE OF PLASMID CONSTRUCTION AND CELL TYPE ON THE GENERATION OF GENETICALLY MODIFIED CELL LINES IN CATTLE

Abstract

Several factors may influence transgenic animal production efficiency, and among them gene construction and the cell type used are of great importance. For a long time, fetal fibroblasts were largely used in generation of transgenic cattle production by nuclear transfer, however adult cells are very useful for cloning once the genotype of the donor nuclei is known, and derivation of such cells is technically simple, efficient, and reproducible. Thus, this study aimed to evaluate the effect of cell type on the percentage of GFP+ cells and fluorescence intensity, using two plasmids constructs encoding for green fluorescent protein (GFP). Transfections were performed in bovine fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) transfected by use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for the internalization of FUGW or pEGFPN₂ plasmid. Forty-eight hours after transfection, the number and the fluorescence intensity (arbitrary units) of GFP⁺ cells was measured by flow cytometry (FACSAria, FACSDiva Software, BD Biosciences, Franklin Lakes, NJ). Non-transfected cells were used as controls. Means were compared by the Student–Newman–Keuls test (SNK; P < 0.05). The FUGW plasmid promoted a higher rate of transfection and fluorescence intensity than pEGFPN2 in all cell types evaluated. When the FUGW plasmid was used, higher transfection rates were obtained with fetal fibroblasts (FF: 17.8 ± 2.82; AF: 10.66 ± 0.65, CC: 3.9 ± 1.97), while higher fluorescence intensity was observed in adult fibroblasts (FF: 4542 ± 497.09; AF: 9367.5 ± 3490.9, CC: 3496 ± 2638.92). The pEGFPN₂ plasmid showed percentage of transfected cells and fluorescence intensity significantly higher than the control only in cumulus cells (pEGFPN2 – FF: 4.9 ± 0.14 and 206.47 ± 755; AF: 760 and 2.4 ± 0.70 ± 330.92; CC: 3.9 ± 1.97, and 1418 ± 36.06, respectively; control – FF: 0.15 ± 0.07 and 249 ± 6 : 36; AF: 0.15 ± 0.07 588 ± 213.54, and CC 0.05 ± 0 214 ± 0.07, respectively). We conclude that the plasmid construction may influence the overall efficiency in transfected cells; however, the transfection percentage and fluorescence intensity is greatly influenced by the cell type. We suggest that transgenesis of a specific cell type may be enhanced by the proper choice of the expression vector.