**3.2.1 Liposome-mediated DNA delivery**

Liposome-mediated DNA transfer can easily transfect a large number of somatic cells without the need of specialized equipment and expertise, compared with other methods. Lee et al. (2005) demonstrated the efficiency of gene transfection with a plasmid containing the enhanced green fluorescence protein gene into fetal-derived bovine fibroblast cells by lipids was significantly higher than that obtained by electroporation. They also validated that transfection efficiency in fetal-derived bovine fibroblast cells, regardless of the delivery methods, was significantly higher than delivering DNA into cumulus-derived fibroblast cells and adult ear skin-derived fibroblast cells, establishing that both delivery method and cell line origin affect the efficiency of gene transfection. Using liposome-mediated DNA delivery followed by SCNT, genetically modified pigs (Hyun et al., 2003a) and sheep (McCreath et al., 2000) have successfully been created.

### **3.2.2 Electroporation**

In contrast to liposome-mediated DNA delivery, electroporation has been widely used for the delivery of exogenous DNA into the cytoplasm of somatic cells to generate genetically modified cell lines for nuclear transfer. Electroporation has been utilized to successfully provide genetically modified donor cells for SCNT to create transgenic cloned domestic animals, including cattle (Kuroiwa et al., 2004), goat (Yu et al., 2006; Zhu et al., 2009), pig (Dai et al., 2002; Lai et al., 2002a; Ramsoondar et al., 2003; Watanabe et al., 2005) and sheep (Denning et al., 2001a). Ross et al. (2010a) indentified optimal electroporation conditions (three 1 ms pulses of 300 V to 200 µL of 1x106 cells/mL in the presence of 12.5µg DNA/mL), which can consistently deliver DNA vector into the 65-80% surviving porcine fetal fibroblasts and have been used to produce healthy, viable transgenic piglets (Ross et al., 2009b). In adult rhesus macaque fibroblasts, it has been demonstrated that electroporation can generate more transfected cells than liposome-mediated methods (Meehan et al., 2008), which is consistent with other similar comparisons (Yáñez and Porter, 1999). Of the numerous delivery methods, electroporation was demonstrated to have the greatest efficiency in generating targeted cell lines via homologous recombination (Vasquez et al., 2001). Targeting the hypoxanthine guanine phosphoribosyl transferase (HPRT) locus, Mir and Piedrahita (2004) demonstrated that the electroporation of a DNA construct with a nuclear localization signal into s-phase synchronized cells can increase targeting efficiency sevenfold and decrease random integration events 54-fold in primary fetal bovine fibroblasts. Later, Meehan et al. (2008) confirmed this method by successful gene targeting of the HPRT locus in adult rhesus macaque fibroblasts achieved by electroporation of Sphase synchronized cells with a construct containing a SV40 enhancer.
