**3.2.3 Viral-mediated delivery**

706 Biomedical Science, Engineering and Technology

clones recovered with the positive selection only (PS) versus the positive and negative dual selections. The PNS strategy can be used to target both active and inactive gene loci. It has been shown that the targeting efficiency at the COL1A1 locus in fibroblasts by the promoter-

Following vector construction and preparation of the DNA construct for delivery into a somatic cell, it is important to identify an efficient method of DNA delivery into somatic cells as efficiency between delivery methods varies greatly. There are numerous methods to introduce an exogenous DNA construct into somatic cells, which can be categorized as liposome-mediated DNA transfer (Hyun et al., 2003a; Lee et al., 2005), electroporation (Dai et al., 2002; Ramsoondar et al., 2003; Watanabe et al., 2005) and viral-mediated delivery (Lai

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

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

trapping strategy is 15.7-fold higher than by PS only (Marques et al., 2006).

**3.2 Delivery of DNA vector into cultured somatic cells** 

et al., 2002b; Rogers et al., 2008).

**3.2.2 Electroporation** 

**3.2.1 Liposome-mediated DNA delivery** 

(McCreath et al., 2000) have successfully been created.

Because viruses have the natural ability to stably transfect somatic cells with high efficiency, utilizing viral particles to delivery exogenous DNA into somatic cells has been widely successful. In contrast to liposome-mediated delivery and electroporation, which deliver linear, double-strand DNA into the cytoplasm of somatic cells, viral delivery of exogenous DNA delivers a high number of the linear, intact single-strand DNA molecules into the nucleus of cells. One report has demonstrated the efficient targeting of the PRNP gene encoding the prion protein PrP in bovine fetal fibroblasts by adeno-associated virus (AAV) vectors (Hirata et al., 2004). Also, the same transfection method followed by SCNT was used to successfully produce the CFTR-null and CFTR-DeltaF508 heterozygous pigs and CFTRdeficient ferrets (Rogers et al., 2008; Sun et al., 2008). Despite high transfection efficiency and production of transgenic cloned animals using viral-mediated delivery method, the need to produce high concentrations of virus particles in addition to limitations of the size of DNA capable of being delivered via virus limits the application of this method.

### **3.3 Selection and characterization of transgenic cell lines 3.3.1 Selection by marker**

Following DNA construct delivery into somatic cells, transfected cells are cultured 24-48h in the absence of selection, followed by selection. Selection agents are chosen according to the DNA construct and added in cell-type specific concentrations into the cell culture medium. Typically, G418 (Geneticin) is used when the neomycin resistance gene is the positive selection marker and gangciclovir when using the TK gene for negative selection. Due to the limited lifespan of many of the somatic cell lines that are typically used for nuclear transfer and the significant amount of time to produce clonal colonies, it is important to perform the genetic screening by PCR or southern blotting as early as possible, prior to cell senescence.
