**2.1 Methods of creating genetically modified animals**

### **2.1.1 Pronuclear microinjection /viral-mediated /sperm-mediated /ICSI (Intractyoplasmic sperm injection)- mediated gene transfer**

Numerous methods have been successfully used to introduce genetic modifications and produce transgenic animals, including pronuclear microinjection of foreign DNA into zygotes (Hammer et al., 1985; Pursel and Rexroad, 1993), viral-mediated gene transfer (Chan et al., 1998; Cabot et al., 2001; Whitelaw et al., 2008), sperm-mediated gene transfer (Castro et al., 1991; Chang et al., 2002; Lavitrano et al., 2002 and 2006) and intracytoplasmic injection (ICSI) of a sperm head carrying foreign DNA (Perry et al., 1999; Osada et al., 2005; Moisvadi et al., 2009; García-Vázquez et al., 2010). Despite the proven successful application of these techniques, some problems, such as inefficiency and mosaicism (transgene not going into the germline) (Table 1) remain to be solved and limit the practical application of these methods.


Table 1. The Advantages and disadvantages of different transgenic methods.

Gene targeting by homologous recombination often offers more precise and site-specific integration, sometimes at single nucleotide level. This is particularly important since single nucleotide changes can be a common culprit for some of the human diseases, which require more precise manipulation to build biomedical models using transgenic animals.

### **2.1.2 Cell-based transgenesis via SCNT**

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**Method Advantages Disadvantages Reference** Pronuclear microinjection The first method successfully used for different animal species. Low embryo survival, low and random integration, multiple copies, high cost in domestic animals. Hammer et al., 1985; Pursel and Rexroad, 1993 Viral-mediated DNA transfer Infect both dividing and non-dividing cells, less damage by co-culture with zona-free zygotes or injection into the perivitelline space compared with pronuclear microinjection, high integration. Limited DNA capacity, random integration. Chan et al, 1998; Cabot et al., 2001; Whitelaw et al., 2008 Sperm-mediated DNA transfer Relatively high efficiency as compared to pronuclear injection, low cost, ease of use. No control of integration site. Castro et al., 1991; Chang et al., 2002; Lavitrano et al., 2002; Lavitrano et al., 2006 Intracytoplasmic sperm injection -mediated DNA transfer Allow introduction of very large DNA transgenes, relatively high efficiency as compared to pronuclear injection. No control of integration site. Perry et al., 1999; Osada et al., 2005; Moisvadi et al., 2009; García-Vázquez et al., 2010

Table 1. The Advantages and disadvantages of different transgenic methods.

The first live animal by SCNT was produced in 1997, "Dolly" (Wilmut et al., 1997), demonstrating the ability of a differentiated somatic cell to produce live offspring following nuclear remodeling and reprogramming by an oocyte. Then, the birth of "Polly" in the same year (Schnieke et al., 1997), the first transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts, demonstrated a route to create transgenic cloned animals. This cell-based method of transgenesis by SCNT can bypass the absence of ES cells, offers the reliability of germline transmission by avoiding mosaic transgene integration, and provides the only currently used strategy to knock out a gene in domestic animals (reviewed by Ross et al., 2009a). Currently, SCNT using transgenic cells cultured *in vitro* as a source of donor nuclei is becoming the most utilized technique to produce the transgenic domestic animals. However, while the advantages and success of this strategy are well documented (Table 2), the procedure is still labor-intensive and inefficient. Recently, some new techniques have been reported that may have the potential to improve the production efficiency of cloned transgenic domestic animals by increasing the efficiency of gene targeting or nuclear remodeling and reprogramming following SCNT.

### **2.2 Utilization of transgenic models**

The potential for transgenic domestic animals to benefit humans is not only in agricultural production by providing more and better agricultural products for human consumption but also in biomedicine, such as for producing recombinant pharmaceutical proteins, making organs suitable for xenotransplantation and establishing human disease models. An overview list of the transgenic domestic animals produced via SCNT is given in Table 2 to demonstrate their applications.

### **2.2.1 Improved animal agriculture production**

Increased utilization of domestic animals and their products requires breeding and selection strategies for specific traits. However, classical breeding and genetic selection have some disadvantages, such as the inability to control gene frequency of desired genotypes coupled with long generation intervals. The application of transgenic technology offers a powerful tool to rapidly improve agriculture production by developing domestic animals that express desired traits via genome manipulation strategies. Previous studies have demonstrated the practical application of transgenesis to improve numerous agricultural traits of domestic animals, including increased growth rate (Pursel et al., 1999), increased meat quality (Saeki et al., 2004), enhanced disease resistance (Lo et al., 1991; Clements et al., 1994), and better milk production and composition (Wheeler et al., 2001; Reh et al., 2004). Nevertheless, utilization of the cell-based method of transgenesis via SCNT has also been successfully used to alter characteristics of pork quality (Lai et al., 2006), enhance disease resistance (Denning et al., 2001a; Wall et al., 2005; Richt et al., 2007) and improve milk composition (Brophy et al., 2003).


Table 2. Overview on successful transgenic domestic animals via SCNT.

### **2.2.2 Xenotransplantation**

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Table 2. Overview on successful transgenic domestic animals via SCNT.

Due to the potential application of human organ transplantation and the growing gap between the demand and availability of organs for transplantation, the pig has long been considered as an alternative source to provide organs for humans. In contrast to organs from other animal species, domestic pig organs share many similarities to those of humans including size, anatomy and physiology. However, despite these similarities, significant immunological barriers exist impeding the success of pig to human xenotransplantation (Cooper et al., 2008), in addition to concerns regarding the transmission of pig specific viruses to the human genome (Magre et al., 2003). The immunological obstacles of xenotransplantation include rapid hyperacute rejection (HAR), delayed acute vascular rejection (AVR), and the cellular immune response that occurs within weeks (Auchincloss and Sachs, 1998). Two transgenic strategies have been successfully applied to overcome HAR. One is to express human proteins that inhibit the complement cascade in transgenic pigs (Fodor et al., 1994; Cozzi and White, 1995; Diamond et al., 2001). For example, transgenic expression of human complement inhibitor CD59, CD46 and DAF (decay-accelerating factor, also named as CD55) in pigs prolongs survival rates from minutes to days and months following heart and kidney transplantation into baboons or monkeys by blocking the damage from HAR (Diamond et al., 1996 and 2001; Byrne et al., 1997; Zaidi et al., 1998; Bhatti et al., 1998; Chen et al., 1999). The second strategy to avoid HAR is to knockout the genes that induce the production of antigenic structures (α-gal-epitopes) on the surface of pig organs (Lai et al., 2002a; Dai et al., 2002; Phelps et al., 2003; Yamada et al., 2005). α-gal-epitopes on endothelial cells of porcine transplanted organs can be recognized by human xenoreactive natural antibodies (XNA) and activate the HAR cascade (Galili, 1993). Genetically engineering of pigs to lower or inhibit the expression level of XNA targets is thought to be a promise way to eliminate the HAR. Following the successful production of α-1,3-galactosyltransferase knockout pigs (Lai et al., 2002a; Dai et al., 2002; Phelps et al., 2003; Yamada et al., 2005), van Poll et al. (2010) recently showed that exposure of isolated xenogeneic pig liver sinusoidal endothelial cells (LSECs) from α-1,3-galactosyltransferase-deficient pigs to human and baboon serum reduces IgM binding and complement activation levels as compared to wild-type pig LSECs. However, Diswall et al. (2010) found a different reactivity pattern of baboon and human serum to pig glycolipid antigens isolated from α-1,3-galactosyltransferase knockout and wild-type pig hearts and kidneys, suggesting that non-human primates may not be an ideal model for modeling pig to human xenotransplantation. If HAR is controlled, the next obstacle to xenotransplantation is AVR which is due to the loss of porcine thrombomodulin in xenograft rejection or the inability of porcine thrombomodulin to activate human protein C. One of genetic engineering strategies to overcome AVR is to express human thrombomodulin in pigs. Petersen et al. (2006) showed the production of transgenic cloned pigs using CD59/DAF and human thrombomodulin triple transgenic adult donor cells.

With regard to risks associated with xenotransplantation, previous studies have shown that the risk of cross-transmission of pig endogenous retrovirus (PERV) to human patients or nonhuman primate recipients is low (Paradis et al., 1999; Switzer et al., 2001), although it has been found PERV can infect human cells in culture (Patience et al., 1997). Never-theless, some investigators have been working to further reduce the possibility by creating pigs with suppressed expression of endogenous retroviruses (Ramsoondar et al., 2009). In addition to PERV, herpesvirus is another concern regarding biosafety in xenotransplantation (Mueller et al., 2011). Overall, dramatically increased knowledge will facilitate the clinical application of transgenic strategies of pig to human xenotransplantation.
