**3. Technical aspects of cell-based transgenesis by SCNT**

The general procedure of cell-based transgenesis via SCNT is to construct a DNA vector, deliver the vector into cultured somatic cells, select transgenic cell lines, utilize SCNT and transfer cloned embryo into surrogates (Figure 1).

Fig. 1. Technical diagram of cell-based transgenesis followed by SCNT in pigs.

### **3.1 Vector construction**

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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

The mammary gland and blood of transgenic domestic animals, including sheep, goats, cows, pigs and rabbits, have been successfully used as bioreactor to produce numerous recombinant proteins, such as antibodies (Grosse-Hovest et al., 2004), growth factors (Schnieke et al., 1997) and pharmaceuticals (reviewed by Melo et al., 2007). Using various mammary gland-specific or blood-specific promoters to drive the expression of specific protein-coding genes, transgenic domestic animals can continuously produce the recombinant proteins in large quantities in their milk or blood. Recombinant proteins, including human von Willebrand factor (Lee et al., 2009), human erythropoietin (Park et al., 2006), human insulin-like growth factor-I (Monaco et al., 2005), human factor VIII (Paleyanda et al., 1997) and bovine alpha-lactalbumin (Bleck et al., 1998) have been produced in the milk of transgenic pigs. Transgenic goats, capable of synthesizing human butyrylcholinesterase (Huang et al., 2007) and human longer acting tissue plasminogen activator (Ebert et al., 1991) in their milk have also been created. Human salmon calcitonin in milk of transgenic rabbits (McKee et al., 1998); human factor IX (Schnieke et al., 1997) and alpha-1-antitrypsin (Wright et al., 1991) in milk of transgenic sheep; and human lactoferrin (van Berkel et al., 2002; Yang et al., 2008), human growth hormone (Salamone et al., 2006) and human α-lactalbumin (Wang et al., 2008) in milk of transgenic cows are all additional examples of using transgenic domestic animals and the mammary gland as a bioreactor for production of recombinant proteins. Table 2 summarizes some recombinant proteins expressed in milk or blood of cloned transgenic domestic animals. One of the major advantages of using domestic animals for this purpose is that the produced protein is thought to undergo more accurate posttranslational processing to ensure their biological activity. While this application of transgenic technology to produce recombinant protein products is rapidly developing, research efforts exploring the efficacy of these products are

Another important application of genetically modified domestic animals is to create better and novel biomedical models of human diseases. Pig models of different human diseases, including retinitis pigmentosa, cardiovascular disease, diabetes, Huntington's disease, cystic fibrosis and Alzheimer's disease have been well discussed by Prather et al. (2008). Many of

The general procedure of cell-based transgenesis via SCNT is to construct a DNA vector, deliver the vector into cultured somatic cells, select transgenic cell lines, utilize SCNT and

xenotransplantation.

still needed.

**2.2.3 Production of recombinant proteins** 

**2.2.4 Biomedical models of human diseases** 

transfer cloned embryo into surrogates (Figure 1).

these biomedical models created by SCNT are listed in Table 2.

**3. Technical aspects of cell-based transgenesis by SCNT** 

Currently, the whole genome sequence of several domestic animals, including cattle, poultry, cats, dogs, horses, pigs and rabbits is available via the ensembl database (www.ensembl.org), providing researchers useful sequence information for a large number of genes useful in designing DNA constructs for transgenic genome modification. Precise design and construction of DNA constructs is critical efficiently creating transgenic domestic animals. Transgenesis involves adding a gene to a host genome (transgenic), physically deleting a specific region of the host genome making a non-functional gene (knock-out), replacing an active gene by another active gene (knock-in), or introducing a point mutation (point mutation knock-in). Depending on the objective of the transgenic modification, different strategies of vector design need to be carefully considered to ensure success.

### **3.1.1 Transgenic vs. Knock-out vs. Knock-in**

The design strategy for the transgenic vector, which is based on random integration, essentially includes a gene ORF (open reading frame), a promoter element and the appropriate RNA processing component(s). The promoter is a major transcriptional regulatory element that normally includes regulatory elements and the transcriptional start site typically located in 5′ sequence of the gene. Utilization of specific promoter with a transgene enables tissue or cell specific expression and can significantly impact the expression efficiency of the transgene. Carefully choosing a well-characterized promoter will enable the precise control of transgene expression. The gene ORF is usually derived from the cDNA for the protein of interest, which includes translational start (ATG) and stop codons. Examination of extra sequence, if any, existing between the transcriptional start site and the translational start codon should be performed to ensure the absent of potential regulatory elements. Furthermore, consideration should be given to remove non-coding sequence of genes to avoid the introduction of the regulatory elements although this may also have consequences impacting mRNA stability. Reliable transgene expression is not only regulated by the sequence in the expression vector, but also by intrinsic factors in the host genome following transgene integration. Several additional points should be considered in transgenic vector construction. (i) How GC rich regulatory sequence are, especially CpG islands in the promoters can have significant implications for the expression of the transgene as methylation of CpG islands can inactivate the promoter and silence the transgene expression. (ii) Inclusion of specific elements that favor mRNA maturation and transfer to the cytoplasm, as it has been demonstrated that the inclusion of an intron in the transgenic vector can increase the transgene expression level (Choi et al., 1991; Duncker et al., 1997). These genomic regions may have critical sequence motifs affecting mRNA splicing and accumulation. (iii) Removal of unnecessary plasmid DNA sequence used in recombinant DNA cloning. In mice, it was found that plasmid sequence existing in the transgenic vector can decrease transgene expression (Kjer-Nielsen et al., 1992). Furthermore, the local chromatin status of the transgenic locus can also affect expression meaning that the integration of the same transgene in different genomic locations can have profound effects on the expression level of the transgene.

The primary strategy for targeted genome modifications, including knock-out and knock-in applications, is to use homologous recombination to introduce precise, site-specific genome alterations. In knock-out targeting vector systems, the primary approach is to delete DNA fragments (entire gene or partial deletion) important for the gene function by homologous recombination. Compared with the knock-out targeting vector, in addition to the targeting arms and positive selection cassette, the knock-in targeting vector includes the extra replacement cassette that will replace the target gene with a new gene (a set of genes or a point mutation). Targeting vectors relying on homologous recombination contain 5′ and 3′ homologous arms flanking a positive selection cassette (Rogers et al., 2008; Sun et al., 2008). Several principles should be considered when designing a successful targeting vector. (i) Avoid excessive repetitive DNA. In mouse ES cells it has been demonstrated that the excessive repetitive DNA within the targeting vector can significantly reduce targeting frequency (Wu et al., 2008). (ii) Use isogenic DNA as the source for producing exogenous homologous arms. While gene targeting in domestic species by using non-isogenic DNA as a source for targeting arms is possible (McCreath et al., 2000; Denning et al., 2001a and b; Kuroiwa et al., 2004; Marques et al., 2006; Richt et al., 2007), the use of isogenic DNA can largely improve the efficiency of gene targeting (te Riele et al., 1992). (iii) Increase the length of continuous exogenous homologous arms. The efficiency of gene targeting was generally found to be increased with the length of targeting arms in mouse ES cells (Hasty et al., 1991a). (iv) Use multiple cell lines; when targeting the CFTR (cystic fibrosis transmembrane conductance receptor) gene in pig fetal fibroblast cell lines, Rogers et al. (2008) demonstrated drastic differences in the targeting efficiencies between cell lines derived from littermate pig fetuses.

### **3.1.2 Tissue specificity and inducible promoters**

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will enable the precise control of transgene expression. The gene ORF is usually derived from the cDNA for the protein of interest, which includes translational start (ATG) and stop codons. Examination of extra sequence, if any, existing between the transcriptional start site and the translational start codon should be performed to ensure the absent of potential regulatory elements. Furthermore, consideration should be given to remove non-coding sequence of genes to avoid the introduction of the regulatory elements although this may also have consequences impacting mRNA stability. Reliable transgene expression is not only regulated by the sequence in the expression vector, but also by intrinsic factors in the host genome following transgene integration. Several additional points should be considered in transgenic vector construction. (i) How GC rich regulatory sequence are, especially CpG islands in the promoters can have significant implications for the expression of the transgene as methylation of CpG islands can inactivate the promoter and silence the transgene expression. (ii) Inclusion of specific elements that favor mRNA maturation and transfer to the cytoplasm, as it has been demonstrated that the inclusion of an intron in the transgenic vector can increase the transgene expression level (Choi et al., 1991; Duncker et al., 1997). These genomic regions may have critical sequence motifs affecting mRNA splicing and accumulation. (iii) Removal of unnecessary plasmid DNA sequence used in recombinant DNA cloning. In mice, it was found that plasmid sequence existing in the transgenic vector can decrease transgene expression (Kjer-Nielsen et al., 1992). Furthermore, the local chromatin status of the transgenic locus can also affect expression meaning that the integration of the same transgene in different genomic locations can have profound effects

The primary strategy for targeted genome modifications, including knock-out and knock-in applications, is to use homologous recombination to introduce precise, site-specific genome alterations. In knock-out targeting vector systems, the primary approach is to delete DNA fragments (entire gene or partial deletion) important for the gene function by homologous recombination. Compared with the knock-out targeting vector, in addition to the targeting arms and positive selection cassette, the knock-in targeting vector includes the extra replacement cassette that will replace the target gene with a new gene (a set of genes or a point mutation). Targeting vectors relying on homologous recombination contain 5′ and 3′ homologous arms flanking a positive selection cassette (Rogers et al., 2008; Sun et al., 2008). Several principles should be considered when designing a successful targeting vector. (i) Avoid excessive repetitive DNA. In mouse ES cells it has been demonstrated that the excessive repetitive DNA within the targeting vector can significantly reduce targeting frequency (Wu et al., 2008). (ii) Use isogenic DNA as the source for producing exogenous homologous arms. While gene targeting in domestic species by using non-isogenic DNA as a source for targeting arms is possible (McCreath et al., 2000; Denning et al., 2001a and b; Kuroiwa et al., 2004; Marques et al., 2006; Richt et al., 2007), the use of isogenic DNA can largely improve the efficiency of gene targeting (te Riele et al., 1992). (iii) Increase the length of continuous exogenous homologous arms. The efficiency of gene targeting was generally found to be increased with the length of targeting arms in mouse ES cells (Hasty et al., 1991a). (iv) Use multiple cell lines; when targeting the CFTR (cystic fibrosis transmembrane conductance receptor) gene in pig fetal fibroblast cell lines, Rogers et al. (2008) demonstrated drastic differences in the targeting efficiencies between cell lines derived from littermate pig

on the expression level of the transgene.

fetuses.

As mentioned, the promoter used in a transgenic DNA construct determines when, where and to what extent the transgene is expressed. Hundreds of promoters can be isolated for the expression of transgenes, and are generally classified as constitutive promoters, tissue/cell-specific or Developmental stage-specific promoters and inducible promoters. Constitutive promoters, such as commonly used SV40 (simian virus 40 promoter), CMV (cytomegalovirus immediate-early promoter), PGK (mouse phosphoglycerate kinase 1 promoter) and CAGG (chicken β-Actin promoter coupled with CMV early enhancer) promoters for mammalian systems (Qin et al., 2010), can continuously drive transgene expression in all tissues and species. Tissue/cell-specific or developmental stage-specific promoters can restrict transgene expression to specific tissue(s) or only during certain developmental stages. For example, the 5.5-Kb osteopontin (OPN) promoter has been used to drive GFP expression in transgenic mice in the same cell-specific and developmental stage-specific manner as endogenous OPN expression (Higashibata et al., 2004). Inducible promoters, as their name suggests, may be activated by the presence of endogenous or exogenous factors. Exogenous factors include chemical compounds such as antibiotics or physical factors such as heat and light. For example, TRE promoter (tetracycline-responsive element promoter) can be activated by the rtTA (reverse tetracycline-controlled transcriptional activator) in a doxycycline-inducible manner (Qin et al., 2010). Antibioticinduced promoters are the most commonly used in animal genetic modification because of easy manipulation. Inducible promoters provide a very useful tool in animal genetic engineering to turn on or off transgene expression in a particular tissue or at certain developmental stages.

### **3.1.3 Positive/negative selection strategies**

Considering the rarity of a homologous recombination event relative to random integration of the targeting vector, an efficient targeting vector design should incorporate a good selection strategy, providing a powerful tool to improve the frequency of targeted colonies and reduce screening cell lines that result from random integration. Promoter-less gene targeting vector, also referred to the promoter-trapping method, has been used to enrich the gene targeting events in somatic cells in pigs and sheep (McCreath et al., 2000; Denning et al., 2001a and b). In a promoter-trapping vector, the selectable gene lacks its own promoter but it becomes activated from the target gene promoter after correctly integrating into the genome. In a fibroblast cell line, promoter-less vectors can enrich targeting frequency 5,000 to 10,000-fold (Hanson and Sedivy, 1995). Despite this potential improvement, the major limitation of the promoter-trapping method is that it requires active transcription of the targeted gene to drive expression of the selectable marker used in the targeting vector. Thus, if the target gene is only active in cell types that are difficult to culture, it is nearly impossible to target the gene locus using this method. Compared with promoter-trapping method, a more widely used strategy includes utilization of both positive and negative selection (PNS) (Jin et al., 2003; Kuroiwa et al., 2004; Richt et al., 2007). A positive selection gene (antibiotic-resistant gene such as neo) in the targeting vector is needed to select cells with an integrated construct a negative selection gene (cytotoxic genes such as the thymidine kinase (TK) gene or the diphtheria toxin A-chain (DT-A) gene) to further select against random integration event. The negative selection marker in the targeting vector usually is placed downstream of homologous arms and recombined away during the process of homologous recombination. The enrichment of the PNS selection is the ratio of 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 promotertrapping strategy is 15.7-fold higher than by PS only (Marques et al., 2006).
