Genetic modification of pig PSCs

To create genetically modified animals by using pig PSCs, genetic manipulation via transgenic technologies has been required in stem cell research. Researchers have successfully delivered transgenes into PSCs using several methods, including electroporation (Eiges et al., 2001), liposomal (Ko et al., 2009) and viral vectors (Ma et al., 2003; Pfeifer et al., 2002), and nucleofection

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(Hohenstein et al., 2008). However, stably introducing transgenes in these cells has proven difficult because of low efficiency and cytotoxic side effects. Delivery of transgenes using viral vectors, which are stably expressed, is considered the most useful tool for inducing low cytotoxicity and inserting transgenes into the host genome (Zhang and Godbey, 2006). Moreover, lentiviral vectors belonging to retroviral families are able to infect several types of cells, as well as nondividing cells (Bukrinsky et al., 1993; Naldini et al., 1996). As vectors carrying transgenes, plasmid vector, virus vectors (including lentiviral vector, retroviral vector, sendai virus vector and adenovirus vector (Zhang and Godbey, 2006)), episomal vector (Van Craenenbroeck et al., 2000) and piggyback transposon system (Zhao et al., 2016) have been widely used. If transfected with short hairpin RNA (shRNA)-expressing vector, expression of targeted gene can be reduced instead upregulation (Xiang et al., 2006). In addition to insert transgenes, replacement and disruption of endogenous genes can be accomplished by gene targeting using homologous recombination, known as repair mechanism of DNA double strand break (Bouabe and Okkenhaug, 2013). Transgenic stem cells using the gene targeting were first reported in mouse ESCs (Thomas and Capecchi, 1987). Although it is possible to selectively modify specific gene via gene targeting technique, it has

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been proven hard since frequency of homologous recombination is low (Gerlai, 2016). Targeting efficiency was improved through artificially inducing DNA double strand breaks by engineered endonucleases such as zinc finger nuclease (Bibikova et al., 2003), TALE nuclease (Miller et al., 2011), CRISPR/Cas9 (Cong et al., 2013a) and CPF1 (Zetsche et al., 2015). These nucleases were effectively applied for gene targeting in human ESCs (Hockemeyer et al., 2009; Hockemeyer et al., 2011; Hou et al., 2013b).

Transgenesis in porcine PSCs was first reported by Piedrahita and colleagues (Piedrahita et al., 1998). Plasmid vectors carrying humanized GFP were introduced into pig embryonic germ cells (EGCs) via electroporation. When transgenic EGCs were injected into blastocysts, the cells were developed into chimeric fetuses with host embryos. In other study, chimeric piglets were born by blastocyst injection using transgenic EGCs harboring transgene, human growth factor (Mueller et al., 1999). Subsequently, various genes including EGFP, hCD46, hCD59 and hPCSK9 have been transfected into pig EGCs, and several transgenic EGC lines could develop to term when used in nuclear transfer as donor cells as shown in Table 5. Gene delivery in porcine ESCs was first reported

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(Yang et al., 2009). In contrast to other reports using somatic cell nuclear transfer (SCNT) with transgenic donor cells (Huang et al., 2011; Tan et al., 2011), the transgene [humanized Renilla green fluorescent protein (hrGFP)] was directly delivered into pESCs via electroporation. Although GFP-expressing pESC lines were established via electroporation, transfection efficiency was very low (only three stably expression lines from 12 trials), and a GFP-expressing line was not obtained by retroviral and liposome-mediated transfection. In other study, EGFP was successfully introduced into pig ESCs via lentiviral vectors under various multiplicities of infection (MOI), with pluripotency and differentiation potential unaffected after transfection (Choi et al., 2013). Recent studies showed that homologous recombination efficiency is higher in PSCs having naïve state than the cells having primed state (Buecker et al., 2010). In non-permissive species such as human, rat and pigs, conversion of pluripotent state from primed state to naïve state have been accomplished by overexpression of exogenous factors including OCT4 and KLF4 and inhibition of cellular signaling pathway through the treatment of signaling inhibition molecules as previously mentioned. In human study, naïve PSCs showed that shorter doubling time and higher plating efficiency colonization rate after single cell dissociation than primed PSCs. Because of these

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characteristics, efficiency of homologous recombination in naïve PSCs was more than 200 times higher than in primed PSCs (Buecker et al., 2010). Porcine naïve PSCs also established by several groups as discussed above. This technique could be applied for improvement of generation of transgenic animals.

Interestingly, it was verified that transgene expression in pig ESCs were gradually declined during extended culture by DNA methylation as determined by bisulfite sequencing (Choi et al., 2013).

Similarly, silencing of the viral transgenes caused by epigenetic modifications and trans-acting factors have been observed in producing transgenic animals (Cherry et al., 2000; Kosaka et al., 2004; Whitelaw et al., 2008). Viral transgenes in transgenic animals were not expressed in some tissues (Hofmann et al., 2006; Park et al., 2010). Silenced transgenes in germ cells did not recover during fertilization and embryo development, and could not be transmitted to the next generation (Hofmann et al., 2006). Because silencing of transgenes are frequently occurred in transgenic animals, to overcome the problems, various approaches have been developed.

Using regulatory elements, including woodchuck hepatitis virus response element (WRE) (Zufferey et al., 1999), HIV FLAP (Arhel

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et al., 2007), and matrix attachment region (MAR) (Bode et al., 2000), which are responsible for transcript stabilization and the translocation of provirus into nuclear and DNA loop formation, respectively, also improve transgene expression. Accordingly, because the expression level of transgenes is dependent upon the vector construct, transfection methods used and cell types, it is therefore important to consider type of vectors and delivery methods for purpose of transgenic animals.

69 Table 5. List of transgenic/cloned pigs using pluripotent stem cells.

Type of PSCs Transfection methods Transgenes Production of transgenic

animal References

EGCs Plasmid vector

(electroporation) Humanized GFP

Blastocyst injection (sacrificed at 25 days of

gestration)

(Piedrahita et al., 1998)

EGCs (isolated from

transgenic pigs) - Human growth

hormone

Blastocyst injection

(developed to term) (Mueller et al., 1999)

EGCs

EGCs Plasmid vector (Effectene

transfection reagent) EGFP

Nuclear transfer (observed at blastocyst

stage)

(Ahn et al., 2007)

EGCs Plasmid vector (Effectene

transfection reagent) Human CD46

Nuclear transfer

EGCs Plasmid vector (Effectene

transfection reagent) Human CD59 Nuclear transfer

(developed to term) (Ahn et al., 2010)

ESCs Lentiviral vector EGFP - (Choi et al., 2013)

70 (Table 5-continued)

Type of PSCs Transfection methods Transgenes Production of transgenic

animal References

iPSCs - - Nuclear transfer

(developed to term) (Fan et al., 2013)

EGCs - - Blastocyst injection

(developed to term) (Dong et al., 2014)

EGCs Sleeping beauty

transposon system Human PCSK9 Nuclear transfer

(developed to term) (Secher et al., 2017)

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