**10.2. Gene insertions**

The most common approach for production of genetically-modified pigs is very similar to the protocol described in the creation of "Dolly the sheep." Briefly, the nucleus from a pig cell carrying the desired genome changes is extracted and introduced into a pig oocyte, which has previously had its own nucleus removed, and then induced to initiate embryogenesis using electrical and chemical induction, a process referred to as somatic cell nuclear transfer (SCNT). The newly-created cells are implanted into surrogate female pigs and allowed to develop to birth. Compared to genetically-modified mouse production, this process is significantly less efficient and more costly, limiting the number of facilities capable of effectively

A key factor in the success of SCNT is the source of the donor nucleus. These cells are typically primary cells derived from fetal sources. Extended culture, transfection, or drug selection of these donor cells can all cause a significant loss of viability for subsequent productive SCNT. Therefore, the approaches commonly used for mouse ES cell manipulation such as multigenic targeting and selections with various drugs over long periods in culture would not allow for production of modified pigs using SCNT. Similarly, any genome manipulations of pig cells must also maintain the viability of the cells for SCNT, which alters the approaches

One of the earliest genome engineering approaches applied to pigs was introduction of gene knockouts (KO). For any given gene, mutations which remove or disrupt the coding sequence can eliminate the expression of the gene and, provided that the KO is not lethal, create an organism which is entirely missing the gene product. The introduction of gene KO technology has been a key factor in the rapid advancement of the field of xenotransplantation [59]. As discussed above, there are several glycan molecules present in pigs which are absent in humans. These glycans are recognized by antibodies present in human serum which leads to rapid and extensive antibody-mediated damage to the porcine cells. Therefore, the elimination of the specific carbohydrate structures should help prevent human antibody recognition of the pig tissues. Unlike protein antigens which are directly coded by the DNA, glycosylation is due to the action of enzymes which create post-translational modifications of a variety of proteins produced by the cell. Therefore, glycosylation pathways must be examined to identify the key enzyme that creates the immunogenic glycan while otherwise leaving cellular metabolism intact. The GGTA1 gene is responsible for creating the Gal alpha (1,3) Gal epitope in pigs. Although the specific reasons for this are unclear, human patients can express high levels of antibody specific for the Gal alpha (1,3) Gal epitope, presenting a major challenge to xenotransplantation [60]. The KO of the GGTA1 gene is one of the earliest genetic modifications of pigs for application in xenotransplantation, and results in greatly reduced human antibody recognition of porcine cells [61]. However, elimination of the GGTA1 gene alone has been shown to be insufficient due to a variety of other xenoantigens present in pig cells which are recognized by antibodies present in human serum. Generation of KO of CMAH [62], B4GALNT2 [28] and other xenoantigen genes have further decreased the reactivity of porcine cells to human serum. However, it is important to keep in mind that the greater the number of gene KO,

especially when made in combination, may lead to detrimental effects on pig health.

carrying out this complex process [58].

344 Organ Donation and Transplantation - Current Status and Future Challenges

available compared with mice.

**10.1. Gene knockouts**

The use of gene KO approaches is highly useful for eliminating xenoantigens but does not address the need for expression of human or synthetic versions of genes necessary for control or proper function of dysregulated pathways. This requires the ability to permanently introduce heterologous DNA into the genome in a manner which maintains gene function.

The initial approach to gene insertions was simply random integration of DNA into the target genome. These genes are introduced from elsewhere and thus termed "transgenes" (TG). Once the ability to introduce DNA into mammalian cells was established using a variety of technological approaches, it became clear that over long-term culture a subset of cells could be isolated which have permanently incorporated the heterologous DNA. Because many transgenes do not provide a straightforward means to identify cells which have incorporated foreign DNA from the population that have not, TGs often include genes encoding drug resistance markers. In order for cells to survive drug treatment they must incorporate the resistance gene, greatly reducing the population to be screened, and increasing the chance of identifying cells which incorporate the TG of interest along with the drug resistance gene [63].

The integration of transgenes is rapid but relatively uncontrolled. Although there may be some preferences for integration site based upon chromatin accessibility, these are hard to predict and may be related to DNA breakage sites at which repair mechanisms fortuitously insert the transgene DNA [64]. The random nature of the insertions can create risks. For example, the same transgene inserted at different sites can yield highly variable results in expression. Furthermore, some insertions may be deleterious to cell function, causing them to grow more slowly or die off, or, if these cells are used for generation of animals *in vivo*, there is a possibility of insertions creating mutations, instability or even lethality.

Due to the risks of random integration, significant effort has focused on protocols to create targeted integration, or gene knock-in (KI), of heterologous DNA into the genome. This is accomplished in mice by taking advantage of ES cells which undergo homologous recombination. In this approach, the transgene of interest is flanked by DNA sequences that are identical to regions of the genome to be targeted. After introduction of the heterologous DNA, the regions of DNA sequence identity are aligned with the target sequence and the homologous recombination machinery creates crossover events to switch the endogenous sequence with the heterologous sequence. This approach is much less efficient than random integration of TG, therefore drug selection schemes often need to be employed to identify the relatively rare targeting events [64].

Homologous recombination is well-established for targeting in mice but requires ES cells which express the enzymes necessary for the targeting event. Unfortunately, porcine ES cells are not available that both possess homologous recombination function and can reliably generate cloned animals. For reasons that are not entirely clear, generation of ES cells competent for homologous recombination and cloning seems to be challenging for most species other than mice [57]. Therefore, alternate approaches are required for targeted integration in the pig genome [65].

#### **10.3. Tools for genome engineering**

A number of novel enzymatic molecules have been created which help resolve the dilemma of targeted integration in porcine cells. Zinc Finger Nucleases (ZFN), Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are all synthetic molecules based on genuine proteins which allow the precise targeting of genomic DNA based upon sequence [66–68]. In each case there are two functional components, a targeting module which recognizes a specific genomic sequence, and an enzymatic module which introduces a double-stranded DNA break at the target site. In the case of ZFN and TALEN, the targeting module is a complex array of protein sequences which have previously been shown to recognize specific DNA sequences and can be mixed in a modular way to bind to any desired sequence. Although both technologies have shown great success, the effort and cost required to identify a single functional molecule can be significant. In contrast, the relatively more recently recognized CRISPR, and related prokaryotic systems, is much more easily applied in mammalian cells. The DNA binding module in this case is RNA base-pairing to provide sequence specificity. The enzymatic module cleaves DNA, creating a double strand break similar to ZFN and TALEN. When heterologous DNA is present, the cellular repair machinery may use the synthetic DNA to repair the break, inserting the TG at the desired genomic site. It is important to note that all of these systems, ZFN, TALEN or CRISPR, are essentially the same in that they introduce double strand DNA breaks at a selected site in the genome and do not directly affect the rate of DNA insertion. Therefore, it is often necessary to include selection schemes for identification of the modified cells. The greater efficiency and ease of use of these systems, CRISPR in particular, has allowed targeted insertion of DNA into genomes that were not previously able to be modified [69].

yet to survive more than a few weeks. This is due to the relative differences in structure and function of organs, the resilience to trauma, and susceptibility to rejection responses. Furthermore, tolerance mechanisms may be able to supplant the need for some genetic modifications, and thus the specific protocols and treatments will govern the ultimate complement of alterations. The immediate need in xenotransplantation is to define the specific genetics required for xenoorgan survival, however, it is possible to project further enhancements such that porcine organs may be superior to human organs for human transplant. Synthetic biology approaches have created novel genetic circuits which can react in real time to human immune responses, inducing counter-reactions in the porcine cells to circumvent and tolerize the xeno-organ against human rejection. Furthermore, xeno-organs may be engineered to express protein therapeutics to further control human immunity while saving hundreds of thousands of dollars in expensive biotherapeutic treatments. Thus, the first version of pigs appropriate for xenotransplantation are likely to be further refined and improved to create increasingly useful rejection-free organs.

Xenotransplantation

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http://dx.doi.org/10.5772/intechopen.76502

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**Author details**

Address all correspondence to: sean\_m\_stevens@yahoo.com

Synthetic Genomics, Inc., La Jolla, CA, USA

Sean Stevens

**References**

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Due to the challenges of creating genomic modifications in porcine primary cells while maintaining their viability for SCNT, more efficient engineering methods are desirable. One approach to enhance efficiency is to target a specific region of DNA, called a landing pad, with multiple genes at once. By inserting a DNA vector bearing multiple therapeutic genes at once, a large amount of breeding and testing can be circumvented using a single event. This approach has the added advantage of avoiding inefficient crossbreeding necessary to bring loci from distinct chromosomes together in one lineage. When combined with the use of tools such as ZFN, TALEN and CRISPR more rapid progress in the genetic modification of animals has been greatly facilitated [70].
