**10. Genome engineering to improve xenotransplantation**

The progress of xenotransplantation research in recent times has closely paralleled the advancement in genome engineering technologies. As the complexity of the engineering toolsets has increased, so too has the complexity of porcine genomic manipulations increased to address the immunological challenges described in previous sections.

Complex mammalian genome engineering has advanced much more rapidly in mice than in virtually any other species, including pigs. The reason for the rapid progress in mice is the availability of embryonic stem (ES) cells which can be maintained in culture for extended periods time and undergo extensive transfection/transduction protocols and drug selection without losing the ability to produce large numbers of fertile progeny via blastocyst injection [56]. Although several labs have made strides in this area, similarly manipulable and viable ES cells are not currently available for routine use in the generation of cloned pigs [57].

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 carrying out this complex process [58].

**10.2. Gene insertions**

resistance gene [63].

lethality.

targeting events [64].

pig genome [65].

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.

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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

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

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

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

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 available compared with mice.
