**4. Genome engineering and xenotransplantation**

A major advantage in using pigs for xenotransplantation is the potential to manipulate the porcine genome to create donor organs that are more compatible for human patients. However, the scope of the engineering challenge in xenotransplantation is extremely large, involving a variety of genes and pathways. With so many potential targets for genetic modification, an assortment of different genome engineering strategies have been applied, including editing or deletion of porcine genes and insertion of human or engineered genes. Because of the great diversity of genome modification efforts being carried out in xenotransplantation research, representative approaches will be highlighted here as examples of the general types of the engineering strategies being employed.

Historically, mice have been subject to more and different genetic modifications than any other mammalian species, and many of the protocols described here were first developed in mice. Aside from their well-established and convenient husbandry, small size, and rapid generation times, mice also have a variety of technological advantages for genome manipulation and production. Although genetic modification has been demonstrated for multiple agricultural species, including pigs, the scale and complexity possible with mice has, until recently, not been available for pigs [23].

One advantage for the creation of mice with multiple genetic modifications is the availability of embryonic stem (ES) cells, which can be cultured *in vitro* for many generations and subject to repeated transfections and selections without loss of competence for production of viable mice [24]. By contrast, pig ES cells have been much more difficult to create, and have not been routinely used for genetic manipulation and production of animals [25]. Cloning of genetically-modified pigs has required use of primary cells, typically fetal, which can be passaged only a short time *in vitro* before losing their competence to produce viable embryos [26]. Therefore, the complex multi-site modifications and selections used in mice are not accessible for use in pigs.

Mouse ES cells not only allow more straightforward and efficient genome engineering, but also facilitate large scale production of cloned mice. The mouse ES cells typically employed for genetic modification can be injected into very early stage embryos (blastocysts) and will aggressively populate the inner cell mass, creating viable chimeric mice which are almost entirely ES-cell derived. Since the ES cells will also contribute to the germ cells of the chimeric mice, the progeny will be highly likely to receive the genetic modifications made to the ES cells [27]. Without readily available porcine ES cells, pig cloning instead relies upon somatic cell nuclear transfer (SCNT), similar to the protocols used to create the sheep

"Dolly". In this approach, pig oocyte nuclei are replaced with nuclei from the modified primary pig cells, and embryonic development stimulated electrochemically. The embryos are transferred to female surrogates and allowed to develop. The level of complexity and effort involved leads to lower efficiencies and higher costs for porcine SCNT relative to mouse ES cell cloning. Additionally, the size and scale of the facilities required for pig cloning is significantly greater compared with mouse cloning, further limiting availability [28].

In the following sections, different types of gene modifications are described with examples of their application in porcine genome engineering for xenotransplantation.
