Targeted genetic modification: xenotransplantation and beyond.

Abstract

The 10th anniversary of the report of the first animal cloned from an adult cell (Wilmut et al., 1997) is an excellent time to look back, and to look forward. The ability to clone domestic animals from differentiated cells not only provided new insight into the processes of development, but also significantly impacted the ability to make targeted genetic modifications to domestic livestock. The early work of nuclear transfer focused on using early embryos as a source of donor nuclei because it was thought that more differentiated nuclei either were more difficult or impossible to reprogram. While the strategy of using early embryonic cells could result in the production of cloned embryos and offspring; expansion of the genotype would require serial nuclear transfer to result in a significant number of clones. In addition the ability to genetically modify the donor nuclei was very limited. The developmental envelope was pushed by transferring nuclei from cells that were cultured from progressively more differentiated stages of embryos and fetuses. The first step was to take an early embryo and culture cells from that embryo and then use those cells for nuclear transfer (Sims et al., 1994). Next it was shown that fetal-derived cells could be cultured and subsequently used to clone individuals (Campbell et al., 1996). Once it was shown that donor cells could be cultured prior to nuclear transfer, it followed that the donor cells could be genetically modified prior to the nuclear transfer and that cloned transgenic animals could be produced (Schnieke et al., 1997). The advantage of using this system for making genetically modified animals is that the exact nature of the genetic modification could be determined in the cultured cells prior to creating the animal. This was very important for domestic animals since embryonic stem cells, which have been so useful for genetic modification in mice, have yet to be isolated from any domestic animal, and it was thus not possible to make targeted modifications. In addition, pre-selection of the donor cells would provide the investigator an opportunity to confirm that integration had occurred and that significant expression could be detected in the donor cells before the animal was made. With the ability to genetically modify the donor cells prior to creating the animal in-hand, both the ideas of transgenic animals created from donor cells selected for high levels of expression of the gene of interest, as well as targeted modification (knock-ins and knock-outs) now entered the realm of imagination. Examples of transgenesis in pigs that used the strategy of selecting the cells prior to cloning the animal include addition of the enhanced green fluorescent protein, hFAT-1, endothelial cell nitric oxide synthase (NOS3), thymidine kinase, and cytosine deaminase (reviewed by (Prather, 2006).