Genetic Manipulation of the Equine Oocyte and Embryo

As standard in vitro fertilization is not a viable technique in horses yet, many different techniques have been used to create equine embryos for research purposes. One such method is parthenogenesis in which an oocyte is induced to mature into an embryo-like state without the introduction of a spermatozoon, and thus they are not considered true embryos. Another method is somatic cell nuclear transfer (SCNT), in which a somatic cell nucleus from an extant horse is inserted into an enucleated oocyte, creating a genetic clone of the donor horse. Due to limited availability of equine oocytes in the United States, researchers have investigated the potential for combining equine somatic cell nuclei with oocytes from other species to make embryos for research purposes, which has not been successful to date. There has also been a rising interest in producing transgenic animals using sperm exposed to exogenous DNA. The successful creation of transgenic equine blastocysts shows the promise of sperm mediated gene transfer (SMGT), but this method is not ideal for other applications, like gene therapy, because it cannot be used to induce targeted mutations. That is why technologies like CRISPR/Cas9 are vital. In this review, we argue that parthenogenesis, SCNT, and interspecies SCNT can be considered genetic manipulation strategies as they create embryos that are genetically identical to their parent cell. Here, we describe how these methods are performed and their applications and we also describe the few methods that have been used to directly modify equine embryos: SMGT and CRISPR/Cas9.