Cloning Research

Taken from "A Review of Cloning in the Horse" by Dr. Katrin Hinrichs

The Equine Embryo Laboratory is at the forefront of cloning research. Having successfully cloned 7 donors for 15 live foals, the staff continues to do research so that their cloning efforts can be used to benefit society as a whole. Cloning can be used to produce breeding animals to help preserve valuable equine genetics.

Introduction

Equine cloning has been discussed in the popular press since the birth of the first cloned equids (three mules and one horse) in 2003. In general, interest has been centered on whether or not the cloned offspring will be normal, how closely they will resemble the donor animal, and what cloning may be used for within the industry. Although equine cloning is still in its infancy, sufficient information is available from other species and from the few equine clones already produced to allow us to start answering these questions.

Technique

The principle of cloning is relatively simple. The chromosomes of a cell from the donor animal are transferred into the cytoplasm of an egg, and the egg is signaled to develop an embryo. The cells from the donor animal are typically grown from a small sample of subcutaneous connective tissue. At the laboratory, the tissue is placed into culture, and fibroblasts are grown from it onto the culture dish. The fibroblasts will proliferate until they cover the bottom of the plate and they may be resuspended in medium and used to "seed" additional dishes. After a sufficient number of cells are obtained, the cells are typically frozen to be used at a later time.

Oocytes used for cloning may be harvested from the dominant pre-ovulatory follicles of live mares or they may be obtained by maturing immature oocytes in vitro. The donor cell is then combined with the enucleated oocyte either by electrofusion or by directly injecting the cell into the cytoplasm of the oocyte. The recombined oocyte is activated to stimulate embryonic development; this is typically done by triggering calcium oscillations within the oocyte that mimic those that occur at fertilization.

After the recombined oocyte has been activated, it may be transferred surgically to the oviduct of a recipient mare, or it may be cultured in vitro to the blastocyst stage for transfer directly to the uterus of a recipient mare as for standard embryo transfer.

How Closely will the Clone Resemble the Donor

A variety of factors will affect the degree of similarity between the cloned offspring and the original donor, but only two are actually related to the cloning procedure. Epigenetic changes compatible with a viable foal may still cause the gene function of a cloned foal to differ somewhat from that of the donor; therefore, the foal may potentially be shorter or taller, have more or less bone, etc. than did the donor animal. The second cloning-related potential cause of differences between the clone and the donor animal is related to mitochondria.

If the cloned embryo was cultured in vitro before transfer to the recipient mare, in vitro culture itself has been shown to cause differences in neonatal size and other phenotype differences in other species.

Other potential causes of differences between the cloned foal and the donor would be seen in any transferred embryo; however, they will be seen in any transferred embryo; however, they will be more obvious in cloned foals because the expected phenotype is known.

These variations in phenotype and in mitochondrial genotype will be useful in identifying individual cloned offspring that are produced from the same genetic donor. The possibility of phenotypic variation in cloned offspring as well as possible health problems associated with cloned neonates makes it unlikely that the cloned offspring will perform at the same level as the donor animal.

How Will Cloning Affect the Industry

Cloning is most accurately viewed as a method for genetic banking, similar to freezing semen or oocytes so that progeny may be obtained from a genetic line after the original horse is no longer fertile or is deceased. However, cloned horses are currently not eligible for registration with most breed registries in the United States.

Even in the United States, cloning is currently applicable to horses in which the value of the progeny does not depend on registration with a breed association. Thus, cloned animals may produce progeny that could compete in dressage, jumping, cross-country, polo, cutting, reining, or other events.

The possibility exists with cloning for misuse and manipulation, and it is difficult to predict the range of these potential problems. The cloned animals themselves will be different from each other and from the original donor by their markings and their mitochondrial genotype. However, not only is cloning inefficient and costly, but it is also unlikely to produce a champion of the same quality horse because of the various factors potentially affecting the performance of cloned foals.

Can the progeny of cloned horses be differentiated from the progeny of the other horses? Progeny of cloned mares will be different from progeny of the original mare by their mitochondrial DNA. However, progeny of cloned stallions may not be different from progeny of the original stallion. Substitution of semen from a clone for semen from the original stallion would need to be monitored by evaluating the mitochondrial DNA from the semen sample. The small number of mitochondria in a spermatozoon presents some problems for efficient genotyping; this is an area that is currently under investigation at our genetics laboratory at Texas A&M University.

Summary

Equine cloning is possible today, and its value to the industry will be determined over the next few years. Cloning should be viewed as a method for producing a breeding animal rather than as a means to "duplicate" a performance horse. To the equine practitioner, cloning provides a procedure that may be offered to clients to preserve valuable genetics in the face of reproductive problems that in the past were insurmountable.