Which species have already been cloned?
Sheep, cattle, pigs, mice, rabbits, goats and cats.

Have there been successful attempts to clone humans?
No one really knows, but probably not.

How difficult is the cloning process? How is it done and how expensive is it?
Commercially, there are some entities that are charging $20,000 per cloned animal produced. Difficulty is a relative thing. Cloning requires great expertise and technique to successfully produce live offspring. Using microsurgery techniques, the nucleus (which houses the cell’s genetic material) is removed from an unfertilized egg in a process called enucleation. The genetic material from a donor’s somatic cell, any cell other than an egg or sperm cell, is then transferred into the enucleated egg. The donor cell and enucleated egg form a couplet surrounded by a “shell” and the couplet is placed between two electrodes where it receives an electrical pulse that induces fusion of the two adjacent membranes. The resulting nuclear transfer embryos are genetically identical to the somatic cell’s donor. The embryos are then chemically stimulated to begin developing.

Why clone animals?
To increase the impact of the genetics of a specific animal in the overall population. Cloning allows the complete and exact duplication of an individual animal’s genetic make-up. Therefore, if you have a genetically exceptional animal, it may be economically desirable to make genetic copies. In addition, valuable animals that die or become diseased can be duplicated through cloning.

The ability to produce offspring from “cell lines” by using cloning technology may provide an important opportunity to efficiently modify the genetic characteristics of animals to make them more disease resistant, produce healthier products, or produce valuable pharmaceuticals. Cloning may also help us increase populations of some endangered species.

What is the future for mule cloning?
Not certain. Clearly, cloning is the only opportunity for mules to be replicated because they are sterile hybrids of horse/donkey crosses. If someone had a champion racing mule that had either outlived its ability to continue racing, due to age or injury, or a valuable animal that died, cloning would be a way to provide a copy of that individual animal.

Can mules be bred?
No, and our data indicates that the reason for reproductive failure in this species is due to a chromosomal odd number rather than an inherent developmental anomaly.

How many years have gone into this project?
We did one year of preliminary work (1999) and have made transfers for three years (2000-2002).

How many attempts has this research team made before a mule was successfully cloned?
There were a total of 305 clones transferred to recipient animals over a three-year period. From these there were 21 two-week, 11 30-day, five 45-day pregnancies, one foal and two additional pregnancies.

How do these numbers compare with successful cloning of non-equine animals?
There are many ways to look at this question, but if you compare it with published data about 30-day pregnancies in cattle (the time at which most cattle recipients are evaluated for pregnancy after receiving cloned embryos) the data for equine recipients looks very promising. Depending on how you calculate it, 35 percent of cattle clones survive to term. Using the same kind of calculation, we have a 27 percent success rate with mules. Cattle have been successfully cloned by somatic cell cloning for 4 to 5 years, while these are the first successfully cloned equine animals.

What do ethicists on both sides of this issue say about cloning?
The major concern is the potential application of cloning technology to humans. There seems to be less concern relative to cloning animals. It may be better received in animals because people can imagine applying the technology to a favorite pet or valuable animals. Our cloned mule is not an engineered animal. No genes were moved within or between species. We are merely taking the genes nature created and trying to make more copies to increase the genes’ impact on the overall population.

Do cloned animals have more health problems than other animals?
Not documented. It is true that an unusually high percentage of clone pregnancies are lost in utero. Most of these losses, at least in cattle, sheep and goats, can be primarily attributed to problems with the placenta’s attachment to the mother. It also appears that cloned offspring can have a difficult first 24-48 hours, during which time they appear diabetic. But this situation is not always observed in cloned offspring. However, if they do experience and overcome this difficulty, they appear to develop normally. Also, much more data has been reported indicating there is no “premature aging” in these animals.

Will cloning become mainstream? What will be its primary uses?
Cloning will become mainstream and used primarily to duplicate valuable animals. For example, to reproduce multiple copies of high production animals and those with very desirable genetic traits. It will also be used to more efficiently produce animals that are genetically engineered to produce valuable proteins or healthier products.

Has any member of the horse family (equine) been cloned before? No, this is the first.

How is a mule different from a horse?
Genetically speaking, a mule has 63 chromosomes, horse has 64, and donkey 62.

What is the source of the cells used in this project?
Tissue recovered from a 45-day-old mule fetus was used to establish a primary cell line. The cell line is composed of fibroblast cells.

How do you know the baby is a clone? Will it look and behave like its siblings? We used genetic markers to show the DNA from the foal was the same as the cell line used as the nuclear donor and different from the DNA of the mare that received the embryo and other control mules. The genetics (DNA) of these foals will be exact matches of each other and the original cell donor. We don’t know if the foals look identical to the cell donor because the cells were collected from a mule fetus with no hair, coloration or distinguishing marks. From a behavior standpoint, they may have some similarities, but no more than other siblings, so they will also have some differences.