Technical Aspects of Cloning

After nuclear transfer, the ooplast must reprogram the DNA from the somatic cell so that it functions like that of a zygote. This is controlled largely by methylation and demethylation of bases in the DNA and by modification of the histones, the proteins around which the DNA is wrapped. Controlling the transcription of DNA in this manner, without altering the structure of the DNA itself, is termed epigenetic control. The oocyte must reprogram the DNA of the donor cell initially at the time of cloning, and then maintain the normal patterns of epigenetic modification through the different stages of development. The amount and accuracy of reprogramming of the donor DNA is probably the major reason for success or failure of fetal development in cloning. Minor changes in epigenetic status may not affect the general health of the cloned individual but may still cause it to vary in phenotype from the donor animal. A visible example of this is seen in the first cloned cat, CC. CC expresses only brown coat color, whereas her genetic donor expresses both orange and brown (eg, calico). Because the X chromosome carries the gene for coat color in cats, this indicates that in CC, X chromosome inactivation is not random; rather, the same X chromosome is inactivated in all cells, presumably because of failure to reactivate the inactive X at the time of cloning.

Epigenetic effects may also influence the phenotype of the nuclear transfer–derived animal after birth; eg, an animal with growth-factor genes transcribed at high levels may grow larger than its cloned sibling with less active genes, even though the actual number and makeup of the genes are the same. However, it has been shown in all species studied that major epigenetic anomalies are not passed on to the offspring of clones because of resetting of epigenetic status during sperm and oocyte development.

The nuclear transfer embryo will have the nuclear DNA of the genetic donor but have the mitochondrial DNA of the recipient oocyte. A small number of mitochondria from the donor cell may also be present, but proportionately few. The impact of the source of mitochondria, or a mixture of mitochondria, on the traits of the progeny is currently unknown. Because mitochondria are the source of energy for the cell, differences in mitochondria could potentially have an effect on production, stamina, or other physical or behavioral traits.

The heterogeneous mitochondria present in a female produced by nuclear transfer will be passed down to the female’s offspring, because they will be present in her oocytes. However, although sperm from a male produced by nuclear transfer will carry the heterogeneous mitochondria, these mitochondria will be eliminated after the sperm fertilize an egg, so a male clone can be considered to produce progeny that are genetically identical to those that the original donor animal would have produced.

Uterine size and health; milk production of the dam; nutritional, exercise, and training programs; and handling to which the neonate is exposed may all influence the animal as an adult. For example, the cloned cat, CC, is outgoing and gregarious, whereas her genetic donor was reserved. However, the genetic donor was a research cat, raised in a cage and unused to attention, whereas CC was raised with an overabundance of attention and stimulation.

Genetic mutations are potentially more likely in cloned animals, because cultured cells are used as the DNA source. The donor cells are grown and passaged in vitro, and this is associated with an increasing number of chromosomal abnormalities with increasing passages. However, cells with chromosomal abnormalities are unlikely to produce viable embryos.

Cell differentiation occurs in cascades, as differentiation of one cell type affects the status of the cells around it. During development, cell multiplication and apoptosis occur in response to many environmental and internal stimuli. Thus, random individual variations will occur in the makeup of tissues, even in individuals of the same genetic background. A visual example of this is in the markings of cloned animals; individuals cloned from the same cell line tend to have white in similar places, but the markings can vary dramatically in size and shape.