Technical Aspects of Cloning of Domestic Animals
Cells recovered from early embryos, up to the morula stage, work efficiently as donor cells for cloning. Cloning from embryonic cells was conducted successfully for more than a decade before the birth of the first mammal produced via nuclear transfer using adult somatic cells (“Dolly,” a sheep reported by the Roslin Institute, Scotland, in 1997). Cloning using differentiated somatic cells as nuclear donors is termed somatic cell nuclear transfer, or SCNT.
The most common tissue used for cloning from adult animals is subcutaneous connective tissue. The tissue is minced and cultured in vitro. Outgrowing fibroblasts are harvested and replated (passaged) in new dishes to continue proliferation until millions of cells have been produced. These are then typically cryopreserved for future use.
Mature oocytes from the same or closely related species (essentially subspecies) are required for the nuclear transfer. The genetic value of the oocytes is not important, although their mitochondrial makeup may be, because the resulting clone will carry the mitochondria of the host oocyte (see below). Oocytes are typically recovered from slaughterhouse material and then matured in vitro, although in some species, meiotically mature oocytes (from preovulatory follicles or from the oviduct after ovulation) are collected.
Nuclear transfer is typically performed using a microscope with micromanipulators. The chromosomes of the oocyte are removed, creating an enucleated oocyte, or ooplast. The somatic cell used for cloning must be synchronized to be early in the cell cycle (before DNA synthesis). The somatic cell is combined with the ooplast, either by membrane fusion using an electric pulse or by direct injection of the donor cell into the ooplast via micromanipulation.
The recombined oocyte, now containing the nucleus of the donor cell, is treated to mimic the activation signal of fertilization, which stimulates it to develop into an embryo. After activation, the developing embryo may be transferred surgically to the oviduct of a recipient female or may be cultured in vitro to a stage at which it can be transferred to the uterus transcervically (nonsurgically), as for standard embryo transfer.
Several factors influence the health and phenotype of the cloned individual, including epigenetic effects, mitochondrial DNA, uterine and postnatal environment, mutations, and individual variation.
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 deficiencies in conferring normal 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 was seen in the first cloned cat, CC. CC expressed only brown coat color, whereas her genetic donor expressed 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 was not random; rather, the same X chromosome was inactivated in all cells, presumably because of failure to reactivate the inactive X at the time of cloning.
One of the major tissues affected by failure of normal epigenetic reprogramming after SCNT is the placenta. Much of the pregnancy loss associated with transfer of SCNT-derived embryos is attributed to abnormal placental function.
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 present in cloned animals are not passed on to the offspring of the clones, because the epigenetic status of cells is reset during sperm and oocyte development.
The nuclear transfer embryo will have the nuclear DNA of the genetic donor but will have the mitochondrial DNA of the ooplast used. A small number of mitochondria from the donor cell may also be present, but typically proportionately few. The impact of the source of mitochondria, or a mixture of mitochondria, on the traits of the progeny is currently unclear. 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. In addition, miscommunication between the transferred nuclear DNA and the mitochondria may effect placental development.
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. Offspring may inherit a different proportion of donor and host ooplast mitochondria than are present in the cloned female due to the bottleneck in numbers of mitochondria present at some stages of oocyte development. For male clones, 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 the male clone's mitochondria are not found in the offspring. Thus, 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. All of these factors affect conventionally produced offspring as well, but their effects are visible in clones because there is an expected phenotype (that of the donor animal). For example, the cloned cat, CC, was 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.