Cloning by Cell Fusion
With nuclear transfer, the main principle is that the ovum, or oocyte is a minifactory designed to produce an embryo, which eventually develops into a term pregnancy. Half of the genetic instructions to make the conceptus normally come from the oocyte, and half from the sperm. With cloning, a complete set of genetic instructions is provided by the nucleus of one embryonic or somatic cell. Of course, those instructions originally were derived from the sperm and oocyte that resulted in the organism that provided the donor cell.
One problem is obtaining oocytes to use as recipients for the diploid nuclei. These cells, the largest in the body (about 1/200 inch in diameter), must be of the same species as the donor nucleus. Usually, they are aspirated from ovarian follicles (large blister-like, fluid-filled structures). In the case of farm animals, oocytes are often obtained from ovaries of slaughtered animals of unknown background. An alternative is to aspirate (remove by suction) oocytes through a large needle inserted into the ovaries in the body cavity of living animals—ultrasound is usually used to visualize the follicles so the needle can be guided into them after piercing the wall of the vagina. This method is used in women to obtain oocytes for routine in vitro fertilization. Oocytes from laboratory animals such as mice are usually obtained after the oocytes are ovulated (released from the follicles) naturally. The oocytes then are located in the part of the reproductive system called the oviduct, and the body cavity needs to be opened to get them out, either via surgery with anesthesia, or after euthanizing the animal.
After oocytes are obtained, they are cultured under specific conditions with specific chemicals until they have matured appropriately. The length of the maturation period may range from less than an hour to two days, depending on the species, the treatments, and the reproductive status of the animal providing the oocytes.
The next step is to remove or destroy the unwanted chromosomes of the oocyte. This usually is done by aspiration of this material with a micropipette (see Figure 2), although there are other options, such as destroying the chromosomes with a laser. Following this step comes transplantation of the nucleus. This can be done by removing the nucleus from the donor cell and injecting it into the cytoplasm of the oocyte. However, in the vast majority of cases the entire donor cell is simply fused with the oocyte using an electric pulse. This incorporates the nucleus into the oocyte, but it also mixes the cytoplasm of the two cells, which also mixes the mitochondria. This is usually not a problem because the oocyte has more than 100 times the volume of the cytoplasm of the donor cell, so the donor cytoplasm essentially gets diluted out.
When a sperm fertilizes an oocyte, it not only adds its 50 percent contribution of genetic material, it also activates, or turns on, the oocyte. Prior to fertilization the oocyte is a large, slowly dying cell. The sperm adds a specific enzyme that chemically activates the ovum, so it comes to life, starts using more energy, and, among other things, duplicates the genetic material in preparation for division to the two-cell stage. This activation function must be duplicated during the nuclear transplantation process for successful embryonic development. It is accomplished in a variety of ways, depending on the species and other details, such as the degree of maturity of the oocyte. A common approach is to apply a strong electrical shock.
The final step is to allow the cloned embryo to develop in vitro, eventually growing from the two-cell stage to a suitable stage for transferring the embryo back to the reproductive tract of a recipient. The length of this culture is usually a few days to a week, depending on the species.
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