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| Cloning Animals |
Asexual reproduction in bacteria and plants allows scientists to obtain genetically identical populations; this does not occur naturally in vertebrates, except in twins. In 1996 Dolly the lamb was born: chromosomal material derived from an adult sheep was used to generate an animal with chromosomal DNA identical to that of the donor animal. Cloning livestock, using the techniques that generated Dolly, may become an economical method for traditional breeders to replicate their superior animals and provide them to farmers. Rather than selling semen, breeding companies might distribute cloned embryos for implantation into surrogate cows. Because Dolly did not possess foreign DNA, she was not transgenic. However, she did represent a valuable step toward the development of transgenic livestock. With donor DNA for cloning derived from cultured recombinant cells, it becomes possible to carry out specific genetic modifications and introduce the modified genes into animals.
Ian Wilmut and his colleagues cloned Dolly using a technique called nuclear transfer 1. In this technique, the nucleus of a recipient egg is removed to make way for the genetic material of the donor (Fig. 5). The donor cell is fused with the enucleated egg cell by subjecting the two cells to pulses of electricity. Earlier studies had suggested that donor nuclei from early embryos were more likely develop properly. The use of an adult cell for the donor nucleus was unique in Dolly's case. Although most differentiated animal cells contain all the genes for making an entire organism, nuclei change as cells differentiate. To dedifferentiate the udder cells used for nuclear transfer they cultured the cells in a nutrient-poor medium. This caused the cell cycle to stop in the GO phase. After fusion, 277 embryos were grown in culture for six days before implanting them in thirteen surrogate mothers. Only one of the embryos completed normal development.
Cloning by nuclear transfer depends on the availability of donor cells with the appropriate genetic information. Somatic cells such as fibroblasts, ovarian cells, muscle cells, and mammary epithelia are grown in cell culture and by genetically modified fusion with the enucleated egg. Commonly, DNA is transferred to the cells using viruses.
Microinjection and Other Techniques
Another technique for cloning animals is microinjection. In this technique, a gene construct is characterized in culture and an adequate quantity of the desired DNA is obtained. The DNA is injected into fertilized ova before the first cell division occurs. This increases the probability that all of the cells of the organisms will harbor the gene. The injection is done soon after fertilization, before the male and female pronuclei have fused. A very thin pipette or needle injects the DNA into the large male pronucleus (Fig. 6). Surrogate mothers are made pseudo-pregnant with hormones and implanted with the injected eggs. After birth, tissue samples of the young are assessed for the presence of the desired gene. DNA from germ line cells is given special attention. If the novel gene is present in these cells, the animal can be used as a founder for breeding.
Genetic constructs that include regulatory regions targeting gene expression to specific tissues are necessary if the gene product is to be harvested readily. For example, GTC Biotherapeutics uses the betacasein promoter to ensure that antithrombin III is secreted in goat milk. Common biochemical procedures, such as filtration and chromatography, are then used to isolate the AT-III from the milk.
Scientists often use southern blots to evaluate DNA extracts from tissue samples (Fig. 7). Southern blotting a type of nucleic acid hybridization test, in which single-stranded DNA from two sources interact. Strands with similar nucleic acid sequences will anneal by base pairing (A with T, and G with C) to form double-stranded molecules. One of the single-stranded DNA molecules is a unique portion of the gene of interest, and is radiolabeled so it can be detected on photographic film (the probe). Southern blotting allows the detection of fragments of genomic DNA, which anneal to the radiolabeled probe. The fragments are generated using restriction enzymes and separated in a gel by electrophoresis. The size of a given fragment relates to the distance it migrates on electrophoresis. The fragments are denatured to single strands, transferred to a special filter paper that is immersed in a solution containing the probe, and then rinsed. If the probe has annealed it will expose the photographic film, resulting in a band.