The following points highlight the three important examples of transgenic animal. The examples are: 1. Cloning Dolly 2. Transgenic Mice 3. Reporter System.
Example # 1. Cloning Dolly:
In February 1996, Ian Wilmut and co-workers from the Roslin Institute and PPL Therapeutics, both in Edinburgh, Scotland, reported in Nature journal that they had successfully cloned a sheep from a cell taken from the udder of a six-year old ewe.
The cloned lamb was named Dolly. In the past, genetically identical animal embi70s had been created only with amphibian cells, and those created from adult nuclei had never successfully reached adulthood. Cloning in which the nuclei came from fetal cells or cells from cell lines had been successful before in mammals. The word clone means to create a genetically identical copy.
To clone an animal, it is necessary to begin with an egg, the only cell known to initiate and support development. In order to clone an individual, it is necessary to get an egg without a nucleus and then to transplant in it a nucleus of known origin.
Techniques for nuclear transplantation had been worked out with frogs and toads in the 1950s. The Scottish scientists succeeded in obtaining sheep eggs, enucleating them (removing their nuclei) and then transferring in donor nuclei by fusing the donor cells and the enucleated eggs with an electric pulse.
The electric pulse also initiated development of the egg. Although only one pregnancy of the twenty nine initiated was successful, the lamb that was born seemed normal in every respect; since it had produced offspring.
Others had tried this type of experiments with many types of animals, including mice. They were not successful for numerous reasons. The most likely explanation for the recent success, according to scientists, is that the donor cells were kept in a non-growth phase for several days, which may have synchronized them with the oocyte.
Thus the nucleus and the oocyte were at the same stage of cell cycle and thus compatible. Other reorganization that had to take place in the donor chromosomes are not really known for certain but one thing is clear: the nucleus of an adult cell in the sheep has all of the genetic material needed to support normal growth and development of the egg. The work has since been repeated with goats, cattle and mice.
There are various ramifications to the success of this work:
1. Mammal cloning could becomes a routine procedure. This would allow us to study mammalian development and to replicate genetically identical individuals, particularly transgenic animals that would have particular genome of value.
2. We can also use these techniques to study aging, since an “old” nucleus in initiating development of a new organism.
3. Also, of interest is the interaction of a particular genome with a particular cytoplasm, since the cytoplasm contains not only the material needed for early development, but also cell organelles, including mitochondria that have their own genetic material.
Example # 2. Transgenic Mice:
Animal cells, like the protoplasts of plant cells, can take up foreign chromosomes or DNA directly from the environment with a very low efficiency (in the presence of calcium phosphate). Directly injecting the DNA greatly improves the efficiency.
For example, transgenic mice are now routinely prepared by injecting DNA either into oocytes or one or two- celled embryos obtained from female mice after appropriate hormonal treatment.
After injection of about 2 picoliters (2 X 10-12 liters) of cloned DNA, the cells are reimplanted into the uteruses of receptive female hosts. In about 15% of these injections, the foreign DNA incorporates into the embryo.
Transgenic animals are used to study the expression and control of foreign eukaryotic genes. In 1988, a transgenic mouse prone to cancer was first genetically engineered animal to be patented. Thus mouse provides an excellent model for studying cancer. (A controversy arose as to whether engineered higher organisms should be patentable; currently they are).
Mice have already been successfully transfected with a rat growth-hormone gene, and transgenic sheep have been produced that express the gene for a human clotting factor. The latest recombinant DNA dispute arose, from cloning of sheep in 1997.
The transfection can also be mediated by retroviruses (RNA viruses containing the gene for reverse transcriptase). For example, a retroviral vector was ritrodused and repaired human white blood cell lacking the enzyme adenosine deaminase.
A retrovirus responsible for a form of leukemia in rodents, the Moloney Murine leukemia viruses was engineered so that all the virus genes were removed and replaced with an antibiotic marker (neomycin resistance) and the human adenosine deaminase gene.
The virus binds to the cell surface and is taken into the cell, its RNA is converted to DNA by reverse transcription and the DNA is incorporated into one of the cells, chromosomes. It is not possible for the highly modified virus to attack and damage the cells.
Example # 3. Reporter System:
Two reporter systems are used to indicate that a transfection experiment was successful. Plants can be transfected with the Ti plasmids of Agrobacterium tumefaciens. When a plant is infected with A. tumefaciens containing the Ti plasmid, a crown gall tumor is induced transferring the T- DNA region.
Those cells transfected with the T-DNA are induced to grow as well as to produce opines that the bacteria feed on. Much recent research has concentrated on engineering Ti plasmids to contain other genes that are also transferred to the host plants during infection, creating transgenic plants. One series of experiments have been especially charming.
Tobacco plants have been transinfected by Ti plasmids containing the luciferase gene from fireflies. The product of this gene catalyzes the ATP- dependent oxidation of luciferin, which emits light. When a transfected plant is watered with luciferin, it glows like a firefly. The value of these experiments is not the production of glowing plants but rather the use of the glow to “report” the action of specific genes.
In further experiments, the promoters and enhancers of certain genes were attached to the luciferase gene. As a result, luciferase would only be produced when these promoters were activated; thus, the glowing areas of the plant show where the transfected gene is active.
One of the more recent reported systems developed uses a gene from jellyfish that produces green fluorescent protein. The value of this system is that it “reports” when ultra-violet light falls on it, rather than it requiring an addition, as in the luciferase system (see Tamarin, 2002).