Transgenic procedures
Recent technology has introduced a way to insert genetic material from one living being into the cellular DNA of a completely unrelated organism. In these cases, genes are inserted into a zygote or fertilized egg. The genes are thus integrated into the DNA of that zygote. This area of genetics is known as transgenic technologies. These procedures are also associated with Xenotransplantation which is the transplantation of animal organs into human beings as Mary has just explained. Transgenic procedures have proved to be more successful than Xenotransplantation at the present time. This is because over time the immune system will reject the animal organs as foreign tissue. Transgenic technologies on the other hand, are not recognized as foreign tissue to the immune system since they are integrated directly into the DNA. In the long run, the many patients who are in need of a heart or liver, blood transfusions or even skin grafts might benefit from such technological advances with transgenic animals.
The generation of a transgene is the first step in performing a transgenic technique. This transgene consists of a DNA segment containing the targeted gene and the elements that control the overall function of that gene. The critical component is the promotor that drives transcription and causes the transgene to be expressed in a specific location. Once these transgenes are produced, they are directly inserted into a zygote or egg in the female’s reproductive tract. Thus, integration into the DNA occurs. Where the transgene actually inserts is extremely difficult to control; therefore, there is a degree of variance in the expression of the gene. Even further, there is a low success rate in this process. Presently, there is a less than 5 percent chance that the DNA will be successfully passed on to future generations.
There are many different types of animals that are being experimented with as far as transgenic animals are concerned. Such animals may include pigs, sheep, goats, cows, and most favorably mice. Mice have provided for extending our knowledge in gene function. When speaking about transgenic mice, there are two main methods of their creation.
The first system is the Embryonic Stem Cell Method, referred to as method number 1. Embryonic stem cells are harvested from the inner cell mass of the mouse blastocysts. They are then grown in culture and retain their full potential to produce all of the mature cell, including its gametes. DNA must then be made consisting of the structural gene of desire, a vector to enable the insertion into the host DNA molecules, and the promotor and enhancer sequences to bring about expression of the gene. The Embryonic stem cells are then exposed to the culture grown cells for some incorporation to occur. These cells are next placed back into the inner cell mass. The embryos are then transferred in the uterus of a pseudopregnant mouse. Finally, testing of the offspring occurs and a transgenic strain is established. Generally speaking 10-20% of the DNA will contain the DNA for the desired gene, thus being heterozygous. The transgenic strain, which is created by mating two of the heterozygous mice, will establish the 1:4 ratio. The homozygous mice will generate the actual transgenic strain.
Method 2 or the Pronucleus Method of creating transgenic mice begins in the same manner as method 1. The DNA must be prepared. At this point, freshly fertilized eggs before the sperm head has become a pronucleus are harvested and placed into the DNA. The pronucleus will thus fused and allow the zygote to divide by mitosis to form a 2-cell embryo. These embryos are next planted into a pseudopregnant mother and the process continues as in method 1.
What is the reason for carrying out transgenic techniques? Besides improving livestock and plants, such methods allow the opportunity to obtain more knowledge about gene functions. These transgenic techniques will also create more desirable properties and traits among those involved in breeding programs, as well as furthering knowledge and perhaps generating possible cures for diseases. For example, transgenic animals engineered to express a mutant form in an over abundance of the gene beta-amyloid protein precursor (APP) has neuropathological changes representing a similarity to Alzheimer’s disease. These experiments provides the opportunity to test methods for prevention or delay of the disease.
Possible dangers of such methods may include new zoonoses or diseases transmitted from animals to humans. Dangers of priors, or self-replicative proteins, causing diseases are also worried about. The concern with transgenic technology is the possible disturbance of the existing balance between organisms. This may inturn have an affect on the environment. Careful examination is essential when experimenting with such procedures.
An area where the transgenic technique has been used is in lung carcinogen experiments involving the human c-Ha-ras gene. This particular experiment uses mice who eventually encode a proto-type p21 gene product. Nine week old mice, both female and male, as well as transgenic and non-transgenic subjects were injected with 6-nitrochrysene (6NC) three times biweekly or administered urethane in their drinking water for 3 weeks. The control mice were given dimethylsulfoxide, the solvent for 6NC. Four out of seven transgenic females or 57% of the mice treated with 6NC showed incidences of lung adenocarcinomas. In the same fashion, three out of three males and females mice, or 100%, receiving urethane contributed the same results. No adenocarcinomas were observed in the control and non-transgenic animals. Although adenomas were noticed in all of the treatment groups, the incidence and multiplicity were higher in the transgenic groups of subjects. Analysis of the DNA sequence revealed point mutations at codon 61 of transgenic human c-Ha-ras from CAG to CTG or CAG to AAG. In regards to the mouse Ha-ras gene, there was no recorded change. The study overall indicated a high sensitivity to lung carcinogens in transgenic mice carrying the human c-Ha-ras gene.
The fastest area of growth dealing with transgenic technologies is in Pharmaceutical production. Presently, the production of human pharmaceuticals in farm animals is in development and should possibly be commercialized by the year 2000. This application is gaining popularity amongst biotechnologists because the cost of these drugs may be much less than for those produced using conventional techniques. For example, the first successful products were insulin and growth hormone. Although these protein drugs aren’t produced in mammals, they can be generated in the inexpensive and easily grown culture of the common bacteria E. coli.
The overall goals of pharmaceutical production in transgenic animals should 1) produce the desired drug at high levels without endangering its own health and 2) pass its ability to produce the drug at high levels to its offspring. Unfortunately, the success of these goals is not so easily obtained. About 10 to 30 percent of mouse embryos produce transgenics, but less than 5 percent of goats, sheep, or cows do. Since there are long time periods involved and low success rates, developing transgenic animals is currently very expensive. In the long run though, these costs could be reduced to where we would be profiting from such manufacturing of drugs. In general, animal pharming is considered to be 5 to 10 times more economical on a continuing basis and 2 to 3 times cheaper in start-up costs than cell culture production methods.
Along with the ideas of this "pharming", there are regulation and ethical issues to take into consideration. These methods of production will have to undergo significant evaluations by the Food and Drug Administration. It is likely that these drugs will also require exceptional levels of safety testing before animal and human health concerns are addressed to the satisfaction of consumers. The ethical issues concern the animal welfare and relationship between animals and humans. These processes are human endeavors to improve the availability, quality, and safety of drugs. Such productions also geared to enhance human health and to improve animal health.
In most of these drug productions, the drug is delivered from the animal in the convenient form of milk. This has become the single most studied transgene system where there is modification of the peptides produced in the mammary gland. In the US alone, dairy cows make over 3 billion kg of protein annually. This dairy milk is to enhance health value and produce better foods. Generally speaking, manufacturing proteins in milk makes these proteins readily available and easily taken in.
The procedure to manufacture proteins in the milk starts by the coupling of the DNA gene for the protein drug with a DNA signal directing production in the mammary gland. This new gene specifically only functions in the mammary gland, resulting in the production of the protein only in the milk. One of the positive aspects of this method is there is virtually no danger of disease or harm to the animal. This is because the mammary glands are essentially located outside of the main life support systems. After coupling, the DNA is inserted into a fertilized animal such as a cow, sheep, goat, pig or mouse. The injected embryos are then implanted into the surrogate mother, followed by birth.
The specific case where Genie, the pig, was used for the production of highly concentrated levels of protein C in her milk is an example of such a transgenic technique. By manipulating the pig’s DNA, humans with an inborn deficiency of protein C could obtain the extra amounts needed through the pig’s milk. Such transgenic methods are favorable because of the reduced costs. At the same time, mass profit could be gained as a result.
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