Genetic Engineering Research Paper

1. Biological Background of Genetic Engineering

The technology has developed in the context of our understanding of genetics. A vital property is the production and maintenance of variability in populations. One of the mechanisms that produces such variability is recombination. Information for a number of peptides as well as the controlling mechanisms for their synthesis are inherited together on a single chromosome. In diploid organisms, exchanges between homologous chromosomes is an important source of variation. This process occurs in the context of DNA replication, including the enzymatic unwinding of the double helix and synthesis of the new, complementary chain of nucleic acids.

Cellular properties may be altered by external mechanisms. One of the first systems that was extensively studied, and provided the tools that are currently used, is the phenomenon of infection of bacteria by bacteriophages, the bacterial viruses. The usual direction of events is followed. The message is coded in the DNA sequence. It is then transcribed into the sequence of the intermediate messenger RNA molecules, and then translated into protein. In the lytic cycle, the viral DNA instructions are used to synthesize new viral DNA and protein that can assemble into new viral particles that destroy the cell and are then released into the environment to infect other host cells. In the lysogenic cycle, the DNA of the virus integrates into the host DNA. In addition, there are a number of other particles called plasmids, which can transfer genetic information from one bacterium to another. They are small, circular DNA molecules that replicate independently of the DNA of the host bacterium. Their role is particularly prominent in the transmission of antibiotic resistance. Most animal viruses are similar to the lytic cycle of bacteriophage, however, there are a number of viruses that can become latent. There is also agents like the retroviruses, whose genome gets transcribed into the host DNA, thereby changing cellular properties. Some of these agents will also incorporate host DNA into their sequences, and then capable of transmitting this sequence into a new host.

The tools that allow us to transfer genes from one organism to another are the restriction endonucleases. These enzymes are found naturally in bacteria and protect them from infection by breaking down foreign DNA. They are chemically very specific, only working on specific palindromic sequences. Consequently, the kind of infection discussed in the last paragraph can only occur if the bacteriophage sequence does not contain elements that will be broken down by the host bacterial system of endonucleases. A number of different enzymes are found in different bacterial species, and the enzymes referred to by an abbreviation of the bacterial species name and a number. Similarly, there are enzymes that are used to connect fragments, the ligases (Griffiths et al. 1996).

2. Cloning Genes

All of the manipulations of genetic engineering require muliple copies of the DNA sequence or gene of interest. The original methods of getting multiple copies relied on bacteriophage or plasmid vectors to introduce the foreign DNA into bacteria to produce these copies, as each modified cell produces multiple copies, and the bacterial culture itself increases. This is done by first physically isolating the vector, opening its DNA with a restriction enzyme and binding in DNA from the organism being studied that has also been cleaved with a restriction endonuclease. A new population of bacteria is then infected with the altered vector. Given an appropriate way of selecting the population of bacteria so that it uniformly has the DNA of interest multiplying within, one can isolate a large population of vector molecules with the desired sequence, which is then freed by enzymatic cleavage once again.

Fragments of DNA are identified by physically separating them by electrical charge and molecular weight through gels. The DNA of the vectors and bacteria are generally in the range of one to ten thousand base pairs, and there are a sufficiently small number so that the fragments can be identified with a simple staining technique, usually a compound that binds to DNA and fluoresces under ultraviolet light. The larger quantity of fragments that would be isolated from more complex organisms produces a smear with such dyes, so the base-pairing property of DNA, the obligate pairing of adenine with cytosine and guanine with cytosine that allows for both recognition and synthesis of the linear sequence, is used to identify the same sequence on the gel by labeling a known fragment with an isotope or fluorescent dye. The labeled molecules are called probes. This is also the basis for identifying genetic variation in organisms, either for basic studies or identification of mutations associated with disease.

Isolation of fragments produced by digestion with several enzymes, used both singly and in combination, allows for the construction of a physical, restriction fragment map. Smaller fragments may be replicated, followed by the chemical analysis of the base sequence within fragments which are then assembled into the final base sequence of the gene. Once the sequence is known, production of useful amounts of a region of DNA may now be done enzymatically in vitro with the polymerase chain reaction (PCR). In this technique, the region between two primers, one from each strand of the final DNA molecule is copied in a logarithmic fashion by a heat-resistant DNA polymerase from a small amount of genomic DNA (it has been done with single cells), using multiple heating and cooling cycles. This technique is also used in diagnostic work (Strachan and Read 1996).

3. Modifying Cells To Produce Proteins

A number of strategies have been used to produce large amounts of a peptide or protein. Multiple techniques have been necessary because of the different kinds of cells that have been used. The production of human insulin, one of the earlier products produced commercially, is a useful model that is an example of expression cloning. The sequences for the A and B chains were introduced into separate vectors in sequence with a bacterial promoter (a signal to turn a gene on) and the enzyme β-galactosidase that produces colored bacterial colonies. This arrangement is necessary because bacteria will not just produce the human protein if only the structural gene is introduced. The bacteria are tricked into producing a fusion protein: a bacterial β-galactosidase with a human insulin chain sequence at the end. After selecting the colonies by color, mass cultures are produced from which the fusion proteins can be purified. The normal insulin dimer is then produced chemically by removing the βgalactosidase, purifying the chains, combining the chains and allowing them to refold and form the sulfide bridges between the two chains.

Expression cloning in bacteria works well if the peptide or protein is composed entirely of the amino acid sequence. Many functional proteins contain specific carbohydrate groups at precise locations on the chain that are attached after translation. These modifications are different in different classes of organisms, so techniques using yeast, plant, cultured insect, other mammalian, or human cells have been used to make the complete molecule. Another problem that is addressed in this approach is the elimination of bacterial toxins that cannot be removed from the desired protein product mixture. The engineered DNA can be introduced into these cells by a variety of methods. The physical techniques like electroporation, which uses an current to move the DNA inside, or coating various kinds of particles, like gold, with the DNA and then bombarding the cells to get the DNA inside has a chemical parallel in the use of calcium salts and heat. Artifical yeast chromosomes have been produced to move gene constructs. For the cultured insect cells, modified Bacloviruses are used (these are viruses that have invertebrate hosts). In mammalian systems, several viruses have been used, including retroviruses (the class of agents involved in acquired immunodeficiency and tumor transformation in experimental organisms), adenoviruses (agents that typically produce respiratory disease, however, some strains are capable of producing more severe systemic disease), and herpes simplex.

The goal of expression cloning is to produce large amounts of protein that can be purified more easily, and in much larger amounts than is possible using chemical techniques starting with cells from the normal tissue. On the research laboratory scale, the three-dimensional structure of the whole protein or the active region can be determined by a combination of nuclear magnetic resonance and x-ray crystallography. This information is useful for understanding function or designing small molecules that are expected to have pharmacological actions. This is then one element of the strategy in rational drug design. If the protein is to be used as a product, the growth of the modified cells has to be done on an industrial scale that is an engineering problem similar to that used in the production of a number of antibiotics (Glick and Pasternak 1998).

4. Modifying Organisms

An organism that has been engineered so that it has a gene from another species integrated into its genome is called a transgenic organism. These techniques have been used to produce plants and animals expressing mutated genes of that species to study function, with mutated or genes introduced from another species with desired agronomic properties, as models of human diseases, and to produce therapeutic products. There is a large and continuously growing literature of studies in the mouse.

Two procedures have been widely performed in mice: the interruption of a mouse gene to eliminate its function, for which the term knockout has been used, and introduction of a transgene. The ‘standard’ procedure employs classical, cellular, and molecular genetic techniques. The gene to be studied is first cloned, and then introduced into a targeting vector in such a way that the coding sequence of the gene is interrupted by the neomycin resistance gene. The vector also has the thymidine kinase gene from herpes. Embryonic stem cells are obtained from a brown mouse and placed in culture. The cells are then exposed to the vector. There are three possible outcomes. The vector does not insert at all, the whole construct inserts at random in a chromosome, or only the desired coding sequence and the neomycin resistance gene insert into the normal location on the chromosome. Treating the cells with a neomycin analogue and the antiviral agent ganciclovir will kill all of the cells except the ones with the integrated sequence. The surviving stem cells are injected into the blastocyst stage embryo of a black mouse and placed in a surrogate mother. If a chimera is formed, it will have a coat of two colors. The chimeras are mated with black mice, and their brown offspring tested for the modified gene. Those that are heterozygous are mated, and one of four will be homozygous for the modification.

In addition to the introduction of genes into stem cells by vectors, injection of the desired sequence has been done by some of the physical methods discussed earlier such as electroporation, coated gold particles, and salt precipitation. Some transgenics have been derived from direct injection of the plasmid into the egg.

The production of genetically identical organisms, cloning in the original sense, was first done in amphibians by nuclear transplantation into fertilized ova from which the original nucleus was removed. This was done widely in mammals by using embryonic cells as the nuclear donor. The production of the sheep named Dolly was noteworthy because the donor nuclei were obtained from adult mammary cells that were maintained in cell culture. This precipitated a round of discussion about the feasibility and the desirability of doing this in human beings. The claim was made that this would be an alternative for infertile couples, and therefore acceptable. It is necessary to point out to people who would propagate themselves for narcissistic reasons that the derived individual would not be absolutely the same because of the developmental environment, both before and after birth (Hubbard and Wald 1997).

Another possible use of this technology that has been proposed is the therapeutic cloning of replacement tissues or organs. Cells would be biopsied from the eventual recipient and placed in culture. Nuclei from these cells would be transplanted into eggs from which the nuclei have been removed; these eggs will be allowed to develop to the blastocyst embryonic stage for the isolotion of embryonic stem cells that are treated to produce the desired cell types. This avoids the problem of using fertilized eggs, but still involves the use of human eggs and early embryonic stages (Gurdon and Colman 1999).

5. Gene Therapy

The goal of gene therapy is to provide a functional protein to an individual whose genotype leads to a disease because the protein is missing or modified so that it is not functional. There are two potential modes. In germ-line therapy, the modification would be heritable, and the individual’s offspring potentially would not be affected. In somatic cell therapy, only the treated individual would have the modification. There have been many arguments against germ-line therapy including our inability to know whether potentially harmful changes in the sequence have been made inadvertantly. All of the trials to date have been in somatic cells for the treatment of inherited disease and cancer by control of cell death.

Two strategies are being attempted. For those disorders for which bone marrow transplantation has been effective, the gene is introduced into marrow cells, the population of which is then expanded and infused into the patient. The techniques for gene introduction are similar to the ones previously discusssed: viral vectors and chemical or physical techniques. An immune deficiency disorder, adenosine deaminase deficiency, was the first attempt reported. Although the modified cells express the enzyme, repeated infusions of treated cells have been necessary, and the patients have continued to receive chemically modified enzyme, so it is not clear whether or not gene therapy alone is sufficient.

Other trials have used viral vectors to treat target organs, such as the lung in cystic fibrosis. The therapeutic trail of an adenovirus-mediated transfer of a gene to the liver has received general media attention because of the death of a participant. Evidently, other participants in gene therapy trials have died, although it is not clear how many of those deaths are a result of the procedure (Strachan and Read 1996).

6. Genetic Engineering Applications To Behavior

The application of these techniques in behavior is loaded with scientific, ethical, and legal complications. Part of the problem stems from the very human desire to have a simple answer to a complex problem. Genetic, environmental, and developmental factors interact to produce behavior, and it is difficult to distinguish the role of each component. One should ask if it is even desirable to do this, because of the mixture of potential benefit and risk. It is clear that each behavioral trait is heterogeneous. There may be families where a single major gene is responsible, and a particular drug, expressed protein or even gene therapy could work, but in the more usual instance of several genes and environmental factors, it is unlikely that such a unitary approach would work. The promise of a solution to disease problems by definition of molecular mechanisms has been difficult to achieve. We still do not have an answer in the case of sickle cell anemia, the first disorder to be described in this manner. Common behavioral traits would be more difficult because of the interactions.

Because of the Human Genome Project, it is likely that a number of genes will be discovered that contribute to the development of a number of behavioral characteristics. The technology therefore will create the possibility of diagnostic tests for the presence of variants at these genetic loci that might be involved in diagnostic confirmation, or even asymptomatic screening without necessarily having the ability to safely and ethically treat those in whom the genetic variant is identified. Furthermore, since it is well known that the genotype does not always predict the phenotype even for such things as emphysema associated with a variant α-1-antitrypsin, one should question this approach with respect to behavioral characteristics (Hubbard and Wald 1997).

Using genetic information to design pharmacologically active agents will probably be useful for drugs interacting with receptors influencing neural function. A similar problem arises here. Will the intervention be beneficial to someone who is suffering or will it be used to control individuals who are designated by those in authority to be deviant? Modifying the environment to enhance development is effective and poses less of a biological risk than drugs or gene therapy (Duyme et al. 1999). The relative economic costs are unknown, but the human ramifications are immense.

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