Genetic manipulation will be the next big leap in science and with it there are limitless possibilities. The first gene therapy trials on humans began in 1990 on patients with severe combined immunodeficiency (SCID). "Gene therapy can be defined as the transfer of genetic material into the cells of an individual resulting in a therapeutic benefit to the individual. It involves the intentional modification of genetic material with the aim of preventing, diagnosing, or curing a disease. These modifications include the correction of a genetic defect resulting from the absence or alteration of a protein, or the addition of genetic information to modify cellular characteristics. Gene therapy allows the modification of specific genes without having to alter the disease phenotype using agents that either interact with the gene products (proteins), or are gene products themselves. The genetic modifications can be done in vitro or directly in vivo by using vectors that are capable of genetic transfer. Products used in gene therapy include viral vectors, genetically modified cells, and free or complex nucleic acids" (Vento). Transgenic primates have been obtained from microinjections of retroviral vector. Sperm-mediated gene transfer is potentially the easiest way to the human germline, but the required methods are underdeveloped. Nuclear transfer is an alternate possibility to germline genetic modification, but there are health concerns with the current methods. There are several techniques that can be used in genetic manipulation.
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Genetic modification is the alteration of an individual's genotype with the aim of choosing the phenotype of a newborn or changing the existing phenotype of a child or adult. Currently research is being done to look for cures of genetic diseases that cannot be treated with traditional methods. The methods used on animal transgenesis could be applied to humans. These methods would allow humans to change anything about themselves. Changes range from physical features to mental capacity. The conditions that need to be met before transgenesis would be considered safe for humans are the ability to deliver transgenes in a highly efficient manner, non-prohibitive cost and expertise requirements, minimal risk of causing insertional DNA damage, low rate of mosaicism, high DNA carrying capacity, the ability to permit adequate and controlled transgene expression, and "the ability to target transgenes to precise genomic loci" (Smith).
The methods for doing research was done through searching internet articles. Articles were found through the Michigan Tech University library databases. To search for related topics the keywords used include genetic manipulation, recombinant DNA, germline, transfection, and gene splicing.
The earliest technique in genetic engineering was created by Joshua Lederberg. He found that bacteria can transfer genetic information through plasmids. In 1968, after the identification of restriction enzymes capable of cutting DNA in specific locations, scientists were able to insert foreign DNA into bacterial cells. The foreign DNA would naturally bond with the host DNA, which made it possible to splice together genes from multiple organisms. This technique is used in recombinant DNA (rDNA) engineering. Recombinant DNA engineering is when genetic material from a donor is isolated and cut using a restriction enzyme and then recombined into the genetic material of the receiver. Recombinant DNA is a form of artificial DNA because the sequences would not normally occur together whereas genetic recombination occurs through natural processes.
Pronuclear microinjection was first demonstrated in 1980 by Jon Gordon. He showed that "DNA could be introduced into the germline simply by the physical injection of a solution of cloned DNA into zygote pronulei" (Smith). The technique is simple, however it requires expensive equipment and high levels of skill. A fine glass needle is filled with DNA solution, then "under a microscope, the needle is guided through the cytoplasm towards one of the zygote's pronuclei" (Smith). A nanoliter of DNA solution is injected, which sends about two hundred DNA molecules into the pronucleus. "This method of producing transgenic animals results in the introduction of a purified double-stranded DNA sequence into the chromosomes of the fertilized egg. The foreign DNA must integrate into the host genome prior to the doubling of genetic material that precedes the first cleavage or a mosaic animal may be produced in which many cells do not possess the new gene, so the transgene DNA is inserted into the zygote at the earliest possible stage" (Smith).
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"A viral vector is a virus that carries a modified or foreign gene. They are commonly used in gene therapy where the viral vector delivers the desired gene to a target cell" (Vento). Retroviral vectors can deliver genes in somatic gene therapy and germline therapy. "The zygote is incubated in media containing high concentrations of the retroviral vector or a retroviral vector producing a cell monolayer can that be used. After zygote transfer into pseudopregnant females, the infected embryos should produce transgenic offspring. Rearrangements of the host genome are usually restricted to short direct repeats at the site of integration" (Smith). Virus uptake is effective for somatic cell lines but germline cells have a low frequency of infection because of a high level of mosaicism. The new generation of lentiviral vectors avoids the mosaicism in germline cells. Lentiviral vectors have a small insert capacity, microinjections are more controllable and a microinjection of retroviral vectors has been successful with primates. The retroviral vector particles has an envelope type known to recognize and bind to the membrane of all cell types. Another virus that can be used is the adenovirus. "Adenovirus can be defined as a group of DNA containing viruses, which most commonly cause respiratory disease. Adenoviruses can also be genetically modified and used in gene therapy to treat cystic fibrosis, cancer, and potentially other diseases." (Vento). These are but a few of the virus types that can be used as viral vectors.
Sperm-mediated gene transfer has been successful in virto uptake of exogene constructs by animal sperm cells. This method has not yet become a reliable form of genetic modification. "The reproductive tracts contain free DNA molecules, so sperm cells are expected to be highly resistant to being used as exogene vectors. The successful results are thought to be responsible by certain factors in the cases in which transgenes were taken up and transferred by sperm" (Smith). One of the factors could be the inhibitory factors with sperm cells which prevent exogenous DNA uptake to protect the genetic integrity of the offspring. The successful instances would be those in which the inhibitory factor was removed. Seminal fluid contains an inhibitory factor that blocks the binding of exogenous DNA to sperm. There are three classes of identified proteins in sperm which have been claimed to have binding properties. The possibility of inhibitory factors would cause the negative results in many attempts to use sperm as a transgene vector.
Nuclear transfer is a technique used in the cloning of adult animals. It requires two cells, a donor cell and an oocyte. The oocyte should be unfertilized because it will be more likely to accept the donor nucleus, and the oocyte must be enucleated. The nucleus of an egg cell is removed and then it is inserted into the enucleated egg cell. After being inserted into the egg, the nucleus is reprogrammed by the host cell. The egg is then stimulated with a shock and it will begin to divide. Many mitotic divisions occur and then the cell forms a blastocyst, an early stage embryo, with almost identical DNA to the original organism.
The result of the research is based off of the work of others. Statements that were found to be of great importance for the results include some the following. Fritz Allhoff wrote, "Somatic cells, such as skin or muscle cells, contain 23 chromosomal pairs and do not transmit genetic information to succeeding generations. Germ-line cells, which are the egg and the sperm cells, contain 23 unpaired chromosomes and provide genetic information to offspring, as well as to the future generations descended from those offspring. Genetic therapy aims at the treatment or prevention of a disease, whereas genetic enhancement aims at the enhancement of some capability or trait" (Allhoff). Bill Pohlmeier and Alison Van Eenennaam said, "To understand the function of individual genes, scientists use a process known as gene knockout, to disrupt the function of a gene of interest. In the laboratory, scientists can develop a non-functional copy of a gene of interest. When this non-functional copy is introduced to cells in culture, it can recombine and replace the functional copy, in a process called homologous recombination" (Pohlmeier). Amy Vento and David Gillum go into great detail about recombinant DNA. "Recombinant DNA, also known as in vitro recombination, is a technique involved in creating and purifying desired genes. The following is a summary of the process of making recombinant DNA: Treat the DNA taken from both sources with the same restriction endonuclease. The restriction enzyme cuts both molecules at the same site. The ends of the cut have an overhanging piece of single-stranded DNA called "sticky ends." These sticky ends are able to base pair with any DNA molecule that contains the complementary sticky end. Complementary sticky ends can pair with each other when mixed. DNA ligase is used to covalently link the two strands into a molecule of recombinant DNA. In order to be useful, the recombinant DNA needs to be replicated many times (i.e. cloned). Cloning can be done in vitro, via the Polymerase Chain Reaction (PCR), or in vivo (inside the cell) using unicellular prokaryotes (e.g. E. coli), unicellular eukaryotes (e.g. yeast), or mammalian tissue culture cells" (Vento). All of these statements from various sources became a part of the results and were all necessary facts to complete the research.
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There are many techniques in which genetic manipulation can be performed. Some methods are better for certain types of modifications. Research in genetic modification is focused on finding cures for genetic diseases but it can also improve other aspects of humans. Genetic enhancement can make humans smarter, faster, stronger, or change almost anything about them. There have been successful changes in mice that have made them stronger and have a significantly longer life span. These mice have a noticeably bigger physique. Soon the genetic modifications that are being tested on animals will be applied to humans.