For thousands of years, man has attempted to improve animal genetics by selective breeding. Targeted mating strategies are based on the presence or absence of specific traits that can be identified and transmitted to offspring. Improvements have been limited to naturally occurring events or mutations. Starting in the early 1970’s, the advent of recombinant DNA technology has introduced a variety of new techniques intended to accelerate and refine the process of genetic manipulation.
Transgenics is the science of intentionally introducing a foreign gene or genetic construct (series of genes and associated regulatory elements) into the genome of a target animal. Initial work involved a splicing technique to insert foreign genetic materials into mammalian cells maintained in culture. This in vitro work rapidly progressed into laboratory rodents, providing a more targeted and proactive approach for the establishment of new animal models for biomedical research. The results have been very successful and provide a unique and precise mechanism for the study of a variety of specific conditions or diseases with a genetic basis or influence.
The development of transgenic applications in livestock is a logical progression for this technology. Insertion of modified human gene constructs into livestock is being utilized to create "designer production animals" capable of producing useful proteins, tissues, and organs for pharmaceutical and biomedical use. Additionally, the manipulation of indigenous gene sequences has the potential to convey enhanced disease resistance and/or improve production in target animals. The primary objective in using transgenic technology in animal agriculture is to improve the quality of livestock by altering the animal’s biochemistry, hormonal balance, or harvested protein products. Scientists hope to produce animals that are larger and leaner, grow faster and are more efficient at using feed, more productive, or more resistant to disease.
There are several techniques for the production of a transgenic animal and new processes are continually being developed or refined. All have the same primary objective, which is the successful integration of a functional sequence of a DNA strand (a transgene) into a chromosome within the host genome. Most of the following methods for introducing transgenes into animals have been used since the 1980’s.
The first genetically altered embryos were created using viral vectors in the early 1970’s. This technique is still in use. A viral vector (or phage) is first modified so that it will not replicate or cause disease in the target cells of the host embryo. The gene(s) of interest is incorporated into the viral genome and the virus is then used to infect an early stage embryo. The viral vector binds uniformly to the embryonic cells and acts as a vehicle to allow transfer and integration of the transgene into the host genome. Many of the experimental human gene therapy trials currently underway use basic viral vectors as a means of "gene-delivery" to susceptible cells or tissues in a very similar procedure.
An advantage to using viral vectors is that usually only a single copy of the transgene is integrated into the genome. If the viral transfection is applied to oocytes prior to fertilization, then the novel gene will be present in all cells of the resulting embryo as though it had been contributed by the maternal germline.
The major disadvantage of this system is the time and labor-intensive process to prepare the viral vector. There is also a remote possibility that the modified viral vector may revert to its original state or recombine with other pathogenic viruses.
Most existing transgenic animal lines have been produced using pronuclear microinjection. This technique involves the injection of genetic material into an early-stage embryo to create what are called germ-line transgenic animals.
The gene (or genes) of interest is first identified and the nucleotides for that segment of DNA is sequenced. Frequently, there is a special segment of DNA in addition to the gene of interest, which is referred to as a promoter. The promoter is a regulatory segment of DNA located on the same chromosome as the gene of interest. It influences or controls expression of the gene. An endogenous promoter may be modified during transgene assembly in order to increase the likelihood that the gene will function in the targeted tissues of the host animal. The promoter can also be used to turn the gene on or off as needed. For example, a promoter sequence that requires a specific dietary "trigger" substance can be used to turn on genes for important hormones in animals so that the hormone is only produced when the animal is fed the appropriate trigger. A majority of the current research focuses on the understanding and development of useful promoter sequences to control transgenes and mechanisms for more precise insertion of the transgene into the recipient animal.
The prepared DNA construct (transgene and promoter) is usually replicated in a plasmid vector to produce multiple exact copies for microinjection into the pronucleus of an embryo. The injection volume is quite small (approximately 2 to 3 picoliters) and is accomplished by means of a very fine glass micropipet which is able to penetrate the cell membrane of the fertilized ovum without damage. Although many copies of the transgene construct are injected, the actual number of copies that eventually incorporate into the host genome will vary. If the transgene successfully integrates into one of the chromosomes of the pronucleus, the transferred genetic material should be present in every cell of the resulting animal and have the potential to be transmitted to future offspring.
A major disadvantage of the pronuclear microinjection system is that the rate of integration of the transgene may not be uniform between cells. Certain cell populations may not include the new DNA or may have multiple copies. Likewise, it is not always possible to specify, or target, the locus (or specific location) of integration for the transgene into the host DNA. Frequently, integration site may be a critical determinant of the transgene’s expression and function may be impaired even though the transgene is present. Similarly, if the integration of the transgene disrupts a functional DNA sequence in the host’s genome, an insertional mutation may result which interferes with the function of the existing gene.
The use of homologous recombination has facilitated the development of targeted transgene insertion and, by consequence, the production of better-defined transgenic research models. The term homologous recombination refers to the exchange of DNA fragments between two DNA molecules at an identical site, which allows insertion of the transgene to be targeted to a specific location on the chromosome.
Undifferentiated embryonic stem (ES) cells have the potential to differentiate into any type of cell within the developing organism. These cells are harvested from a blastocyst stage embryo and cultured in an in vitro environment. The transgene is attached to a DNA construct that is analogous to a segment of the host DNA (except for the presence of the transgene). The new DNA-transgene is then introduced into the nuclei of ES cells in culture by means of a vector or by electroporation (the application of an electric current to enhance cell membrane permeability). As cell division occurs, the novel DNA replaces the existing segment and is incorporated into the nucleus of some of the new cells. Transgene positive cells are identified and sorted using various selection techniques, including survival selection (positive-negative selection) and polymerase chain reaction (PCR) amplification. The modified ES cells are then injected directly into a normal blastocyst embryo.
The advantage of this system is that only a single copy of the transgene is incorporated into the new cells. Also, the site of integration is highly controlled. Unfortunately, the system is very time-consuming, in that the DNA sequence of the segment of interest for the host cell must be known in order for the transgene to be constructed.
The recently much-publicized successful cloning of livestock has raised interest in the use of this technology for the production of transgenic animals. Cloning is the process of nuclear transfer, as opposed to single gene transfer, and results in the production of genetically identical animals (clones).
The nucleus of an unfertilized oocyte is removed and replaced with nuclear material harvested from a cell from an existing animal of the same species. The donor cells are frequently of epithelial origin and contain a full complement of DNA (diploid), in contrast to the unfertilized oocyte with half the total (haploid). A modified gene sequence can be introduced into the cultured donor cells prior to nuclear exchange. The nuclei of cells that successfully incorporate the transgene are identified, isolated, and inserted into recipient oocytes. Cell division is activated and the resulting embryos are implanted into the uterus of a foster recipient.
The major advantage of this system is that the generation of a large number of animals from a single donor is possible. Of course, the technology is still in the early stages and specific procedural elements are frequently being modified or evolving. Ethical issues related to the transfer of this technology into human medicine are the source of much debate.
Although these technologies are primarily restricted to research settings at the current time, it is inevitable that they will be incorporated into more traditional situations in the near future. It is very difficult to anticipate or comprehend the ultimate impact that will result. Consequently, a basic understanding and appreciation of the science involved is essential to the critical assessment of these ideas.
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