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Transgene

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Transgene

A transgene is a phenotype of an organism.

In its most precise usage, the term transgene describes a segment of germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum. This technique is routinely used to introduce human disease genes or other genes of interest into strains of laboratory mice to study the function or pathology involved with that particular gene.

The construction of a transgene requires the assembly of a few main parts. The transgene must contain a promoter, which is a regulatory sequence that will determine where and when the transgene is active, an exon, a protein coding sequence, usually derived from the cDNA for the protein of interest, and a stop sequence. The way that all these parts are typically combined is in a bacterial plasmid and the coding sequences are typically chosen from previous transgenes with previously known function.[1]

History

The idea of shaping an organism to fit a specific need isn’t a new science, selective breeding of animals and plants started before recorded history. However, until the late 1900s farmers and scientist could breed new strains of a plant or organism only from closely related species, because the DNA had to be compatible for offspring to be able to reproduce another generation.

In the 1970 and 1980s, scientists passed this hurdle by inventing procedures for combining the DNA of two vastly different species. The organisms produced by these procedures were termed transgenic. genome. Transgenesis will therefore not only change all the somatic cells, but also the germ cells so when the organisms reproduces, the transgenes are passed down to the offspring. Transgenes alter the genome by blocking the function of a host gene; they can either replace the host gene with one that codes for a different protein, or introduce an additional gene.

In 1978, yeast cells were the first organisms to undergo transgenes. Mouse cells were first transformed in 1979 followed by mouse embryos in 1980. Most of the very first transmutations were performed by microinjection of DNA directly into cells. Scientist however were able to develop other methods to perform the transformations, such as incorporating transgenes into retroviruses and then infecting cells, using electroinfusion which takes advantage of an electric current to pass foreign DNA through the cell wall, biolistics which is the procedure of shooting DNA bullets into cells, and also delivering DNA into the egg that has just been fertilized.[2]

The first transgenic animals were only intended for genetic research to study a specific genes function, and by 2003 thousands of genes had been studied. Today transgenic organisms serve a great purpose to all kinds of research and even for agricultural purposes. A large variety of transgenic plants have been designed for agriculture to produce genetically modified foods

Current Use: Mice

Genetically modified mice are the most common animal model for transgenic research.[3] Transgenic mice are currently being utilized to study a variety of diseases including cancer, obesity, heart disease, arthritis, anxiety, and Parkinson’s disease.[4] The two most common types of genetically modified mice are knockout mice and oncomice. Knockout mice are a type of mouse model that uses transgenic insertion to disrupt an existing gene’s expression. In order to create knockout mice, a transgene with the desired sequence is inserted into an isolated mouse blastocyst using electroporation. Then, homologous recombination occurs naturally within some cells, replacing the gene of interest with the designed transgene. Through this process, researchers were able to demonstrate that a transgene can be integrated into the genome of an animal, serve a specific function within the cell, and be passed down to future generations.[5]

Oncomice are another genetically modified mouse species created by inserting transgenes that increase the animal’s vulnerability to cancer. Cancer researchers utilize oncomice to study the profiles of different cancers in order to apply this knowledge to human studies.[5]

Current Use: Drosophila

Multiple studies have be conducted concerning transgenesis in P element, Cre-loxP, and ΦC31 insertion. The most practiced method used thus far to insert transgenes into the Drosophila genome utilizes P elements. The transposable P elements, also known as transposons, are segments of bacterial DNA that are translocated into the genome, without the presence of a complementary sequence in the host’s genome. P elements are administered in pairs of two, which flank the DNA insertion region of interest. Additionally, P elements often consist of two plasmid components, one known as the P element transposase and the other, the P transposon backbone. The transposase plasmid portion drives the transposition of the P transposon backbone, containing the transgene of interest and often a marker, between the two terminal sites of the transposon. Success of this insertion results in the nonreversible addition of the transgene of interest into the genome. While this method has been proven effective, the insertion sites of the P elements are often uncontrollable, resulting in an unfavorable, random insertion of the transgene into the Drosophila genome.[6]

To improve the location and precision of the transgenic process, an enzyme known as Cre has been introduced. Cre has proven to be a key element in a process known as recombination-mediated cassette exchange (RMCE). While it has shown to have a lower efficiency of transgenic transformation than the P element transposases, Cre greatly lessens the labor-intensive abundance of balancing random P insertions. Cre aids in the targeted transgenesis of the DNA gene segment of interest, as it supports the mapping of the transgene insertion sites, known as loxP sites. These sites, unlike P elements, can be specifically inserted to flank a chromosomal segment of interest, aiding in targeted transgenesis. The Cre transposase is important in the catalytic cleavage of the base pairs present at the carefully positioned loxP sites, permitting more specific insertions of the transgenic donor plasmid of interest.[7]

To overcome the limitations and low yields that transposon-mediated and Cre-loxP transformation methods produce, the bacteriophage ΦC31 has recently been utilized. Recent breakthrough studies involve the microinjection of the bacteriophage ΦC31 integrase, which shows improved transgene insertion of large DNA fragments that are unable to be transposed by P elements alone. This method involves the recombination between an attachment (attP) site in the phage and an attachment site in the bacterial host genome (attB). Compared to usual P element transgene insertion methods, ΦC31 integrates the entire transgene vector, including bacterial sequences and antibiotic resistance genes. Unfortunately, the presence of these additional insertions has been found to affect the level and reproducibility of transgene expression.

Future Potential

The study of application of transgenes is a rapidly growing area of transplantation into humans, and the production of pharmaceutical products products such as insulin, growth hormone, and blood anti-clotting factors from the milk of transgenic cows.

There are currently five thousand known genetic diseases, and the potential to treat these diseases using transgenic animals is, perhaps, one of the most promising applications of transgenes. There is a potential to use human gene therapy to replace a mutated gene with an unmutated copy of a transgene in order to treat the genetic disorder. This can be done through the use of cre-lox or knock out. Moreover, genetic disorders are being studied through the use of transgenic mice, pigs, rabbits, and rats. More recently, scientists have also begun using transgenic goats to study genetic disorders related to fertility[9]

Furthermore, transgenes for may soon be used for xenotransplantation from pig organs. Through the study of xeno-organ rejection, it was found that an acute rejection of the transplanted organ occurs upon the organs contact with blood from the recipient due to the recognition of foreign antibodies on endothelial cells of the transplanted organ. Scientists have identified the antigen in pigs that causes this reaction, and therefore, are able to transplant the organ without immediate rejection by removal of the antigen. However, the antigen begins to be expressed later on, and rejection occurs. Therefore, further research is being conducted.

Transgenes are also being used by manufactures to produce goods such as milk with a high levels of proteins, silk from the milk of goats, and proteins that contain enzymes that increase the rate of industrial reactions [9]. Further applications of transgenes include agricultural applications that aim to selectively breeding of animals for particular traits and animals that are resistant to diseases. With further research in the field, the applications of transgenes can continue to grow rapidly and become a crucial part of microbiology.

Ethical Controversy

Transgene use in humans is currently fraught with issues. Transformation of genes into human cells has not been perfected yet. The most famous example of this involved certain patients being treated for X-linked severe combined immunodeficiency (X-SCID) developing T-cell leukemia.[10] This was attributed to the close proximity of the inserted gene to the LMO2 promoter, which controls the transcription of the LMO2 proto-oncogene.[11] There are other issues involving ethics. In common with most forms of genetic engineering, the use of transgenes for purposes other than to correct life-threatening genetic abnormalities is a major bioethical issue.

See also

References

  1. ^ [1], A. J. Clark, A. L. Archibald, M. McClenaghan, J. P. Simons, R. Wallace and C. B. A. Whitelaw Philosophical Transactions: Biological Sciences , Vol. 339, No. 1288.
  2. ^ [2], Bryan D. Ness. (Feb 2004) Transgenic Organisms. Encyclopedia of Genetics Reference Edition. ,
  3. ^ [3], "Background: Cloned and Genetically Modified Animals". Center for Genetics and Society. April 14, 2005.
  4. ^ [4]
  5. ^ a b Genetically modified mouse#cite note-8
  6. ^ Venken, K. J. T., Bellen, H. J. (2007). Transgenesis upgrades for Drosophila melanogaster. Development Vol 124. Pgs 3571-3584. Doi:10.1242/dev.005686.
  7. ^ Oberstein, A., Pare, A., Kaplan, L., Small, S. (2005). Site-specific transgenesis by Cre-mediated recombination in Drosophila. Nature Methods. Vol 2. Pgs. 583-585. Doi:10.1038/nmeth775.]
  8. ^ Houdebine, L.-M. (2005), Use of Transgenic Animals to Improve Human Health and Animal Production. Reproduction in Domestic Animals, 40: 269–281. doi: 10.1111/j.1439-0531.2005.00596.x
  9. ^ Kues WA, Niemann H, 2004: The contribution of farm animals to human health. Trends Biotechnol 22, 286–294
  10. ^ Woods, N.-B., Bottero, V., Schmidt, M., von Kalle, C. & Verma, I. M. Nature 440, 1123 (2006) DOI: 10.1038/4401123a
  11. ^ Hacein-Bey-Abina, S. et al. Science 17 October 2003: Vol. 302 no. 5644 pp. 415-419 DOI: 10.1126/science.1088547

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