General principles of transgenesis
Transgenic organisms contain foreign DNA that has been introduced using biotechnology. Foreign DNA (the transgene) is defined here as DNA from another species, or else recombinant DNA from the same species that has been manipulated in the laboratory then reintroduced. The terms transgenic organism and genetically modified organism (GMO) are generally synonymous. The process of creating transgenic organisms or cells to be come whole organisms with a permanent change to their germline has been called either transformation or transfection. (Unfortunately, both words have alternate meanings. Transformation also refers to the process of mammalian cell becoming cancerous, while transfection also refers to the process of introducing DNA into cells in culture, either bacterial or eukaryote, for a temporary use, not germ line changes.) Transgenic organisms are important research tools, and are often used when exploring a gene’s function. Transgenesis is also related to the medical practice of gene therapy, in which DNA is transferred into a patient’s cells to treat disease. Transgenic organisms are widespread in agriculture. Approximately 90% of canola, cotton, corn, soybean, and sugar beets grown in North America are transgenic. No other transgenic livestock or crops (except some squash, papaya, and alfalfa) are currently produced in North America.
To make a transgenic cell, DNA must first be transferred across the cell membrane, (and, if present, across the cell wall), without destroying the cell. In some cases, naked DNA (meaning plasmid or linear DNA that is not bound to any type of carrier) may be transferred into the cell by adding DNA to the medium and temporarily increasing the porosity of the membrane, for example by electroporation. When working with larger cells, naked DNA can also be microinjected into a cell using a specialized needle. Other methods use vectors to transport DNA across the membrane. Note that the word “vector” as used here refers to any type of carrier, and not just plasmid vectors. Vectors for transformation/transfection include vesicles made of lipids or other polymers that surround DNA; various types of particles that carry DNA on their surface; and infectious viruses and bacteria that naturally transfer their own DNA into a host cell, but which have been engineered to transfer any DNA molecule of interest. Usually the foreign DNA is a complete expression unit that includes its own cis-regulators (e.g. promoter) as well as the gene that is to be transcribed.
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When the objective of an experiment is to produce a stable (i.e. heritable) transgenic eukaryote, the foreign DNA must be incorporated into the host’s chromosomes. For this to occur, the foreign DNA must enter the host’s nucleus, and recombine with one of the host’s chromatids. In some species, the foreign DNA is inserted at a random location in a chromatid, probably wherever strand breakage and non-homologous end joining happen to occur. In other species, the foreign DNA can be targeted to a particular locus, by flanking the foreign DNA with DNA that is homologous to the host’s DNA at that locus. The foreign DNA is then incorporated into the host’s chromosomes through homologous recombination.
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Furthermore, to produce multicellular organisms in which all cells are transgenic and the transgene is stably inherited, the cell that was originally transformed must be either a gamete or must develop into tissues that produce gametes. Transgenic gametes can eventually be mated to produce homozygous, transgenic offspring. The presence of the transgene in the offspring is typically confirmed using PCR or Southern blotting, and the expression of the transgene can be measured using reverse-transcription PCR (RT-PCR), RNA blotting, and Western (protein blotting).
The rate of transcription of a transgene is highly dependent on the state of the chromatin into which it is inserted (i.e. position effects), as well as other factors. Therefore, researchers often generate several independently transformed/transfected lines with the same transgene, and then screen for the lines with the highest expression. It is also good practice to clone and sequence the transgenic locus from a newly generated transgenic organism, since errors (truncations, rearrangements, and other mutations) can be introduced during transformation/transfection.
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