Agricultural Biotechnology Strategies for National Competitiveness (1987) / Chapter Skim
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Appendix: Gene Transfer Methods Applicable to Agricultural Organisms
Appendix: Gene Transfer Methods Applicable to Agricultural Organisms
Pages 149-192
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From page 149... ...
The major differences between traditional agricultural breeding and molecular biological methods of gene transfer lie neither in aims nor in processes, but rather in speed, precision, reliability, and scope. When traditional, or classical, breeders cross two sexually reproducing plants or animals, they mix tens of thousands of genes in the hope of obtaining progeny with the desired trait or traits.
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From page 150... ...
Scientists learned in the 1950s and 1960s to exploit these mechanisms to study gene regulation in bacteria and in
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From page 151... ...
The goals of gene transfer experiments with other organisms are the same as those of earlier work with bacteria to study gene regulation and to obtain stable inheritance and expression of new characteristics. The difference is that these other organisms are more complicated biological entities than are bacteria.
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From page 152... ...
Special vectors can improve the efficiency of gene transfer. Foreign genes attached to the vector will be carried by it into the host cell.
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From page 153... ...
The final state of foreign genes inside the host cell is also important. Genes can be maintained on vectors that are independent, self-replicating "minichromosomes," or they can be integrated into the larger chromosomes of the host cell.
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From page 154... ...
The transferred genes can also be reisolated from the anneal or plant cells, put back into bacteria, and grown in quantity again for further use. DIRECT DNA UPTAKE The earliest and still most widely used method for introducing DNA into animal cells grown in culture in the laboratory is direct uptake- of DNA from the surrounding culture medium.
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From page 155... ...
Direct DNA uptake thus quickly reveals the function of newly isolated or engineered genes during this period of "transient expression." For long-term studies the genes must integrate into the cellos own chromosomes, or be carried ~ by the uptake of new chromosomes, to ensure that they are stably inherited. Integration occurs at a high frequency after direct DNA uptake into animal cells because so many copies of the foreign genes have been introduced.
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From page 156... ...
Direct uptake procedures simply place foreign genes inside the cell; vectors can help to integrate the genes into the cell's chromosomes or stably maintain the genes within the cell on the vector's minichromosome (see the section on Vector-Mediated Gene Transfer)
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From page 157... ...
As with chemical or electrical uptake methods, foreign genes can be in the form of isolated molecules or attached to vectors. A disadvantage compared to direct uptake is that relatively few individual cells can be injected; however, the frequency of successful incorporation of DNA per injected cell is higher.
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From page 158... ...
by microinjection. The same foreign gene for growth hormone used to produce transgenic mice was used for these other species.
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From page 159... ...
Plants Microinjection can be used to deliver genetic material into plant cells. Segments of DNA, whole chromosomes, and even cellular organelles such as chioroplasts, which contain their own DNA molecules, can be micro~jected by methods used for animal cells, although certain physical properties of plant cells complicate the technique.
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From page 160... ...
techniques. Attempts are being made to transform plant cells by microinjection of isolated chromosomes (Greisbach, 1983, 1987~.
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From page 161... ...
However, this problem can be surmounted by creating hybridomas by direct DNA uptake. DNA from B-celIs and myeloma cells is simultaneously introduced into recipient cells by calcium phosphate coprecipitation or by electroporation (Gamble, 1986~.
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From page 162... ...
Gene mapping is expected to play an important role in finding genes for transfer of complex traits in livestock, such as lactation, fertility, growth, and disease resistance. Plant Cells In eucaryotic cells the cytoplasm that part of the cell surrounding the nucleus-contains organelles that have their own separate DNA.
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From page 163... ...
Vectors offer many advantages: high frequency of gene transfer, transfer into specific cell types, more control over the final copy number of a transferred gene, and certain properties that make them easy to track, permit them to be stably maintained in the target cell, and enable them to express foreign genes. Vectors can, therefore, greatly improve gene transfer.
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From page 164... ...
With adenovirus-mediated transfer, one to three copies of foreign genes were transferred intact at very high frequency and maintained stably in the host cells' chromosomes. This low-copy number, stable
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From page 165... ...
The researchers made these "expression vectors" by connecting viral regulatory sequences that normally cause high-level production of proteins needed in huge quantities by the virus (e.g., coat proteins, which encase the thousands of viruses produced during infection of a cell) to genes for commercially desired proteins such as the hormone human choriogonadotropin, which is important in maintaining pregnancy.
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From page 166... ...
Therefore, when Vaccinia is used as a vector for foreign genes, these genes are expressed only if they are hooked up to vaccinia's own regulatory sequences. Among its advantages is vaccinia's ability to grow easily in cell culture.
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From page 167... ...
Second, animal cells are infected with normal vaccinia virus, and then insertion vector DNA is added to the infected cells by direct DNA uptake. Inside the cells an exchange occurs between the thymidine kinase sequences on the insertion vector and the identical (homologous)
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From page 168... ...
The HPRT gene functioned in both mouse and human cells in culture, as well as in live mice. Further experiments demonstrated efficient transfer of a rat growth hormone gene into mouse cells by retroviruses and correct expression of the gene by its own regulatory sequences (Miller et al., 1984b)
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From page 169... ...
Foreign genes transferred in by these vectors are expressed from their own regulatory sequences. Retroviral gene transfer vectors applicable to agricultural animals have been developed.
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From page 170... ...
Foreign genes cloned in baculoviruses can also be expressed from their own promoters. A bacuTovirus, high-level expression system could be used to manufacture commercially useful proteins, as bacuToviruses can be mass-produced in insect cell cultures.
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From page 171... ...
furthermore, powerful CaMV gene regulation sequences can promote high-level expression of foreign genes. In fact, CaMV promoters are being used to augment the expression of plant genes transferred via other systems, as most plants recognize these promoters even when they are cletached from the rest of CaMV.
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From page 172... ...
These researchers achieved transfer and expression of a bacterial drug resistance gene in barley protoplasts. The vector replicated rapidly within the cells, and the foreign gene, under the control of the p owe rfu]
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From page 173... ...
Viral satellites might also serve to transfer foreign genes into plants directly, in the manner described for BMV. There are several potential advantages to RNA virus vector systems for plants.
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From page 174... ...
This might cause mutations detrimental to the foreign genes or to the vector itself during its replication cycle (van VIoten-Doting et al., 1985~. Transposable Elements Transposable elements (also called "transposons")
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From page 175... ...
Researchers are trying to adapt these elements as vectors, particularly because they are so prevalent in the monocot crop corn, which has resisted most efforts to transfer genes via the most highly developed plant vector, the Ti plasmid. Engineered as vectors, transposable elements might be microinjected into corn embryos to transfer genes into the germ line, bypassing problems encountered with the introduction of DNA into cultured corn cells and the subsequent regeneration of plants.
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From page 176... ...
A strategy similar to that used for vaccinia is used to insert foreign genes within the T-DNA of the large Ti plasmid: transfer of engineered genes from a small plasmid insertion vector to the Ti plasmid by in viva homologous DNA recombination within Agrobacterium cells. The T-DNA containing the foreign genes is then transferred from the Ti plasrnid within Agrobacterium into the chromosomes of plant cells by its natural process (de Block et
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From page 177... ...
In this case two separate plasmids are placed within Agrobacterium, one containing foreign genes cloned within the T-DNA's border sequences that enable the DNA segment to move, the other providing the helper functions that catalyze movement. Again, foreign genes contained between T-DNA border sequences are transferred into the plant cell (An et al., 1985; Bevan, 1984; Hoekema et al., 1983~.
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From page 178... ...
Once inside the cell they replicate and stably maintain themselves and can express foreign genes that have been engineered into them. Furthermore, under certain conditions plasmids can transfer the foreign genes they carry into the host cells' chromosomes, where the genes can also be maintained and expressed.
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From page 179... ...
Work on other pathogenic fungi is also progressing. The systems for pathogenic fungi rely on elements of a plasmid vector developed for the laboratory model fungus Aspergitius nidulans (Yelton et al., 1984~.
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From page 180... ...
Corynebacteria have their own plasmids, into which foreign genes could be inserted, and their proteins produced by the corynebacteria could easily be secreted through bacterial cell walls. Thus, foreign proteins expressed within corynebacteria might be made reaclily available to the plant host.
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From page 181... ...
This review has described major gene transfer methods with immediate potential for agricultural research. Diverse techniques are available: direct uptake of DNA, microinjection of DNA, cell fusion, and gene delivery by an array of vectors.
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From page 182... ...
However, inserted genes are not always controlled correctly, even when their own regulatory sequences are still attached to them. For example, an inserted growth hormone gene controlled by its own promoter was not regulated correctly in transgenic mice, causing the female mice to be sterile.
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From page 183... ...
For example, a salt-tolerant cell in culture may not yield a salt-tolerant plant when regenerated. The problem of selecting traits in cell culture extends to all plant gene transfer techniques performed on cultured cells, including direct DNA uptake, DNA microinjection, cell fusion, and vector-mediated methods.
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From page 184... ...
It should also be noted that current methods are applicable only to dominant or c>dominant genes, since transfer of a recessive gene cannot change a trait within an organism unless the normal, dominant gene can be inactivated. In summary, a variety of gene transfer methods is needed to accomplish diverse goals, which include fundamental studies of gene regulation, isolation of genes whose function and location are unknown, production of ~rc~t~in.~ in loran r,,,~nit.i"e arch ;._ tion of new traits.
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From page 185... ...
1985. Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype.
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From page 186... ...
1984. Expression of foreign genes in regenerated plants and in their progeny.
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From page 187... ...
1984. Inheritance of functional foreign genes in plants.
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From page 188... ...
1982. Vaccinia virus: a selectable eukaryotic cloning and expression vector.
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From page 189... ...
1986. An insect baculovirus host-vector system for high-level expression of foreign genes.
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From page 190... ...
1986. Co-transformation of unlinked foreign genes into plants by direct gene transfer.
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From page 191... ...
1984. Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene.
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From page 192... ...
1985. Overproduction of the protein product of a nonselected foreign gene carried by an adenovirus vector.
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