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Gene Transfer Techniques: Biolistics, Bacterial and Viral TransformationBY: Hannibal Musarurwa | Category: Biotech-Research | Submitted: 2010-08-05 17:20:24
Article Summary: "Genetic engineering is largely depended on the gene transfer techniques. This paper gives a brief description on the most used methods in biotechnology laboratories. The techniques vary from biolistics, bacterial and viral transformation..."
Since the discovery of the genetic code, the world of science has benefited immensely through subsequent innovations that blossomed thereafter. One of the major driving forces behind the success of gene manipulation is the ability to transfer nucleotides from one organism to the other across taxa, or horizontal gene transfer. This presented us with an interesting possibility to transfer traits between species. This phenomenon is now referred to as genetic engineering or genetic modification. Naturally such transfers are usually either pathogenic (as in the case of Agrobacterium infection and plant viruses) or result in the formation of new species as the case when we have hybridization of species. Today there are a number of protocols that are used to facilitate gene transfer. Below is a brief discussion of some of the methods used in most laboratories all over the world.
This is the most common method for introducing foreign DNA into a plant cell. Gold or tungsten particles are coated with DNA prior to propulsion into a mass of callus cells using a gadget called the gene gun, designed by John Sanford at Cornell University in 1987. Usually the genetic material will be carrying a marker gene that will be used to assess success of transformation. The beauty of using this technique is that it can be used different kinds of cells since it is not governed by the biochemistry of the cell membrane. Thus it can be used for both plant and animal cell transformation. However, the downside is that in most cases more than one gene is introduced and this may result in gene silencing. There is also the use of callus material which may sometimes fail to readily regenerate into plantlets.
This method exploits the ability of bacteria to transfer DNA into plants they are attacking. The most commonly used is Agrobacterium tumafaciens. Infection of some dicotyledonous plants result in the formation of cancer like cells and the disease is call gall sickness. Upon infection, the bacteria transfers its tumor inducing plasmid into to host plant and it is integrated by the host plant resulting in the tumor growth. In biotechnology Agrobacterium strains used are not capable of inducing tumors and are also equipped with another plasmid that will carry the gene to be transferred. Insertion of the second plasmid into Agrobacterium is achieved either by using electroporation or heat shock method. Electroporation also known as electropermeabilization is basically giving the Agrobacterium cells an electric shock to increase cell membrane permeability and at the same time introducing the plasmid solution into the cuvette. Transformed cell are then plated out on selection medium prior to carrying out a colony PCR on the putative transformants to confirm presence of the desired gene. Positive cells are then grown in liquid media prior to incubation of plant tissue to be transformed. With heat shock method, the cell membrane is opened up by incubating the cells on ice on ice to chill. These followed by a rapid transfer to 37˚C water bath, addition of plasmid and lastly incubation on ice to allow the cells to chill. From here the procedure is the same as for electroporation.
Virus Induced Transformation
In many ways this method is similar to the Agrobacterium transformation. Here the gene is introduced into the plant using a viral vector. Similar to the gene gun method, virus induced transformation has a high likelihood of inserting more than one copy of the gene, a result which is not always favorable and the construction of viral vectors is usually difficult. Method of choice is largely depended on the goal of the researcher material to be transformed, but of all the methods and protocol s around, it is Agrobacterium transformation that most biologists are moving towards for plant genetic modification
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