DNA Mediated Gene Transfer (Vector Mediated Gene Transfer)
- Competent host is essential for transformation with recombinant DNA.
- Transformation" a process by which a cell takes up naked DNA fragment from the environment, incorporates it into its own chromosomal DNA and finally expresses the trait controlled by the incoming DNA ".
- The tools described earlier in this chapter will result in the generation of recombinant DNA ( rDNA ) molecules in the laboratory.
- Finally, the propagation of these DNA molecules must occur inside a living system or a host.
- Many kinds of host cells, including E. coli, yeast, animal and plant cells, are available for gencuc engineering and the kind of host cell to be used mainly depends on the aim of the cloning experiment.
- For the expresssion of some eukaryotic proteins, eukaryotic cells may be the preferred hosts.
- Yeasts have been used extensively for functional expresssion of eukaryotic genes because they offer several advan tages.
- Yeasts are the simplest eukaryotic organisms and like bacteria are single - celled, genetically well - characterize, easy to grow and manipulate.
- They can be grown readily in both small culture vessels and large scale bioreactors. Plant and animal cells may also be used as hosts in gene manipulation experiments and for protein expresssion either in tissue culture or as cells in the whole organism to create genetically modified plants and animals.
- Since DNA is a hydrophilic molecule, it can not pass through membranes, so the bacterial cells must be made capable to take up DNA.
- This is done by treating them with a specific concentration of a divalent cation, such as Calcium which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
- Recombinant DNA (rDNA) can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42 ° C ( heat shock ) , and then putting them back on ice This enables the bacteria to take up the recombinant DNA .
- Transfer of DNA into eukaryotic cell is called transfection
- It is the process of gene transfer into the host cell without using a vector.
- This is possible by the following four important methods
- In this method foreign DNA is directly injected into the nucleus of animal cell or plant cell by using micro needles or micro pipettes.
- It is used in oocytes, eggs and embryo.
- Alec Jeffreys ( 1993 ) of Human Genome Centre, Michigan University U.S.A has cured mice that inherited a neuromuscu lar disease which is like muscular dystrophy of humans.
- Electroporation is the formation of temporary pores in the plasma membrane of host cells by using lysozyme or calcium chloride.
- These pores are used for introduction of foreign DNA.
- In this method certain chemicals such as polyethylene glycol ( PEG ) help foreign DNA to enter the host cell.
- Biolistic is a means of introducing DNA into cells that involves bombardment of cells with high - velocity microprojectiles coated with DNA.
- In biolistic method tungesten or gold particles, coated with foreign DNA are bombarded into target cells at a very high velocity.
- Although this method is suitable for plants yet this technique is also used to insert genes into animal that promote tissue repair into cells (particulary cancer of mouth) near wounds.
- This method failed to make an impression in treat ment of genetic disorder but made great impact in the field of vaccine development.
- After being introduced briefly to the tools in re combinant DNA let us describe the processes to create recombinant DNA .
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