TRANSFECTION

The introduction of foreign DNA into eukaryotic cells is called transfection. And the eukaryotic organism which has up the foreign DNA is referred to as transgenic.

Animal cells, or plant cells with their walls removed (protoplasts), can take up foreign chromosomes or DNA directly from the environment with a very low efficiency. It is done usually in the presence of calcium phosphate or DEAE dextran, which raises the usual low transformation frequencies to 1-5%.
Calcium phosphate precipitates are used which are obtained by adding a DNA /calcium phosphate solution to an isotonic buffered phosphate solution. The precipitates which from after approximately 30 minutes are used to transfer suitable tissue culture cells.

Directly injecting the DNA improves the efficiency. For example transgenic mice are now routinely prepared by injecting DNA into either Oocytes or one or two called embryos obtained from female mice after appropriate hormonal treatment. After injection of about 2 picoliters (2-10¯12) of cloned DNA the cells are re-implanted into the uterus of receptive female hosts.

In 1988, a transgenic mouse prone to camera was the first genetically engineered animal to be patented. This mouse provides an excelled model for studying cancer.

Transgenic sheep have been produced that express the gene for a human clothing factor.
Transfection can also be medicated by retro viruses (RNA viruses containing the gene for reverse transcriptase) for example a retro viral vector infected and repaired human white blood cells lacking the enzymes adenosine deaminase. A retro virus responsible for form of leukaemia in rodents, the Maloney murine leukaemia virus, was engineered so that all the virus genes were removed and replaced with antibiotic market (neomycin resistance) and the human adenosine deaminase gene. The virus binds to the cell surface and is taken to the cells, its RNA is converted to DNA by reverse transcription and the DNA is incorporated in to one of the cells chromosomes. It is not possible for this highly modified virus to attack and damage the cells unless a helper virus is added. Unlike the SV40 virus, the modified Maloney viruses cannot irritated a successful infection without the helpers because vital genes have been removed.

Chu and Sharpe (1981) observe red that tranfection are more efficient when cell are grown in suspension rather than monolayer cultures.

In 1982, chenetal recommended shock treatment with 60-70% polyethylene glycerol.
These authors also pointed out that the nature and the physiological state of the cells used for trasfection may be an important variable.

Wigler et al in 1979 demonstrated that the calcium phosphate technique also can be used to transfect non selectable genes. For example a selectable marker such as thymidine kinase and non select able gene, which may be physical unlinked, are co transferred, i.e. transfected simultaneously up to 90% of the thymidine kinase positive transform ants may contains the non selectable gene. The ratio of non selectable and selectable maker DNA for transfection is usually 1000:1.

TECHNIQUES INVOLVED IN TRANSFER OF RECOMBINANT DNA IN THE EUKARYOTIC CELLS.
Some of the recent methods are,
1) Electroporation
2) Liposome
3) Biolistic transfer

ELECTROPORATION

A physical procedure of the introduction of nucleic acid into mammalian cells known as electroporation vitalises short electric pulses of a certain field strength which alter the permeability of membranes in such a way that DNA molecular can enter the cell. This method recently permitted the introduction of DNA

DNA & RNA molecules also can be incorporate into phospholipid vesicles, which usually contain phosphatidyl serine and cholesterol. These vesicles known as leptosomes are generated by suspending lipids and nucleic acids in ether and subjecting the mixture to ultra sonic treatments which converts the micelles into liposome containing DNA molecules. The DNA is introduced into by fusing the liposome with plasma membrane.

ADVANTAGES

These prevent the DNA against attack by nucleases.
They are comparatively non toxic and allow surprisingly high transformation yield.
Efficiency of transfect ion by this method is high compared to other method to gene transfer.

BIOLISTIC TRANSFER

This technique is developed to deliver foreign DNA into mitochondria and chloroplast. These have proven different targets for genetic engineering because they have double membrane walls that have not proven amenable to delivery recombinant DNA.

This method (biological ballistics) involves literal shooting of recombinant DNA coated on tungsten micro projectiles into this organelle.

Schaffer (1980) has devised a method which allows a direct and efficient transfer of DNA between bacterial and mammalian cells.

The advantage of this technique is that SV40 DNA and other vector DNAs do not have to be isolated and purified from bacteria.

STEPS INVOLVED

1) Bacteria containing the recombinant plasmid are first converted into protoplasts by treatments with polyethylene glycol.
Protoplasts + Eukaryotic cells. {Polyethylene glycol treatment}
(Fusion)
2) A careful observation of optimal fusion condition and the use of suitable bacterial strains stimulates with chloramphmiol is said to result in the transfer of SV40 and polymer vectors which is our efficient as that observed in viral infection. This method is approximately ten to twenty times more efficient than the calcium phosphate method.
3) Another important technique Employs glass capillaries to microinject nucleic acid molecules directly in to the nuclei of tissues culture cells.

ADVANTAGES

Transformation frequencies reach almost 100%, the use of carries DNA becomes unnecessary transform ants are stable and the amount of injected DNA can be exactly controlled. Obviously the number of cells that can be injected is limited.

To overcome this limitation, the techniques widely used in conjunction with other sensitive times detection system such as immune fluorescence, radio immune assays and auto radiography.

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