Introduction
of rDNA into host cells
The next step after a recombinant molecule has been generated
is to introduce it into a suitable host. There are many methods to introduce
recombinant vectors and these are dependant on several factors such as the
vector type and host cell. Some commonly used procedures are discussed below.
Transformation:
In rDNA technology, the most common method to introduce rDNA
into living cells is called
transformation. In this procedure, bacterial cells take up DNA from the
surrounding environment. Many host cell organisms such as E. coli, yeast and mammalian cells do not readily take up foreign
DNA and have to be chemically treated to become competent to do so. In 1970,
Mandel and Higa found that E. coli
cells become markedly competent to take up external DNA when suspended briefly
in cold calcium chloride solution.
Transfection:
Another method to transfer rDNA into host cells involves mixing
the foreign DNA with charged
substances like calcium phosphate, cationic liposomes or DEAE dextran and
overlaying on recipient host cells. Host cells take up the DNA in a process called
transfection.
Electroporation:
An electric current is used to create transient microscopic
pores in the recipient host cell
membrane allowing rDNA to enter.
Microinjection:
Exogenous DNA can also be introduced directly into animal and
plant cells without the use of
eukaryotic vectors. In the procedure of microinjection, foreign DNA is directly
injected into recipient cells using a fine microsyringe under a phase contrast
microscope to aid vision.
Biolistics: A
remarkable method that has been developed to introduce foreign DNA into mainly plant cells is by using a gene or
particle gun. Microscopic particles of gold or tungsten are coated with the DNA
of interest and bombarded onto cells with a device much like a particle gun.
Hence the term biolistics is used.
Another method of introducing foreign genes is by the natural genetic engineer Agrobacterium tumefaciens.
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