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.