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  Section: General Biotechnology / Genes & Genetic Engineering
 
 
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Techniques of Genetic Engineering

 
     
 

Direct transformation
Like plant cells, the mammalian cells can be transformed by the foreign DNA fragments. For transforming the mammalian cells, it is necessary to precipitate the DNA with calcium phosphate and mix the cells to be transformed. DNA molecule passes through cell membrane and integrates randomly with mammalian chromosome. Using this technique selective marker can be linked up with DNA fragment to be cloned and can be introduced into mammalian cells. The transformed cells are, thereafter, separated from cell line after plating them on selective medium. Many techniques are used to recover the selectable marker gene from the biochemically transformed cell line. Following these biochemical processes. Perucho et al. (1980) successfully isolated chicken thyamidine gene (tk gene) by ligating with pBR322 and transforming the tk' (deficient of thyamidine gene) mouse.

 

Content

Gene cloning in prokaryotes

 

Isolation of DNA to be cloned 

 

Insertion of DNA fragment into vector 

 

 

Use of restriction Linkers

 

 

Use of homopolymer tails

 

Transfer of recombinant DNA into bacterial cells

 

Selection of clones

 

 

Colony hybridization techniques

 

 

In vitro translation technique

 

 

Immunological tests

 

 

Blotting Techniques

 

Recovery of cells

 

Expression of cloned DNA

 

 

Shine-Dalgano sequence

 

 

Expression vectors

Gene cloning in eukaryotes

 

Plant cells

 

 

Yeasts

 

 

Filamentous fungi

 

 

Agrobacterium plasmids

 

 

Plant cell transformation

 

 

Plant cell transformation by ultrasonication

 

 

Liposome mediated gene transfer

 

Animal cell  

 

 

Animal viruses

 

 

Electroporation

 

 

Particle bombardment

 

 

Microinjection

 

 

Direct transformation

Site directed mutagenesis

 

Methods of mutagenesis

 
     
 
 
     



     
 
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