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

 
     
 

Animal viruses
A variety of animal viruses are known, for example, simian virus 40 (SV40), adinovirus, retrovirus (ssRNA), vaccinia virus, etc. They can increase the efficiency of animal cell transformation if they are used as vector. A special feature of viruses is that they contain the strong promoters which can possibly bring about expression of inserted DNA fragment of the viruses. The most commonly used virus is SV40 which contains a circular DNA of about 5.2 Kb. In addition to ori region, the DNA contains early genes and late genes. Early genes are required for DNA replication, whereas late genes encode coat protein of the virus.

Like phage λ, the amount of DNA to be packed into virus capsid is limited. The genome has 3 identified non-essential regions; hence the use of non-defective viral vectors is limited. The defective virus vectors have missing genes whose infection process has to be helped by viral DNA constituting functional copies of missing genes. If the cos cells (the host monkey cells) are used as host, they can be transformed by a plasmid carrying the early region of SV40 with a defective replication origin, cos cells express wild type large T antigen and can support the growth of SV40. Recombinants containing foreign DNA inserted into early region can be propagated without the need of helper virus (Glover, 1984). In this way, the recombinant and helper can be separately recovered from the host.

 

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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