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  Section: General Cell & Molecular Biology » Recombinant DNA Technology
 
 
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Host Cells

 
     
 
Content
Recombinant DNA Technology
     ⇒ Cloning
     ⇒ Restriction Endonucleases
     ⇒ Vectors
     ⇒ Host Cells

A number of bacterial and yeast strains have been developed for recombinant DNA experiments. In order for a given plasmid to be replicated by a host cell, the cell must recognize its origin of replication site (oriC). Recombinant plasmid vectors are normally introduced into competent cells by transformation and then selected using appropriate cell culture media. For example, if the vector contains an ampR gene that encodes resistance to ampicillin, the culture media would include that antibiotic to ensure that only transformed cells will grow.

Another method for introducing recombinant DNA molecules into host cells is electroporation. In this method, a suspension of exponentially growing host cells is mixed with a solution of recombinant DNA molecules and exposed to a high electric field for a few milliseconds. The high voltage alters the structure of the membrane so that pores are temporarily formed, allowing plasmid DNA to enter the cell. This method is fast and efficient.



If bacteria are used as the host to clone eukaryotic genes, certain steps must be taken to make it possible for the bacteria to make sensible mRNAand functional proteins, since bacteria do not possess mechanisms for processing eukaryotic pre-mRNA molecules. To do this, it is necessary to isolate already processed mRNA from the donor eukaryotic cells and convert the single-stranded RNA to double-stranded DNA . Reverse transcriptase from retroviruses uses RNAtemplates for synthesizing DNA . The resulting DNA molecules, known as cDNA (for complementary DNA ), can then be used for cloning in bacteria since they posses only intron-free protein-coding genetic information.



Notes
Common hosts are E. coli and S. cerevisiae.
 
     
 
 
     



     
 
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