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Retrovirus Vectors - Rectroviruses have single stranded RNA genomes which are reverse transcribed by reverse transcriptase to yield DNA double strand copies inside the host cells. The DNA copy integrates into the host genome to become a provirus, which causes permanent transfection of the cells. The provirus genome is transcribed and expressed; virions are formed and extruded into the medium.

Retroviral vectors have the following three features.

(1) The vector has viral sequences for replication, gene expression and packaging (ψ sequences).

(2) DNA inserts may either replace or be located in the nonessential coding region of the viral genome.

(3) The vector and the recombinant DNAs are packaged into virions and used as transducing phages.

(4) The viral proteins are usually provided by a helper virus or a provirus.

(5) DNA copies of the retrovirus genomes are used as vectors, generally as shuttle vectors.

A typical vector has the following sequences:

(1) pBR322 ori and a selectable marker,

(2) retroviral 5'-LTR and 3'-LTR (long terminal direct repeats),

(3) R, US, U3, P and Pu encoding sequences (involved in reverse transcription),

(4) ψ sequence (necessary for packaging into virions),

(5) S sequence needed for splicing to produce functional mRNA for envelope protein synthesis, and

(6) at least one unique restriction site for insertion of DNA fragment without interrupting any of the essential sequences.

The recombinant DNA is constructed and first cloned in E. coli; it is then isolated and introduced into animal cells where the entire vector, except the E. coli plasmid sequence, is transcribed. This transcript contains RNA copy of the DNA insert, and is packaged into virions, which is infective to animal cells.

Since the vector does not encode the viral capsid proteins, they has to be supplied by a helper virus or provirus. If the provirus or helper virus lacks the ψ sequence, its genome will not be packed into virions. As a result, all the virions recovered from the medium contain the recombinant DNA.

The recombinant DNA so recovered can be used in further experiments. The recombinant DNA can integrate as provirus into the host genome; the transfected cells can be easily selected either as piled up colonies of cells (if the recombinant DNA retains an oncogene) or due to the expression of an E. coli gene, e.g., gene neo conferring resistance to the drug G-418.
 

 
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