Retrovirus Vectors

REctroviruses have single stranded RNA genomes which are transcribed by reverse transcriptase in a DNA double strand copy inside the host cells. THe DNA copy integrates into the host genome to become ap 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 haveb the folowing three features. (1) the vector has viral sequences for replication, gene expression and packaging sequences (2) DNA inserts may either replace or be locaed in the nonessential coding region of the viral genome. (3) The vector and the recombinant vectors 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 genome are used as vectors. generally as shuttle vectors.
A typical vector has the following (1) pBR 322 ori and a selectable marker (2) retroviral 5'LTR and 3" LTR (3) R,U5, U3, p and pu encoding sequences (involved in reverse transcription ) (4) sequences necessary for packaging into virions (5) sequences needed for splicing to produce functional mRNA for envelope protein synthesis, and (6) atleast one unique restriction site for insertion of DNA fragment without interrupting any of the essential sequences.
THe recombinant shuttle vector is constructed and first cloned in E.coli it is then isolated and tintroduced into animal cells where the entire vector, expect the E. coli plasmid dequence, is transcribed. This transcript contain RNA copy of the DNA insert, and is packaged into virions and is infective on animal cells. Since the vector does not encode the viral capsid proteins, it has to be supplied by a helper virus or provirus. If the provirus or helper virus lacks the sequence, it genome is not packed into virions. As a result, all the irions recovered from the medium contain the recombinant vectors. The recombinant vector so recovered can be used in further experiment. The recombinant vector can integrate as provirus into the host genome the transfected cells can be easily selected either as piled up colonies of cells if the vector retains an oncogene or due to the expression of an E. coli gene e.g gene neo conferring resistance to the drug G-418.