Molecular Genetics

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• A cell line is constructed that contains all the functional genes of a retrovirus (w.t.) except the sequence of nucleotides (Y sequence) needed to package the genome into viral particles. This line is called a helper cell line. Helpers may also be made by removing other parts of the retroviral genome, parts that are essential for replication. Parts (gag and env) required for packaging of RNA into particles must be retained.
• A plasmid DNA is constructed that contains a Y sequence between retroviral long terminal repeats (LTR's). Functions needed to package the retrovirus are absent. The pol gene may also be omitted since retroviral particles carry the reverse transcriptase enzyme (pol gene product) inside them.
• The plasmid is transfected into the helper cell line. The sequences between the plasmid LTR sequences are replicated. The gag and env products from the helper cell line package the product RNA. Particles are released. These particles resemble cosmids in that they can deliver their payload into target cells but will not produce infectious particles. The RNA payload of the retroviral vector can, in the cells, form defective proviral DNA and integrate it in the cells' chromosomes, thus transforming the cells with YF (your favorite) gene.

Implications and Further Information:

• Retroviral vectors are useful for efficiently delivering genes into cells of an organism.
• Theoretically, the pseudoretroviral particles are safe, incapable of initiating a retroviral infection.
• Similar gene therapy schemes have been devised using other viruses, such as adenovirus, adeno-associated virus and herpes simplex virus.