Production of retroviral vectors

In this unit, the basic protocol generates stable cell lines, which produce retroviral vectors that carry selectable markers. Alternate Protocol 1 applies when the retroviral vector does not carry a selectable marker. Alternate Protocol 2 presents rapidly generating retroviral vector preparations by transient transfection. Support Protocol 1 describes construction of the retrovirus vectors. The methods for generating virus from retroviral vector plasmids rely on the use of packaging cells that synthesize all of the retroviral proteins but do not produce replication-competent virus. One packaging cell line is transiently transfected to produce virus carrying the vector RNA that can be used to infect a second packaging cell line. Virus that carries a selectable marker can be titered and viral stocks can be tested for the presence of helper virus, as described. Vectors that contain sequence for alkaline phosphatase can be assayed histochemically. In this unit, the basic protocol generates stable cell lines, which produce retroviral vectors that carry selectable markers.