Wild-type retrovirus test

For testing the presence of a wild-type retrovirus expressing the lacZ gene (see Note 27) Stain with X-gal when the 2nd batch of 3T3 cells become confluent. If the test supernatant contains only replication defective virus, there should be no /acZ-positive cells in the 2nd batch of 3T3 cells. 

If the titer of wild-type retrovirus in the test supernatant is low, very few 3T3 cells will become infected. Continued growth of the cultures allows time for the wild-type retrovirus to infect other 3T3 cells in the dish increasing the likelihood of detecting its presence. Low levels of polybrene in the media help the viral infection of neighboring cells. 

If the test supernatant contained wild-type retrovirus, then the 3T3 cultures now produce the wild-type retrovirus themselves after several days. Thus, the supernatant from the 3T3 cells is capable of infecting a naive set of 3T3 cells. 

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