Production and design of more effective avian replication-incompetent retroviral vectors

Retroviral vectors have been invaluable tools for studies of development in vertebrates. Their use has been somewhat constrained, however, by the low viral titers typically obtained with replication-incompetent vectors, parti cularly of the avian type. We have addressed this problem in several ways. We optimized the transient production of avian replication-incompetent viru ses in a series of cell lines. One of the optimal cell lines was the mammal ian line 293T, which was surprising in light of previous reports that avian viral replication was not supported by mammalian cells. We also greatly in creased the efficiency of viral infection. Pseudotyping with the vesicular stomatitus virus G (VSV-G) protein led to an over 350-fold increase in the efficiency of infection in ovo relative to infection with virus particles b earing an avian retroviral envelope protein. To further increase the utilit y of the system, we developed new Rous sarcoma virus (RSV)-based replicatio n-incompetent vectors, designed to express a histochemical marker gene, hum an placental alkaline phosphatase, as well as an additional gene. These mod ified retroviral vectors and the VSV-G pseudotyping technique constitute si gnificant improvements that allow for expanded use of avian replication-inc ompetent viral vectors in ovo, (C) 1999 Academic Press.