Production of high-titer RCAS retrovirus

RCAS (B/P) is a replication-competent avian retrovirus engineered by Hughes et al. (J Virol 61:3004-3012, 1987) and is referred to in this chapter as RCAS for simplicity. The RCAS retrovirus has been widely used as a vehicle for stable transduction of a gene into cells both in the developing chick embryo and tissue/cell culture. It can be used for both gain- and loss-function experiments. The ability of this virus to spread among proliferating cells makes it possible to achieve widespread gene transduction in the developing retina. The transduction efficiency of RCAS is highly depending on the titer of the viral stock, particularly for experiments involving solid tissues such as the developing retina. Here, we describe the procedure that we have used for 15 years to generate RCAS viral stocks with a titer of 1-5 × 10(8) pfu/ml.