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... The available RCAS vectors allow one to introduce a variety of genes into any cultured mammalian or avian cell. There are some advantages to using the RCAS system to introduce genes into cultured cells, particularly into avian cells. Because the vector replicates, it spreads rapidly to essentially every cell in the dish. If an efficiently replicating vector (such as RCASBP) is used, this usually takes about 1 week. No selectable marker is used, and there are no problems related to picking out one cell or a few cells that may not share all of the properties of the cells in the original culture. Chicken embryo fibroblasts (CEFs) are easy to prepare; experiments with CEFs can be carried out in primary cells as well as in cell lines. If you are willing to work with a continuous cell line, DF-1 is relatively easy to handle and is available through the ATCC. The RCAS vectors are available with several distinct envelopes, so that it is possible to doubly (or even triply) infect a culture. This allows one to look at the interactions of two (or more) genes in cultured cells. Similar experiments can be done in vivo (Givol et al., 1994, 1995, 1998). We have also developed RCAS vectors that use an IRES to express two proteins (see Figure 1 below).



(click on the image for a larger view)


Figure 1. DF-1 cells expressing GFP and mouse heat-stable antigen (HSA). HSA is visualized by Texas Red immunofluorescence.


There are a few obvious pitfalls in experiments of this type. If you are trying to overexpress a gene that will strongly interfere with the replication of the host cell (or the viral vector), you are likely to have problems. Basically, the expression of a gene that strongly interferes with the replication of the host cell sets up a strong selection for the viral vector. Among the variant viruses that arise, any that do not express the gene of interest will replicate much more rapidly than viruses that express the gene. This gives rise, usually quite rapidly, to virus stocks that fail to express the gene. We have been able to do some experiments with RCAS vectors expressing genes that block cell division (Givol et al., 1995, 1998), but the experiments and the cell culture have been much more difficult than usual.

Over the years, a number of laboratories have tried to use the RCAS system to block the expression of host cell genes by providing antisense transcripts. So far as we know, all of these attempts failed; therefore, we do not recommend using the RCAS vectors for expressing antisense transcripts. However, the vectors can be used to express siRNAs (Bromberg-White et al., J. Virol. 78: 4914-4916, 2004).




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