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To avoid recombination with any of the closely related endogenous retroviruses commonly found in chickens, we recommend the use of cells derived from the EV-0 strain of white leghorns. This strain is maintained at the Regional Poultry Research Laboratory (RPRL) in East Lansing, Michigan (telephone 517-337-6828; fax 517-337-6776). Fertilized eggs can be obtained from the RPRL for a nominal charge. In many cases, it is convenient to use the permanent EV-0-derived chicken cell line DF-1. DF-1 cells were prepared by Doug Foster (University of Minnesota) and can be obtained from the American Type Culture Collection (ATCC; catalog #CRL-12203). Note that this cell line is not listed in the ATCC website, but is available through telephone orders (800-638-6597 or 703-365-2700).

We have tried a number of transfection protocols. Most will work; the standard calcium phosphate protocol is efficient and reliable. Start with 5-10 micrograms of purified RCAS plasmid DNA. Originally, we used cesium chloride-banded DNA; for most purposes, we now purify the DNA by using Qiagen columns. (A detailed transfection protocol will be provided soon at this website.)

The virus is unstable; treat it gently. Freshly harvested virus can be stored for a day or two at 4°C; for longer periods, freeze at -70°C. We usually prepare fresh virus by transfection rather than by passaging a virus stock. All retroviruses are genetically unstable; therefore, if the viral stock is passaged, the inserts will be lost. This is not (usually) a problem if the vector carries a gene for a nontoxic protein and the vector stock has been freshly prepared by transfection.

In some cases, it is convenient to use cocultivation to transfer virus from one cell to another. This is a particularly useful and efficient approach if the target cell is a mammalian cell in which the RCAS viruses will not replicate. The avian cells that were used to produce the virus can be removed from the culture if the target (mammalian) cells have a selectable marker or a cell surface antigen that can be used to distinguish them from the avian cells. Alternatively, the avian producer cells can be treated with mitomycin C. This blocks further cell division, but the treated cells continue to produce virus.