The parental ASLV vectors are doubly defective in mammalian cells: the viruses cannot efficiently enter mammalian cells; if this barrier is overcome, mammalian cells do not release infectious ASLV particles. We have resolved the first of these two problems and are now working on the second. Basically, the problem with entry is that mammalian cells lack functional receptors for any of the standard ASLV envelope glycoproteins. This limitation can be overcome in two ways. First, it is possible to introduce a cloned ASLV receptor into mammalian cells, which can then be infected by RCAS vectors having the cognate envelope glycoprotein (Bates et al., 1993). Second, it is also possible to derive RCAS vectors that use the envelope glycoproteins normally found in murine retroviruses. These modified RCAS vectors will replicate in appropriate chicken cell lines, and can efficiently infect (but will not replicate in) mammalian cells that express the appropriate murine leukemia virus (MLV) receptors.
Although the fact that ASLVs cannot replicate in mammalian cells is not always useful, in some experiments the replication-defective nature of the vector is quite helpful. The defect is absolute; there are no problems with recombination with a helper virus when the vector stock is derived, nor are there problems with the vector recombining with or being mobilized by endogenous murine viruses, either in cultured mouse cells or in the animals themselves.
There are two different systems that can be used to introduce the RCAS vectors into mice. First, RCAS vectors with MLV envelopes can be used. Vectors of this type can be used with any of the available mouse strains or mouse mutants. The second system involves transgenic mice that express the tva receptor. The advantage of this system is that the distribution of the receptor can be controlled by the experimentalist. We have prepared two sets of transgenic tva mice. The first has the tva cDNA linked to a beta-actin promoter and expresses the tva receptor in all the tissues we tested. The second has the tva receptor linked to the alpha-actin promoter and expresses tva primarily in striated muscle. Other laboratories have produced mouse lines that express tva under a variety of tissue-specific promoters. This system makes it possible to direct an RCAS vector to a specific cell type or tissue type. Because it is both slow and expensive to prepare a new transgenic mouse line, we often suggest that people try the beta-actin tva mouse strain first. This strain can be used for preliminary experiments; if the preliminary experiments look promising, it is easier to justify the work of preparing a new transgenic line. The beta-actin tva line is also a good source of primary cells/cell lines that can be efficiently infected in culture. The original strains of tva transgenic mice were Black 6 backcross animals; we are now characterizing new transgenic lines prepared with pure Black 6 animals and are doing additional backcrosses with the original lines.
When we infect mice (or mouse blastocysts) with RCAS vectors, we usually use infected avian cells rather than a viral stock. The infected cells can be delivered in a small volume, and the subsequent infection of the mouse is more efficient if virally infected cells are used rather than a virus stock.