We use a series of replication-competent retroviral vectors based on Rous Sarcoma Virus (RSV), a member of the Avian Leukosis Virus (ALV) family, to study the role of different genes in tumor initiation and progression. RSV is the only known naturally occurring, replication-competent retrovirus that carries an oncogene, src. In the RCAS vectors, the region encoding src, which is dispensable for viral replication, has been replaced by a gateway cassette. Foreign genes inserted into this region are expressed from the viral LTR promoter via a subgenomic splice site (just as src is in RSV). RCAN vectors differ from RCAS vectors in that they lack the src splice acceptor; the gene of interest is inserted along with an internal promoter. Higher-titer viruses subsequently have been generated by replacing the RSV pol gene with the pol gene of the Bryan strain of RSV. These vectors are termed RCASBP or RCANBP. The ability of these vectors to infect non-avian cells relies on expression of the corresponding receptor, TVA, on the cell surface.
The TVA receptor is typically introduced into mammalian cells (or mice) via an inducible and/or tissue-specific promoter. Therefore, this system allows for tissue- and cell-specific targeted infection of mammalian cells through ectopic expression of the viral receptor. Alternatively, when targeted infection of mammalian cells is not required (e.g., in cell culture), infection can be achieved through the use of non-avian envelopes, such as the amphotropic envelope from murine leukemia virus. The receptor for this envelope is endogenously expressed on almost all mammalian cells. We have used the RCASBP/ RCANBP family of retroviral vectors extensively in both cultured cells and in vivo systems for studies of viral replication and for cancer modeling. Most of these studies have analyzed gain-of-function phenotypes by delivering and overexpressing a particular gene of interest.
Recently, we engineered the RCASBP vector to reduce the expression of specific genes through the delivery of short hairpin RNA sequences in the context of an endogenous microRNA (miRNA). We also engineered the RCANBP vector to control the expression of the inserted sequences using the tetracycline (tet)-regulated system. Sequences inserted into this region are transcribed from a tet-responsive element and not the viral LTR. This virus allows inserted sequences to be turned on and off, in order to determine if their expression is required for tumor initiation, maintenance, and progression. The ability to turn off expression after tumors develop helps determine whether that gene or miRNA is a good target for therapy.
Diagram of the RCAS/TVA mouse model and associated procedures.
Expression of the viral receptor TVA is driven by a tissue specific promoter. Newborn mice are injected with viral producing cells, which are cleared by the host immune system within one week. Animals are then monitored for tumor development. Cells and tumors can be isolated and established in culture for further analysis.