For successful infection, avian sarcoma leukosis virus subgroup A (ASLV-A) requires its receptor, tumor virus A (TVA), to be present on the surface of target cells. This is the basis of the RCAS-TVA gene delivery system: Mammalian cells lack the gene encoding TVA and are normally resistant to infection by ASLV; however, transgenic targeting of TVA to specific cell types or tissues in the mouse renders these cells uniquely susceptible to infection by ASLV-A-based RCAS viruses. The RCAS-TVA system is a powerful tool for effectively modeling human tumors, including pancreatic, ovarian, and breast cancers, gliomas, and melanomas. RCAS viruses can deliver cDNAs (≤2.8 kb), as well as short hairpin RNAs (shRNAs), microRNAs (miRNAs), and other noncoding RNAs. Compared with traditional transgenic and knockout mice, the RCAS-TVA system has several strengths. First, virus delivery is generally performed postnatally and results in a relatively low infection rate of target cells; the sporadic postnatal expression of the gene of interest mimics the situation in developing human tumors. Second, a single transgenic mouse line can be used to compare the consequences of specific genes on tumor development, with viruses encoding oncogenes or shRNAs targeting specific tumor suppressor genes. TVA mouse strains can also be easily combined with transgenic, knock-in, and knockout mouse models to study cooperating genetic events.
THE RCAS VIRUSRCAS stands for replication-competent avian sarcoma-leukosis virus (ASLV) long-terminal repeat (LTR) with splice acceptor. The virus enters cells via the TVA receptor, which mammalian cells do not natively express. Following entry into a mammalian cell, a cDNA copy of the viral genome is made. When the cell undergoes division, the viral cDNA copy incorporates into the mammalian host genome.The RCAS virus retains all of the necessary viral genes for replication. Therefore, in an avian cell culture, there is no need for the use of helper plasmids to generate virus as is commonly done with retroviruses that have tropism for mammalian cells. The immortalized chicken fibroblast cell line DF1 is commonly used to propagate RCAS viruses in culture (Himly et al. 1998;Schaefer-Klein et al. 1998). Chicken embryo fibroblasts may also be used. In addition, high-titer virus can be produced for several days or weeks following a single transfection of a viral plasmid into avian producer cells. (See Protocol: Generation of High-Titer RCAS Virus from DF1 Chicken Fibroblasts [Ahronian and Lewis 2014].) The virus glycoprotein binds to its receptor to gain entry into a cell. Because only avian cells naturally express the TVA receptor to the RCAS glycoprotein, in the absence of engineered TVA expression infection of mammalian cells is very limited. Inserts in the RCAS retrovirus are stable up to 2.8 kb but less stable when >2.8 kb. It is common beyond this insert size that RCAS will lose its insert, generate truncation mutants, or produce low-titer virus. The limited carrying capacity of RCAS viruses is one of the majo...