We recently developed a melanoma mouse model based on the RCAS/TVA system. This system utilizes a viral vector, RCAS, derived from the avian leukosis virus (ALV). The receptor for subgroup A-ALV is encoded by the tv-a gene and is normally expressed in avian cells. Expression of TVA in cells that are normally resistant to infection by ALV confers susceptibility to infection by subgroup A-ALV. In avian cells, the viral vector is replication-competent and can deliver experimental genes of reasonably useful size (up to ~3-4 kb). Defective vectors, which lack the envelope gene, can deliver genes up to ~5 kb when the envelope protein is supplied in trans. High titer viral vector stocks can be generated in the DF-1 cell line, an immortalized chicken fibroblast cell line. In replicating mammalian cells that express TVA, the viral vector is capable of stably integrating into the DNA and expressing the inserted experimental gene at high levels, but the virus is replication-defective.
Therefore, the viral vectors cannot spread in the target animals and there is no interference to superinfection, so theoretically, there is no limit to the number of experimental genes that can be introduced into a TVA-expressing mammalian cell. To model human melanoma using the RCAS/TVA system, we obtained mice that express TVA from the dopachrome tautomerase (DCT) promoter, also known as tyrosinase-related protein 2 (TRP2). This promoter was chosen to drive expression of the viral receptor TVA specifically in melanocytes since this gene is expressed early in melanocyte development when the cells are mitotically active. The DCT-TVA mice were crossed to Ink4a/Arf lox mice and then intercrossed to generate DCT-TVA/Ink4a/Arf lox/lox mice. As proof of principle, subcutaneous injection of RCAS-NRASQ61R and RCAS-Cre viruses postnatally led to infection of somatic melanocytes expressing TVA (resulting in NRasQ61R expression and Ink4a/Arf loss) and resulted in tumor formation at the injection site in 36% of the mice in less than 14 weeks. The schematic of the DCT/TVA mouse model we developed and will employ in this study is shown below.
|Schematic of the DCT-TVA melanoma mouse model, RCAS vectors and expression profile in subsequent melanomas derived from DCT-TVA mice.
(A) RCAS vectors (encoding experimental genes) are transfected into avian DF-1 cells to produce high titer viral stocks. Any combination of these viruses can then be delivered to newborn DCT-TVA/Ink4a/Arf lox/lox mice or TVA expressing cells. In the case of NRASQ61R and Cre delivery, melanomas developed as early as four weeks.
(B) The RCASBP(A) vectors (designated as RCAS in the text) we used to initiate melanomas in DCT-TVA/Ink4a/Arf lox/lox mice. These vectors are Gateway cloning compatible to aid in cloning efficiency. Although there is a limit in gene size, we have successfully expressed genes up to 4kb (e.g., MET and EGFRvIII).
(C) Tumors (designated by a number) were isolated from DCT-TVA/Ink4a/Arf lox/lox mice to verify viral gene delivery. All melanomas were positive for NRASQ61R expression (N-terminal HA tag) and Ink4a/Arf loss (Cre expression results in loss of p19Arf expression). No tumors developed in mice lacking TVA (the viral receptor) or in mice that received NRASQ61R or Cre alone. Normal mouse melanocytes served as a control.