The focus assay determines the ability of a virus to induce oncogenic transformation.
The virus is applied to a cell culture, which is spread onto a culture dish. Oncogenically transformed cells will replicate faster and under more difficult conditions; a focus will form, a colony of piled-up cells, that can be distinguished from the surrounded, un-transformed cells. The focus assay was used to ascertain the oncogenic potential of mouse polyoma viruses, simina virus 40 (found in African green monkeys) and human adenoviruses.
- Infect cultured rat fibroblasts with Py, SV40, or Ad2. These are used because they are easy to culture and will adhere to the bottom of the culture dish.
- Culture for 2 weeks.
- Normal fibroblasts will stop growing at a certain density, but transformed fibroblasts will form colonies (focuses) that are highly visible against this normal background.
|Primary Cells||Primary cells are prepared from embryonic tissues. They generally require attachment to a substratum to grow, such as the bottom of a culture dish. Fibroblasts are easiest to culture in medium containing ~10% bovine serum. The attach to bottom of culture dish and flatten out.|
|Normal vs Transformed||>Normal fibroblasts stop growing when available surface is filled. This is a property called contact inhibition. When primary rat kidney cells are infected with polyoma virus, they are not killed because viral replication does not take place. A rare cell (up to 1/100 depending on moi) becomes oncogenically tranformed. These cells form a focus of tranformed cells which pile up on top of each other. All cells in focus are descendant from a single transformed cell. Oncogenically transformed cells can be grown in culture. They will often proliferate in medium with a much lower concentration of serum (reduced dependence on serum growth factors). They do not flatten out like normal fibroblats, but tend to pile up on top of each other.|
|Assay for Focuses||A focus can be readily observed in background of normal fibroblasts that are contact inhibited. The cells from a focus can be recovred, cultured, and studied. They are said to be oncogenically transformed because the form tumors when injected into a rat.|