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Focus Assay

By Levi Clancy for Student Reader on

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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 CellsPrimary 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 FocusesA 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.