Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

The overall goal of this procedure is to perform the focus formation assay in mouse fibroblast cell lines, using a retrovirus to determine the oncogenic potential of a gene. This is accomplished by first cloning the sequence for the gene of interest into a retroviral vector. The second step is to produce retroviral particles using the viral vector and PLA e packaging cells.

Next, the retrovirus is used to infect NIH three T three mouse fibroblast cells in order to assess the transforming potential of the gene of interest. The final step is to allow the NIH three T three cells to divide for two to three weeks, followed by crystal violet staining to quantify the number of foci formed. Ultimately, the focus formation assay is used to show if overexpression of a gene of interest is sufficient to induce density independent growth in mouse fibroblast cells.

This method can help answer key questions in the field of cancer biology, such as screening for the oncogenic potential of a gene. To begin, make the viral vector as described in the text protocol. Then establish plat E cultures from frozen cells by quickly thawing plat E cells in a 37 degree Celsius water bath, and then transferring to a 15 milliliter conical tube.

Slowly add nine milliliters of PLA E medium to the 15 milliliter conical tube. Centrifuge the tube at 180 times G for five minutes, and discard the supernatant. Resuspend the cells with 10 milliliters of plat E medium, and transfer to a 10 centimeter culture dish.

Then incubate the cells in a 37 degree Celsius 5%CO2 incubator until they are between 80 and 90%Confluent. Perform cells splitting by first aspirating the medium, then wash the cells once with PBS before adding two milliliters of 0.05%tripsin EDTA and incubating for one minute at room temperature, detach the cells by finger tapping. Then add 10 milliliters of plat E medium and transfer the cell suspension to a 15 milliliter tube.

Centrifuge the tube as before and resuspend the cells with 10 milliliters of plat E medium. Next seed, 2 million cells per 10 centimeter culture dish using plat E medium without any antibiotics and leave cells to incubate overnight. The next day, transfer 300 microliters of optimum into a 1.5 milliliter micro tube.

Add 27 microliters of polyethylene amine to the prepared tube with Optum. Mix gently by finger tapping and incubate for five minutes at room temperature. Next, add nine micrograms of transfection grade plasmid DNA into the tube mixed gently by vortexing and incubate for 15 minutes at room temperature.

Then add the mixture dropwise into the plat E dish before incubating overnight. The next day aspirate the medium containing the transfection reagent and add 10 milliliters of fresh plat E medium before returning the cells to the incubator to establish NIH three T three cultures. First, quickly thaw the cells in a 37 degree Celsius water bath and transfer to a 15 milliliter tube.

Slowly add nine milliliters of NIH three, T three medium to the cells, centrifuge the tube at 180 times G for five minutes and discard the snat. Then resuspend the cells with 10 milliliters of NIH three, T three medium, and transfer to a 10 centimeter culture dish. Incubate the cells in a 37 degree Celsius 5%CO2 incubator, keeping them under confluent as the frequency of spontaneous transformation increases.

Once a culture reaches co fluency, once grown seed three times 10 to the fifth nih, three T three cells per 10 centimeter dish and incubate overnight for infection the next day. The next day, harvest the retroviral supernatant from the plat E dish by using a 10 milliliter disposable syringe, filtering it through a 0.45 micron pore size nylon membrane filter, and transferring it into a 15 milliliter tube. Add 10 milliliters of fresh plat E medium to the cells before returning them to the incubator.

Then aspirate the medium from the NIH three T three cell dish to be infected and add five milliliters of regular NI H three, T three medium, and five milliliters of virus containing filtered supernatant. Next, add poly brain to the dish. Add a concentration of six micrograms per milliliter.

Incubate the cells overnight before repeating the infection steps for a second round of infection. Following infection, replace the virus, contain medium with regular IH three T three medium. Allow the IH three T three cells to grow for two to three, replacing the medium as necessary for crystal violet staining.

Aspirate the NIH three T three medium and place the dishes on ice. Wash the dishes twice with ice cold PBS. Fix the cells with ice cold methanol for 10 minutes.

Then remove the dishes from ice and aspirate the methanol. Add three milliliters of 0.5%crystal violet solution made in 25%methanol and incubate the dishes for five minutes at room temperature. Following incubation, aspirate the crystal violet solution and carefully rinse the dish with Milli Q water until no color comes off in the rinse.

Allow the dishes to dry uncovered overnight on a bench top. Once the dishes are dry, use a ruler to measure the foci and count those that are greater than five millimeters in diameter record. The number MXD three is a basic helix loop helix leucine zipper transcription factor that is an atypical member of the MAD family, and it has been reported to be involved in carcinogenesis when compared to the negative control and the positive control.

The NIH three T three dishes where MXD three was overexpressed had significantly less foci. The data were collected from multiple experiments to determine significance and the same trends were observed from this assay. Results suggest that MXD three does not function as an oncogene Following this procedure.

Other methods like focus formation assay with gene combinations, growth in soft auger proliferation and cell cycle analysis can be performed in order to answer additional questions To assess the role of a gene of interest in oncogenesis pathogenesis.