Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Overexpressing genes in cells is a important way to study gene function. Stable transfection with the retrovirus allows endogenous genes to be integrated into the host genome and be expressed continuously. We picked up single colonies of the retroviral infection and generated stable cell lines overexpressing FLAG-tagged DR3.

The cell lines compensated for the unavailability of good DR3 antibody and provided excellent tools for DR3 function study post in vitro and in vivo. We generated the DR3 overexpression cell lines by picking up a single colony of the retroviral infection. Cell lines from single colony could maintain the homogeneity and purity.

In addition, the protocol is easy and simple to handle. To begin this procedure, grow plat-A cells overnight in 60-millimeter dishes with four milliliters of DMEM. When cells reach 80 to 90%confluence, use transfection reagent to transfect them with two micrograms of the constructed plasmid as outlined in the manufacturer's manual.

After 72 hours, collect the supernatant. Using a 0.45 micrometer sterile filter, filter the media, and keep the viral suspension in the dark at four degrees Celsius. Next, grow HT29 cells overnight in a 60-millimeter dish with DMEM, incubating the cells at 37 degrees Celsius with 5%carbon dioxide.

When the cells reach 30 to 50%confluence, infect them with two milliliters of viral suspension in the presence of eight micrograms of polybrene per milliliter of viral suspension. Incubate the infected cells at 37 degrees Celsius for four to six hours. Then, aspirate the viral suspension.

Add four milliliters of fresh DMEM and return the dish to the incubator. At 24 hours post infection, remove the media from the dishes and carefully wash the cells with two milliliters of pre-warmed PBS. Add one milliliter of trypsin EDTA, and incubate the cells at 37 degrees Celsius for three minutes.

Using a microscope at 10 times magnification, observe the cells to ensure that most of them have detached. After this, add two milliliters of DMEM containing 10%FBS to stop the trypsinization, and collect the cell suspension in a 15-milliliter tube. Centrifuge at 200 times g for five minutes at room temperature, and remove the supernatant.

Re-suspend the cell pellet in 10 milliliters of DMEM and gently pipette up and down to mix. Next, dilute the cells by factors of 30, 100, and 300. Seed the cells is 150-millimeter dishes that each contain 20 milliliters of DMEM containing blasticidin at a concentration of one microgram per milliliter.

Incubate at 37 degrees Celsius with 5%carbon dioxide for approximately one to two weeks. During this period, use an inverted microscope at 10 times magnification to observe the colonies each day. Mark the well-isolated colonies on the bottom of the dish when the colonies'diameter is approximately one to two millimeters.

When the incubation period is complete, aspirate the medium and wash the cells with three milliliters of pre-warmed PBS. Add two milliliters of trypsin EDTA in a 60-millimeter dish. Use sterile forceps to pick up autoclaved sterile cloning cylinders and place them in the dish.

Then, pick up the cylinders which will now each contain approximately 30 microliters of trypsin EDTA, and gently place them over the marked colonies. Return the dish to the incubator for three minutes. After this, check the cells under a microscope to ensure that they've detached.

When the cells have lifted up, at 70 microliters of culture medium to each cylinder to inactivate the trypsin. Use a 200-microliter pipette to gently mix the cell suspension, making sure not to move the cylinder while mixing. Set out two 24-well plates, and label them plate A and plate B.Add one milliliter of DMEM to each well.

Add 30 microliters of cell suspension to each well of plate A, and add 70 microliters to the corresponding wells of plate B.Place the plates back into the incubator. When the cells in plate B are at 90%confluence, remove the media and carefully wash the cells with one milliliter of PBS. Removed the PBS completely, and add 50 microliters of 1X SDS-PAGE loading buffer to lyse the cells.

After five minutes, transfer the cell lysate from the 24-well plate to 1.5-milliliter centrifuge tubes. Boil the samples at 100 degrees Celsius for 10 minutes. Run the samples on 10%SDS gel at a constant voltage of 80 volts for 15 minutes and then at 120 volts for one hour.

Then transfer the protein to a polyvinylidene fluoride membrane by wet transfer at a constant current of 400 milliamps for two hours. Dilute the primary antibody by a factor of 5, 000 in 5%nonfat milk that is dissolved in TBST. Add the FLAG antibody to the membrane, and incubate at four degrees Celsius overnight.

Using a decoloring shaker set to 200 RPM, wash the membrane with TBST for 15 minutes, refreshing the TBST every five minutes. Then, dilute an anti-mouse secondary antibody by a factor of 10, 000 in 5%nonfat milk. Incubate the membrane with diluted secondary antibody at four degrees Celsius for five to six hours.

Wash the membrane on the decoloring shaker again as previously described. After this, use Western enhanced chemiluminescence and a gel documentation system to image the membrane and detect the FLAG expression. First, collect both HT29 and HT29-DR3 cells by trypsinization, and use an automatic cell counter to measure the cell density.

Seed the cells into the wells of 12-well plates with DMEM at a density of 30, 000 cells per well, and incubate overnight at 37 degrees Celsius with 5%carbon dioxide. The next day, add 10 nanomolar of diazonamide to each well that contains cells using 1%DMSO as a negative control, and transfer the plates to an incubator at 37 degrees Celsius with 5%carbon dioxide. After 48 hours of treatment, image the cells under a microscope at 10 times magnification.

Next, seed both HT29 and HT29-DR3 cells into the wells of 96-well plate with DMEM at a density of 3, 000 cells per well, and allow them to grow at 37 degrees Celsius overnight. The next day, add dose-escalating diazonamide concentrations as outlined in the text protocol. After 48 hours of treatment, use a luminescence-based cell viability assay kit to measure the cell viability.

Remove the 96-well plate from the incubator, and let it stand at room temperature for approximately 30 minutes. Then, add 50 microliters of assay reagents to each well, and shake the plate for two minutes at room temperature to lyse the cells. Incubate the plate at room temperature for 10 minutes, and then use a microplate reader to determine the luminescence of each well.

Characterization of HT29 cells stably expressing DR3 reveals that the clones express varying levels of DR3 while the wild-type cells do not show exogenous gene expression. Genes one and five are seen to express the highest levels of DR3, and so are chosen for further experiments. The morphology of HT29 and HT29-DR3 cells are observed after being treated for 48 hours with diazonamide.

HT29-DR3 cells display obvious apoptosis with a broken cell membrane and cell debris. However, HT29 cells show only mitotic arrest with intact cells that exhibit a round-up cell shape. The cell viability of these cell types is then determined after being treated for 48 hours with three nanomolar of diazonamide.

HT29-DR3 cells are seen to have a cell death of over 80%while the parental HT29 cells demonstrate only a slight response in the form of mitotic arrest. This indicates that the overexpression of DR3 reconstituted the diazonamide-induced apoptotic pathway in these cells. Proper serial dilutions are important to get optimum density of single clones.

It is also important to make sure every clone cylinder only contains one colony and to avoid contaminating nearby colonies. The DR3 overexpressing cell lines facilitated the biochemical study of the molecular mechanisms in vitro. We generated HT29-DR3 xenografts through subcutaneous injection of HT29-DR3 cells, and it helped elaborate the mechanisms of antimitotic agents-induced apoptosis in vivo.

This protocol can be used to study genes of interest in other cell lines. In addition, gene knockout is another way for functional study, and our protocol can be adapted to the gene knockdown systems. Thus, it is generally applicable to elucidate gene functions.

Remember to drop the virus relay to the garbage to specific containers for safety disposal.