The overall goal of this experiment is to validate the use of the five FC FCU one system as a negative selection method In Entera Histolytica in 48, well plates seed E histolytica, transfect lines expressing either FCU one gene or appropriate vector control treat cells with the pro-drug five FC in cells expressing high levels of FCU one five FC will be converted into effective quantities of toxic end products. The surviving cell population is stained with C-M-F-D-A to demonstrate selective elimination of FCU U one expressing cells in the presence of five fc and to quantitatively compare the efficiency of the negative selection. This negative selection system will allow us to cure for the first time episomal plasmids from transected TBA histo litic atrophy.
Given the challenge in anti presence to conventional genetic manipulation, this method might allow us to disrupt and delete genes and then to test target VIR factors in our drug discovery program. Trophozoites can vary in how well the stock cultures thrive in culture. An error at the start can produce artifacts and therefore a critical early step is quality control of the starting culture.
Seed the inba hisa lines under study from stock tubes into T 25 flasks and maintain selection by adding appropriate antibiotic in TYIS 33. Medium incubate flasks at 37 degrees Celsius for 16 hours for cultures to reach around 80%confluence. For this procedure, always prepare a fresh 50 millimolar stock of five fluorine or FC in warm TYIS 33.
Medium vortex rigorously for several minutes to dissolve five FC completely and filter, sterilize the stock solution through a 0.22 micron filter to harvest the trophozoites. Place the flasks on ice for three to five minutes and collect the cells by centrifuging at 200 times gravity for five minutes at room temperature. Re suspend the pellet in fresh TYI medium, and count the cells in order to facilitate downstream fluorescent measurements.
Perform experiments in triplicate and use a separate 48 well plate for each strain per time point seed, 0.5 times 10 to the four cells per well in a volume of 0.5 milliliters for each test. Five FC concentration, and maintain the antibiotic selection pressure in the TYI medium. Now add the required amount of five FC stock solution followed by TYI medium to a final volume of one milliliter to each well and incubate the plates in an anaerobic bag at 37 degrees Celsius.
Prepare a two milligram per milliliter stock solution of cell tracker green C-M-F-D-A in DMSO. Then to make the working solution dilute the stock 1000 fold in serum free TYI medium at specified experimental time points. Remove the medium completely from Tropho Xite culture plates and replace with 150 microliters of serum free TYI containing C-M-F-D-A.
Continue incubation at 37 degrees Celsius for one hour, aspirate the DI solution from the wells. Finally read the plate in the spectra max M two microplate spectro fluorimeter, using an excitation wavelength of 492 nanometers and a fluorescent emission of 517 nanometers. Compare the fluorescence values for the control cells versus the test transfect.
Continue reading the plates at each time interval. Always use appropriate, positive and negative control wells in each plate. Western BLO analyses demonstrate successful generation of lya transfect expressing FCU one, the yeast cytosine deaminase, and uracil phospho rib transferase chimera.
These results are also reflected in quantitative PCR measurements. In the presence of five fluorine control cells grow normally as up to five millimolar concentration and reach confluence between 72 and 96 hours. By contrast, FCU one expressing e histolytica cells are selectively eliminated in the presence of 0.5 millimolar of the prodrug under brightfield microscopy.
The selective response of FCU expressing E histolytica cells to the five FC Pro drug is visible by clearing of the cells. In this case, 72 hours after transfect are cultured. While attempting this experiment, it's important to remember to monitor the growth at every time point.
This method can be used in hit and run mutagenesis, which we will use to answer important questions on the virulence of Inba Hisa.
