The overall goal of this procedure is efficient and reliable gene replacement using type one and type two delta two 80 strains of Toxoplasmic ndii. This is accomplished by first constructing a targeting DNA molecule that fuses the HX GPRT selectable marker in the forward orientation between distinct five prime and three prime gene targeting DNA flanks. The second step of the procedure is to transfect the targeting DNA molecule into a type one or type two toxoplasma DYI strain.
The third step is to patiently select for double crossover homologous recombination events and isolate individual clones. The final step is to validate the presence of the gene targeting event in the isolated mutant clones. Ultimately, results of this procedure can show the function of parasite genes by the analysis of targeted gene knockouts, gene replacements, and tag genes through a variety of downstream applications.
The main advantage of this technique over existing methods using wild type strains of this parasite is that the type one and type two delta two 80 parasite backgrounds now enable extremely reliable selection of targeted double crossover homologous recombination events to produce precise genetically defined strains. This technique will greatly speed up the discovery of new targets for antitoxin plasmas drug therapy and the development of safe and effective attenuated vaccine strains, especially when paired with the well-conceived genomic resources located online@toxodb.org. Generally, individuals new to this method may struggle with engineering targeted DNA molecules or genetic manipulations with Toxoplasma Gandhi.
However, patients practice and the design of reliable targeting molecules will ensure success. Begin the protocol by PCR amplifying the five prime and three prime genomic targeting flanks for the gene of interest from genomic DNA. Then PCR amplify the HX GPRT selectable marker with its five prime and three prime DHFR flanks from the HX GPRT CD NA mini gene cassette.
Use yeast recombination or a similar method to fuse the 3D NA molecules in their correct five prime to three prime orientation with plasmid PRS four 16, thus generating plasmid p delta GOI that will target the deletion at the gene of interest. Next, prepare a 100 microliter sample containing 15 to 30 micrograms of linearized targeting plasmid for transfection into T gia 68 to 72 hours after inoculating a 25 centimeter flask of confluent HFF cells. Verify the presence of freshly lyed parasites by visually confirming that 90 to 95%of the HFF cells have been lyed viable parasites are essential for gene targeting.
Agitate the viable parasites into solution by vigorously shaking the closed flask without splashing liquid onto the inside of the lid or the neck of the flask. Harvest the parasites from the flask and prepare them for transfection as described in the text protocol. Electroporated the targeting plasmid into the delta CO 80 toxoplasma strain.
Transfer the electroporated mixture to a 150 centimeter flask containing confluent HFF cells and 30 milliliters of infection.Medium. Incubate the infected culture overnight at 37 degrees Celsius about 20 hours post transfection. Replace the medium with MPA plus X selection medium.
Now allow the parasites to incubate at 37 degrees Celsius for about eight days.Undisturbed. Once plaques are visible, shake the flask to dislodge extracellular parasites from the monolayer generating a parasite solution. Transfer about half a milliliter of the parasite solution to a new 25 centimeter flask of confluent HFF cells containing five milliliters of MPA plus X selection medium.
Designate this culture as PAs one A roughly three days later. Repeat the transfer of half a milliliter of the parasite solution from the original flask to another flask of HFF cells with the same selection media. Designate this culture as PAs one B over the next five days.
Monitor passes one A and one B for pfu. Select either pass one A or one B for sub cloning based on an adequate number of greater than 25 zones of infection. Whenever parasites lyce out the HFF cells, pass the culture to a new 25 centimeter flask of confluent HFF cells with selection medium three weeks post transfection sub clone freshly laced parasites by diluting parasites to the appropriate concentration in MPA plus X medium, and evenly distributing the parasite solution into a 96 well tray containing confluent HFF cells.
Prepare one tray at a concentration of one parasite per well and prepare a second tray at a concentration of two parasites per well. Incubate both trays at 37 degrees Celsius after the original transfection date. Do not subclone the parasites before day 20 for type one parasites or day 25 for type two parasites.
Continue passing the 25 centimeter flask on a weekly schedule as described in the online text until the knockout strain has been validated. After about a week score the 96 well trays under 40 to 60 x magnification. Selecting only wells that contain a single PFU identified as a single zone of infection using a pipette set at 50 microliters.
Mix the contents of these wells by directing fluid flow over the PFU to disperse the parasites around the well. Four to five days after mixing the selected wells, choose a dozen clones with lyed cells. Transfer 12 clones from the 96 well tray to 12 wells of a 24 Well tray Do this by scratching with a pipette tip across the bottom of the well and drawing up six microliters.
Transfer the solution into a well of a 24 well tray with confluent HFF cells in one milliliter of MPA plus X selection. Medium incubate the 24 well tray at 37 degrees Celsius and monitor for lysis of the HFF cells. Passage the parasites from a lysed well in the 24 well tray on a weekly schedule.
Use the scratch and draw technique to transfer two microliters of the parasite solution to a new plate with MPA and X selection. After passage, harvest the parasites from the original 24. Well tray and transfer to small tubes for DNA collection.
To finish, use PCR to test for the deletion of the gene of interest to check the integration of the five prime and three prime target flanks. And to check for the HX GPRT selectable marker. This protocol is for the removal of the HX GPRT marker from the manipulated delta Q 80 strain and is outlined in the online text.
P delta GOYC targets the removal of HX GPRT from the parasite by negative selection using six TX media. Then PCR is used to confirm the loss of HX GPRT as shown by a 1.2 Kilobase pair band where HX GPRT was not removed from the locus. There is a 3.4 kilobase pair band.
The final part to this protocol, C terminal tagging of proteins is also described in detail in the online text after following the outlined protocol to make a targeted gene deletion, PCR was used to validate the deletion of a knockout of type one R 18. A low side that is generally difficult to target. PCR one shows the absence of the R 18 gene PCR product.
PCR two shows the presence of the three prime R 18 genomic targeting flank PCR three and PCR four show the presence of the selectable marker properly integrated between the five prime and three prime genomic targeting flanks, which defines the R 18 deletion. In order to reuse the selectable marker to make subsequent deletions in a strain, the original HX GPRT must first be removed from the locus. After following the outlined protocol.
HX GPRT was removed from the deleted GRA two locus in strain delta two 80 delta GRA two HX.G-P-R-T-P-C-R was used to confirm the loss of HX GPRT shown by a 1.2 KILOBASE per band. When H-X-G-P-R-T was not removed, there was a 3.4 kilobase per band Following the initial gene targeting step. Additional gene targeting methods can be applied to construct multiple knockouts in the same type one or type two strain, or to tag or complement a mutant strain to answer additional questions like, where does this protein localize in the cell?
Or what is the key domain or activity of this protein or pathway that explains its biological function? This technique paves the way for researchers in the field to ramp up the exploration of parasite gene function in the model AP complex and parasite toxoplasma. Gandhi eye.
Don't forget that working with toxoplasma can be hazardous and precautions such as following all BSL two guidelines should be maintained throughout the procedure.
