This protocol is key for generating strains that can be used in high-throughput screening assays to evaluate the potential and traditional activity of molecules Demonstrating the procedure will be Juana Quintero and Laura Pineda, research assistants from my laboratory. To begin, amplify the target gene using a high fidelity polymerase to preserve the coding sequence. Add the primers to the reaction mix in a final concentration of 0.2 micromolar and run a PCR cycling protocol as described in the manuscript.
Purify the amplified PCR fragment using a conventional PCR product purification kit. To trim the ends of the eGFP PCR product, set up a reaction with Kpn I enzyme according to the manufacturer's instructions and incubate at 37 degrees Celsius for one hour. Then add 100 millimolar of sodium chloride and 10 units of Bgl II to the reaction and incubate for 15 minutes.
After digestion, purify the reaction as shown earlier. Next, digest pLEXSY expression vector with Bgl II and Kpl I following the sequential digestion as demonstrated earlier. Ligate 100 nanograms of digested pLEXSY vector and 28 nanograms of digested eGFP in a 20 microliter reaction containing ligase buffer and three units of T4 DNA ligase.
Incubate overnight at four degrees Celsius. At the end of the incubation, transform competent Escherichia coli cells with the ligation product using standard transformation protocols. Plate the transformed cells in LB ampicillin agar and incubate for 24 hours at 30 degrees Celsius to select recombinant clones.
After screening the colonies, purify plasmid DNA from a positive clone for subsequent transfection using a commercial plasmid isolation kit. To produce linearized fragments of the plasmid, take at least 10 micrograms of the purified pLEXSY eGFP plasmid and digest it with SWA1 for three to four hours at 25 degrees Celsius. At the end of the incubation, heat inactivate the digestion product at 65 degrees Celsius for 20 minutes.
Then run the digestion product in agarose gel electrophoresis to separate the resulting fragments. Purify the expression cassette using a commercial agarose gel extraction kit according to the manufacturer's instructions. Grow the parasite cultures until there are enough log phase or early stationary phase promastigotes.
Pool the contents of multiple culture flasks if required. Centrifuge the parasite culture at 2, 000 G for three minutes at room temperature. Resuspend the pellet in electroporation buffer at four degrees Celsius to get a concentration of one times 10 to the eighth parasites per milliliter and keep it on ice for 10 minutes.
Simultaneously, pre-chill tubes with 2 to 10 micrograms of linearized pLEXSY eGFP construct in 50 microliters of water or 10 millimolar tris buffer with pH 8.0 and electroporation cuvettes of two millimeter diameter. Next, add 350 microliters of pre-chilled parasites to the tube with the linearized plasmid and transfer the entire 400 microliters to the electroporation cuvette on ice. Electroporate the parasite cells with plasmid and in parallel, parasite cells without plasmid DNA as a negative control.
Put the cuvette on ice for 10 minutes. Transfer the electroporated parasites to five milliliters of the appropriate culture medium and incubate at 26 degrees Celsius for 20 hours. Approximately 20 hours post-electroporation, observe the cultures under a microscope and add the appropriate selective antibiotic depending on the pLEXSY plasmid used.
Follow the cultures microscopically until there is a clear difference between parasites electroporated with and without plasmid DNA. To verify parasite fluorescence through flow cytometry, centrifuge one milliliter of the stationary phase culture at 2, 000 G for three minutes at room temperature. After washing the cells twice in PBS, resuspend the final pellet in one milliliter of PBS.
Run samples and collect 20, 000 events at a speed of 0.5 microliters per second. Set gain values for the forward scatter, side scatter, and fluorescence one channels as 225.0, 200.0, and 520.0 respectively. Create a gate G1 containing the parasite population.
Filter the FL1 channel through that gate to determine the autofluorescence of promastigotes and set a range gate G2 for the FL1 channel. Run the transfected parasites to verify if there is fluorescence. Record the percentage of parasites in G1 that are fluorescent and the mean of fluorescence intensity in G2.Purify genomic DNA from two to five milliliters of a stationary phase parasite culture by conventional phenol chloroform extraction or with a commercial kit.
Perform diagnostic PCR for pLEXSY-sat2.1 using a cycling protocol as described in the manuscript. After confirmation of fluorescence through flow cytometry, prepare a dilution of recombinant parasites at a concentration of five promastigotes per milliliter. Add 100 microliters of this dilution into each well of a 96-well plate and leave the plate undisturbed for at least 12 hours.
Follow the plate microscopically at a minimum magnification of 100X until wells with parasite growth are detected. When a well is full of parasites, use the content to inoculate a five milliliter culture. Once the clone seeded cultures reach the stationary phase, verify fluorescence using flow cytometry as described earlier.
Select clones with 98%to 99%of fluorescent parasites and the highest mean fluorescence intensity for in vitro and in vivo assays. Colony PCR of the E.coli cells transformed with pLEXSY eGFP construct generated an 859 base pair product. Total digestion of the plasmid using SWA1 resulted in two fragments, a 2.9 kilobase pair fragment containing all the necessary elements for replication and selection in E.coli and a seven to eight kilobase pair fragment representing the linearized expression cassette that is used for transfection.
Flow cytometry analysis revealed that 82%of the transfected L.panamensis parasites expressed eGFP. After polyclonal selection, 98.44%of L.donovani parasites analyzed by flow cytometry were fluorescent in comparison with 82.0%of L.panamensis. The successful integration of pLEXSY eGFP into the SSU locus resulted in a 2.3 kilobase pair product in the diagnostic PCR.
Clones with the highest percentage of fluorescent parasites and mean fluorescence intensity was selected for further use in drug screening assays. This method allows the production of recombinant Leishmania parasites with the
