We recommend to first attempt a selectable gene disruption of the target gene (using SLI-TGD) to assess its importance for parasite growth before embarking on inducible methods of target inactivation (knock sideways or diCre based gene-excision).
Procedure for selection linked integration
For localisation and knock sideways, clone the last 500 – 1000 bp of your target gene (start the sequence with an in-frame stop codon but omit the stop at the end of the gene) into pSLI-sandwich (this plasmid usually is superior to pSLI-2xFKBP-GFP). For selectable gene disruption clone ~250 – 500 bp of your target gene into pSLI-TGD. The chosen sequence should be situated in the 5’ coding sequence of the target gene and should also start with an in-frame stop codon (see Figure 1).
- Transfect 50 µg of plasmid DNA using standard procedures (alternatively transfection can be carried out with an Amaxa machine as described8)
- Select transfected parasites with 4 nM WR99210 to obtain a cell line containing the episomal plasmid
- Once the transfected parasites are obtained and the parasitemia exceeds 1%, generate 1 - 2 cryostabilates
- From the parental culture prepare a 5 ml dish (60 x 15mm) with a parasitemia of 1 - 4 % and a hematocrit of 5% without WR99210. Add 40 µl of G418 solution (400 µg/ml final) to start selection for integrants. In the case of SLI-TGD, prepare three 5 ml culture dishes in parallel for G418 selection and if no correct integration is obtained in these cultures, repeat once (resulting in 6 total attempts)
- Change medium daily for the first 10 days, from then on every other day. Prepare Giemsa smears the first 3 days after starting selection to ensure that parasites do not overgrow. If parasitemia exceeds 10%, moderately (e.g. 1:1) dilute the culture or add 7 ml of RPMI medium instead of 5 ml). Parasites will disappear usually before day 10 after Neomycin selection. Note that occasionally integrants are obtained before day 10. Stop G418 selection on day 14-16 and continue culturing without drug
- Wait until parasites re-emerge (usually 1-3 weeks)
- Optional: after obtaining the G418 resistant parasites, add 4 nM WR99210 to the culture for another 2 cell cycles, then again remove drug pressure
- Prepare gDNA of integration cell line using a commercial kit (we routinely use DNeasy Blood & Tissue Kit) and check by PCR if integration occurred at the correct locus. Use genome and vector specific primers for the 5’ and 3’ region so that the PCR product spans the plasmid/genome junction. Vector primers used in this work are: ==GFP_85_rv (ACCTTCACCCTCTCCACTGAC) and pArl_sense_55== (ggaattgtgagcggataacaatttcacacagg). Use both genome specific primers to check that no parasites with the original locus are still present in the culture
- If integration is correct, check localisation of the GFP tagged protein with a fluorescence microscope.
• If the integration check PCR shows a faint band for the original locus, a small number of parasites that entirely lost the transfected DNA may be present in the culture. In this case perform the optional step 7.
• Especially if the target of the integration is a widely worked on gene in the lab, the product DNA may already be omnipresent in the lab. In this case we recommend extra caution when preparing the parasite DNA. The integration check PCR may need to be carried out in a different lab.
• If integration still shows a non-homogenous parasite population after WR99210 treatment, it is possible that the plasmid also integrated into an incorrect locus. In this case thaw cryopreserved parasites and start again with step 3.
• If the PCR integration check did not show positive bands for the 5‘ and 3‘ integration junction, C-terminal fusion may ablate the function of the target protein. In this case thaw cryopreserved parasites and start again with step 3 but use 3 dishes as described for SLI-TGD. If no correct integration can be obtained after this, C-terminal tagging likely is not possible and the plasmid for N-terminal tagging should be used.
• The truncated fragment of a TGD may still be functional. To reduce the likelihood of this (particularly for small genes), the use of short homology regions of ~250 bp may be necessary (note that time to integration increases with shorter targeting regions).
Procedure for knock-sideways
- Transfect 50 µg of mislocaliser plasmid DNA into the knock-in integration cell line created with pSLI-sandwich (preferable) or pSLI-2xFKBP-GFP; use the nuclear mislocaliser (p1xNLS-FRB-mCherry) for targets outside of the nucleus and the PPM mislocaliser (pLyn-FRB-mCherry) for nuclear targets
- Select parasites with 2 µg/ml Blasticidin S or 0.9 µM DSM1, depending on the resistance on the chosen mislocaliser plasmid
- When transfectants are obtained, prepare cryostocks, then assess expression of the mislocaliser using a fluorescence microscope
- Optional: if a mislocaliser with Blasticidin S resistance was chosen, the concentration of this drug may be raised to increase expression of the mislocaliser
- To start the knock sideways experiment, prepare two identical 2 ml cultures (in 35x10 mm dishes) from the same parental culture; to one culture add vehicle (control), to the other 20 µl rapalog working solution (resulting in 250nM final) and place back into culture incubator
- Check mislocalisation with a fluorescence microscope after 1 h and after overnight growth; the kinetics of mislocalisation varies with the target but is often rapid (1-8 h). If mislocalisation is efficient, carry out FC growth assay and phenotypic analyses.
If the protein is not or only poorly mislocalised, check expression of the mislocaliser. If expression is low, increase the drug concentration selecting for the mislocaliser plasmid. Some proteins are refractory to mislocalisation, in this case SLI can be used to disrupt the target and at the same time add a codon-changed, floxed copy of the gene. Transfection with pSkipFlox (encoding split Cre) can then be used to inducibly excise the gene of interest.
FC growth assay
The flow cytometry assay is based on a previously published procedure9. If no flow cytometer is available, counting of Giemsa smears can be used as a substitute.
Part 1: assessing of the parasitemia of the culture to be analysed
- Add 80 µl of RPMI medium to a 1.5 ml Eppendorf tube
- Add 1 µl of HO33342 working solution and 1 µl of DHE working solution to the 1.5 ml tube
- Thoroughly resuspend the parasite culture to be analysed by pipetting up and down and transfer 20 µl into a flow cytometry tube
- Add 80 µl of RPMI dye mix to the flow cytometry tube with the parasite suspension and mix by shaking the tube
- Incubate for 20 min in the dark and afterwards add 400 µl of FC stop solution, shake the tube
- Measure the parasitemia to second decimal place using the LSRII, gate as described9
Part 2: FC growth assay over 5 days (2.5 development cycles)
- Based on the parasitemia obtained, calculate the volume of parasite culture needed for a parasitemia of 0.1 % in a total volume of 5 ml
- In a 15 ml Falcon tube prepare 5 ml of the culture at 0.1 % parasitemia with 5 % hematocrit; keep on selecting for the mislocaliser by adding the appropriate drug for the mislocaliser plasmid. Prepare 2 identical 2 ml cultures from the mixture in the Falcon tube; to one culture add vehicle (control), to the other 20 µl rapalog working solution (250 nM final) and place into culture incubator
- To assess the exact starting parasitemia of the experiment (day 0) carry out steps 1.-6. with the left over culture mixture remaining in the Falcon tube
- The next day (day 1) carry out the following for both, the cultures grown in the presence of rapalog and the culture grown with vehicle control: change medium, add new rapalog (250 nM final) and assess the parasitemia as described in steps 1.-6.
- Repeat step 10 for three more days (days 2-4).
If multiple cell lines are analysed in parallel, 6 well culture plates can be used and a master RPMI/dye mix (80 µl per sample) can be prepared.