5-FU/FUDR Selection of Toxoplasma (UPRT-targeting)

By targeting a knock-in to the UPRT (uracil phosphoribosyltransferase) locus, one can negatively select against Toxoplasma and other microbes using 5-fluorouracil (5-FU) or the its analog 5-fluorodeoxyuridine (FUDR). Resistance to these drugs was first described by Elmer Pfefferkorn in 1977. Targeted deletion of the UPRT locus was first described for Toxoplasma by the Roos lab, and is an excellent way to test the efficacy of chemical or insertional mutagenesis experiments.

We use the pUPRT plasmid as the backbone for our constructs. This was used, for example, to complement the RHΔrop5 strain.

Make a 50 mM stock of FUDR or 5-FU and use a working concentration of ~10-20 μM (2.5 - 5 μg/ml) added to media and filter sterilized at the time of use.

Day 0

  1. For fast growing parasites, e.g., RH, pass parasites at ~0.5ml into a T25.

Day 1

  1. Prepare in cytomix as per normal, but use ~1/4 of a T25 per transfection.
  2. Transfect w/ 15 μg of linearized plasmid, place transfected parasites into a T25 (no selection).

Day 3

  1. After ~2 days, syringe release, wash, and pass all parasites into a T75.

Day 4

  1. Switch the media to +5-FU.

Day 5

  1. Parasites may be completely lysed out. Regardless, syringe release, sediment.
  2. 5 μm filter the pellet into a fresh T75 in +5-FU media.
  3. Allow parasites to invade for ~3 hours (might go slightly longer if using non-RH strains).
  4. Wash 3 - 4 times with PBS to remove uninvaded parasites.
  5. Incubate in +5-FU media. (These usually take 3-5 days to completely lyse out).
Day 8-10
  1. At this point the population can be PCR tested for positive clones.
  2. Scrape cell layer, syringe release.
  3. 5 μm filter (optional) and add 1 - 3 mL parasites to a fresh T25 in +5-FU media.
  4. Allow parasites to invade for 3 - 4 hours.
  5. Wash 3 or 4 times with PBS to remove uninvaded parasites.
  6. Incubate in +5-FU media.
  7. (It could take anything from 2-7 days to see good lysis)
Finish
  • At this point, parasites should be able to be passed without selection.
  • Screen by parasites by immunofluorescence / western blot for expression of appropriate construct.
Notes:
  • Fluorouracil is also toxic to host cells, but much less so if they are not replicating (i.e., a confluent monolayer).
  • As with many Toxoplasma protocols, the above timescale assumes a lab-adapted RH strain, and may be much longer (2-3x) for other strains.
  • Anecdotally, when UPRT knockout works (20-80% of the time, depending on construct and strain), 50-90% of parasites are positive for an introduced construct.
  • Also anecdotally, multiple transfections of identical constructs on the same day doesn't improve probability of success for UPRT knockout. It's better to stagger transfections.
  • Keep in mind that the initial wait without selection is required to allow both for integration and reduction of UPRT protein level. 5-FU will kill parasites that still have residual UPRT even if they no longer have the gene encoding for the protein.
  • A main issue is parasite destruction of the monolayer, even after selection has been applied. These “zombie” parasites may be able to invade multiple times, even though they cannot replicate. The above protocol has been optimized to reduce this problem.
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