Transient Transfection of Procyclic form T. brucei.

Notes:

1. Use vigorously growing log phase cells (I shoot for 2 - 4 x 10⁶ cells/ml at time of harvest for transfection. We grow at 28 deg C, 5 % CO₂ in Cunningham’s SM medium (Cunningham, I. 1977. J. Protozool. 24:325-9) supplemented with 10% heat-inactivated fetal calf serum. (The 5% CO₂ is not required, that’s just how our 28 deg incubator is set up, and I don’t know if it gives insect-form cells a growth boost or not.)

2. YTAT1.1 is the strain I have used most and it has a doubling time of about 6.5 - 7 hours under these growth conditions. I usually dilute a log culture 18 - 36 hours before tranfxn to an appropriate density that will give me 2 - 4 x 10⁶ cells/ml at the time of transfection.

3. I use Qiagen prepped DNA, and spin the DNA to clarify before transfecting.

4. I use 10 - 100 ug of DNA. There is clearly an increase in transfection efficiency when going from 10 to 50 ug, and maybe a little increase from 50 to 100 ug. I haven’t tried above 100 ug.

5. Electroporation cuvettes = BioRad 0.4 cm gap (BioRad part# 165-2088).

6. All buffers are at Room Temp. The fresh medium (step 6) can be cold or at room temp-I’ve done both and haven’t seen a difference.

“EM” = Electroporation medium = (120 mM KCl_2, 0.15 mM CaCl₂, 9.2 mM K₂HPO₄, 25 mM HEPES, 2 mM EDTA, 4.75 mM MgCl₂, 69 mM sucrose, pH 7.6)

Protocol:

1. Harvest log-phase cells at 1000 x g for 3 - 5 min.

2. Wash once with “EM” (electroporation medium, see notes for recipie).

3. Resuspend in EM at 5 x 10⁷ cells/ml.

4. Add DNA to cuvette (0.4 cm gap BioRad electroporation cuvette, the blue ones!). Use 10 - 100 ug of DNA in 100 ul. If necessary, use sterile water to bring DNA volume to 100 ul.

5. Add 0.45 ml of cells to each cuvette Total volume = 0.55 ml.

6. Zap twice in BioRad Gene Pulser 1500 V, 25 uF, with 10 seconds in between pulses.

7. *** The time constant should always be between 0.5 and 0.7 msec. If it isn’t, something went wrong. ***

8. Remove 0.15 - 0.2 ml of electroporated cells and add to 5 ml of fresh medium (SM medium + 10% HI-FCS). It’s ok if the medium is fresh from fridge, or Rm. Temp. Return transfected culture to the 28 deg incubator. (For transient GFP expression, we have been putting the cells at Room Temp on a rotator for aeration----something I started doing way back... and haven’t changed even though it’ll probably work just fine to put the cells back in the incubator.)

9. For GFP expression: we see GFP within 6 - 8 hours and out to 36 hours post transfection. Optimal GFP (% expressors and intensity) is generally at about 16 - 18 hours post transfection, but is really pretty steady from 12 - 28 hours.