Biolistic Transformation of Tetrahymena

 

Growth of Tetrahymena for Transformation:

  1. A pre-growth is started for the stock culture by inoculating 25 ml 2%PPYS+PSF with ~0.5 ml of the stock culture and then it is grown overnight.
  2. The pre-growth is counted the next day and a 100 ml culture is started so that the culture will be at 1-3x105 cells/ml the next day at around 2 p.m.
  3. The next day at 2 p.m. the culture is starved using 10mM Tris pH 7.5 as follows:
    1. Spin the 100 ml culture in a pear shaped flask at 1/2 to 3/4 speed (1000-1500 rpm) on the IEC centrifuge for 3 minutes. 
    2. Then decant off the media and resuspend the pellet in 100 ml 10mM Tris pH 7.5 and spin again for 3 minutes.
    3. Decant off Tris and resuspend in fresh 10mM Tris pH 7.5 (100 ml) and place in clean flask, then add PSF (1x-1ml).
    4. Place in 30°C incubator without shaking for 14-20 hrs (ideal is 18hrs.).

Transformation of Tetrahymena:

Preparing the DNA-Beads:

  1. The preparation of the gold (AU) beads is as follows:
    1. Weigh out 30 mg of AU particles (1.0µm-Somatic;0.6µm-Germline) into a 1.5 ml tube
    2. Add 1 ml of 70% ETOH
    3. Vortex for 3-5 minutes and then spin for 5 seconds and discard the supernatant.
    4. Resuspend in 1 ml sterile water, vortex for 1 minute, spin for 2 seconds, and then remove supernatant.
    5. Repeat step “d” 3 times
    6. After 3rd wash add 0.5 ml sterile 50% glycerol
    7. Aliquot into 25 µl samples in 1.5 ml tubes and store in the -20°C
  2. The preparation of the DNA is as follows:
    1. The DNA (plasmid) is digested overnight to linearize it for better efficiency of transformation (usually with SacI and KpnI) using around 80µg of DNA in a 200 µl reaction and using 5µl of each enzyme (with SacI and KpnI BSA is also added).  The reaction is then incubated overnight at 37°C. 
    2. The next day the Digest is phenol:Chloroform, Chloroform, and ethanol precipitated for 2 hrs-overnight.  The precipitation is then centrifuged at max speed for 10-20 minutes.  The precipitate is washed with 70% ETOH and centrifuged for 5 minutes and the ethanol removed. 
    3. The pellet is then dried to remove any ethanol and rehydrated in 15 µl of TE and then the Digested DNA is Quantified and the concentration is adjusted to 1.0 µg/µl.
  3. The preparation of the DNA coating onto the AU beads is as follows:
    1. To  the tube containing the 25 µl of AU Beads 2.5 µl of the DNA (1.0µg.µl) is added.  Then 25 µl of 2.5 M CaCl2 and 10 µl 100 mM spermidine is added.
    2. The mixture is then mixed on a vortex at setting “4” for 10 minutes.
    3. Samples are then centrifuged 2-3 seconds and the supernatant removed and 100 µl of 70% ETOH is added
    4. Sample are then centrifuged 2-3 seconds and the supernatant removed and 100 µl of 100% ETOH is added
    5. Sample are then centrifuged 2-3 seconds and the supernatant removed and 25 µl of 100% ETOH is added and samples are then ready for transformation
  4. Coating the DNA-Beads onto the macrocarriers:
    1. Sterilize Steel Macrocarrier holder and yellow plastic macrocarrier by dipping in 100% ETOH and then allowing to dry in the hood.
    2. Also the metal stopping screens can be sterized by 100% ETOH or autoclaved in a glass Petri dish.  The brown plastic rupture disks are sterilized using 100% isopropanol and dried in the hood. (also the red plastic cap for placing the macrocarrier in holder is sterized with 70% or 100% ETOH)
    3. Once the stuff is dry the yellow plastic macrocarrier is inserted into the steel holder.  Then 12 µl of the DNA-Bead mixture from above is pipette onto the macrocarrier and allowed to dry (2 shots can be done with one DNA-Bead preparation) (Note:  Do not let sit dry more than 30 minutes)
    4. Keep in hood until cells are ready to transform.
  5. Preparation of the Tetrahymena for biolistic Transformation:
    1. The starved cells are counted and then centrifuged in the pear shaped flasks for 3 minutes and resuspended to give 1x107 cells/ml.
    2. 2 ml of 10mM HEPES is placed into a upside-down petri dishes (10mm) and then a round 9mm filter paper (autoclaved) is placed into the Petri dish (making sure no air bubbles are present and it is full moistened).
    3. 1 ml of cells is placed onto the wet filter paper and is ready to be inserted into the Gene Gun for transformation
  6. Preparation of the Gene Gun for Transformation:
    1. Clean everything with 70% ETOH before, between shots, and after done.
    2. Settings for Tetrahymena Biolistic Transformation are as follows:

                                                               i.      Rupture disk has a 3/8 inch gap to the macrocarrier assembly.

                                                             ii.      The stopping screen is in the 3rd position (furthest away from rupture disk).

                                                            iii.      The cell plate station is in the bottommost position.

  1. Transformation of Tetrahymena
    1. Pre-warm 50 mls of 2% PPYS+PSF in 125mL flasks for at least 1hr (can be done overnight)
    2. 1 ml of cells (1x107 cells) is placed on the Petri/filter paper and placed onto the cell plate station.
    3. The stopping screen is placed in the macrocarrier assembly followed by the macrocarrier with the dried DNA-Beads on it and the top tightened.
    4. Then the rupture disk is placed in its holder and tightened.
    5. The helium tank is turned on at set to 200 PSI above the rupture disk (1100 PSI for 900 PSI disks).
    6. The Gene gun is turned on and the vacuum pump is turned on.
    7. Then the chamber door is closed and a vacuum is created and pressure to 27 mmHg is set and held there.
    8. Then the cells are shot by pressing the shoot button until the rupture disk ruptures.
    9. The vacuum is released and the cells/Petri are rinsed with a few mls of the warmed media in the 125ml flasks and then poured back into the flask.  The filter paper is then folded and placed in the flask too.
  2. Cell Recovery and Drug Selection
    1. Cells are allowed to recover at 30°C without shaking
    2. Then Drug selection for transformants is done as follows:

                                                               i.      For Pmr drug after 4 hrs at a final concentration of 100 µg/ml (stock at 100 mg/ml in Water)

                                                             ii.      For Paclitaxel (Pac) drug after 3 hrs at a final concentation of 20µM. (stock at 20 mM in DMSO)

    1. Then the cells are plated 100 µl per well in 96 well plates (five plates).
    2. Then the cells are placed at 30°C in humidity chambers for 3-7 days until growth can be seen in the wells (Pmr may need to be replicated to fresh 100 µg.ml Pmr if all wells have growth).
    3. Complete assortment occurs when cells are able to grow in 1.0 mg/ml Pmr or 40µM Pac (takes about 2-3 weeks).  One can speed up assortment by picking single cells while increasing drug concentration.
    4.  

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