Transformation of Dictyostelium by calcium phosphate precipitation

Published in: Nellen, Silan, and Firtel RA. (1984), Knecht, Cohen, Loomis, and Lodish (1986), Cohen, Knecht, Lodish, and Loomis (1986).

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[MATERIALS] - [INDEX]

Procedure

1. Prepare the cells: grow D. discoideum in HL5 medium on Petri plates for 3 to 4 days. The day before the transformation, replace HL5 medium with 12.5 ml Bis-Tris HL5.
2. 
   
3. The next day, change the medium be aspirating off and replacing with 10 ml fresh Bis-Tris HL5 to further reduce the phosphate.
4. 
   
5. Prepare the DNA:
   The calcium phosphate precipitate of the DNA to be transformed into the cells is made in a plastic test-tube by adding (in order) to a final volume of 0.6 ml:
6. 
   
   • 0.24 ml sterile water
   • 10 µg DNA (about 10 µl of a 1 µg/µl solution)
   • 0.3 ml 2x HBS
   • 60 µl 1.25 M CaCl₂
   • 
     
7. Transformation:
   Remove the medium from cells and add drops of the suspension of calcium phosphate precipitated DNA (0.6 ml). Let stand 30 minutes while the cells take up the vector. Add 10 ml Bis-Tris HL5 and let stand several hours (4-8) to allow the cells to flatten and stick onto the dish.
8. 
   
9. Glycerol shock:
   
   • Carefully aspirate off the medium and gently add 4 ml of 18% glycerol in HBS by letting it run down the side of the dish.
   • Let stand 5 minutes (accurate to 10 seconds).
   • Gently aspirate off the glycerol solution and replace with 10 ml HL5.
   • Incubate overnight for phenotypic expression.
10. 
    
11. Selection of transformants:
    
    • Aspirate off the medium and replace with 10 ml fresh HL5 containing 5 to 10 µg/ml G418 and 200 µg/ml Streptomycin.
    • Aspirate the medium up and down to remove cells from the plate and distribute into microtiter well plates.
    • Save original plate and replace medium.
    • Feed cells two times per week.

Transformant should come up in two to four weeks, although some are apparent earlier.

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Materials

• Bis-Tris HL5 (1 liter)
  • 2.1 g Bis-Tris
  • 10 g proteose peptone (Oxoid)
  • 5 g yeast extract
  • 10 g glucose
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  Add distilled water to 1 liter.
  Adjust pH to pH 7.1 with HCl.
  Autoclave to fully solubilize, readjust pH to 7.1 with NaOH (it will have gone down to about pH 6.8).
  Filter sterilize; store at room temperature.
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• 2x HBS (250 ml)
  • 4.0 g NaCl
  • 0.18 g KCl
  • 0.05 g Na₂HPO₄
  • 2.5 g HEPES
  • 0.5 g glucose
  • 
    
  Adjust pH to 7.1 with NaOH.
  Filter sterilize, store frozen.
  
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• CaCl₂ (10 ml)
  • 1.84 g CaCl₂
  • 10 ml distilled water
  • 
    
  Filter sterilize, store at room temperature.
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• 18% Glycerol (4 ml)
  • 1.44 ml of autoclaved 50% glycerol in water
  • 0.56 ml sterile water
  • 2.0 ml 2X HBS
  • 
    
  Note: The glycerol concentration is critical for the transformation. Too much will kill the cells; not enough will fail to permeabilize them sufficiently. It is therefore necessary to make a fresh solution for each transformation.

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