Electroporation

We use two protocols to transfect linearised DNA constructs into bloodstream form T. brucei, depending on the integration target and the desired transformation efficiency – both rely on a nucleofection apparatus running progamme X-001 (Lonza)

Protocol 1: standard

This is particularly useful for transfecting pRPa constructs, which integrate at the ‘landing pad locus’ in 2T1 cells, or similarly efficient construct-target combinations

  1. Digest 10 µg construct with the appropriate restriction enzyme(s) to generate a linear DNA molecule with termini to allow integration into the T. brucei genome by homologous recombination
  2. Clean the digested construct by phenol:chloroform:isoamylalcohol extraction and ethanol precipitation (do not include any salts such as sodium acetate, as these will interfere with electroporation); resuspend the precipitated DNA in 10 µl sterile distilled water
  3. Pellet 25 million bloodstream form T. brucei at 1000 g for 10 minutes
  4. Pour off the supernatant, remove as much of the residual media as possible without disturbing the cell pellet, and resuspend in 100 µl cytomix (pre-warmed to 37°C) plus 10 µl DNA solution
  5. Transfer cell-cytomix-DNA mixture to a 2 mm gap cuvette and apply nucleofection programme X-001
  6. Transfer cells to 60 ml media and add appropriate selective drugs 4-6 hours later
  7. Transfer to two 48 well plates: ‘neat’ and ‘5-fold diluted’; positive wells should be transferred to 10 ml cultures five days post-electroporation

Cytomix (filter sterilise and store at 4°C)

KCl 120 mM
HEPES pH7.6 25 mM
1K2HPO4/KH2PO4 pH7.6 10 mM
MgCl2.6H2O 5 mM
EGTA pH7.6 2 mM
CaCl2 150 µM
Glucose 0.5%
Albumin, bovine serum 100 µg/ml
2Hypoxanthine 1 mM

1 10x K2HPO4/KH2PO4 pH7.6: 8.66ml 1 M K2HPO4, 1.34 ml 1M KH2PO4, 90 ml H2O (store at room temperature)
2 100x Hypoxanthine: 0.4 g NaOH, 100 ml H2O, 1.36 g hypoxanthine (store at -20°C)

Recommended drug concentrations

Drug Stock (mg/ml)
Selective (µg/ml)
Maintenance (µg/ml)
Phleomycin 10 mg/ml 2 1
G418 10 mg/ml 2
1
Puromycin 2.5 mg/ml 2
1
Hygromycin 5 mg/ml 2.5
1
Blasticidin 10 mg/ml 10 5

 

Protocol 2: nucleofection

We use this protocol to integrate cassetes at RNA polymerase II transcribed loci; these typically represent much less efficient integration targets, and this approach is generally used for transfecting disruption or native tagging constructs

  1. As per Protocol 1
  2.             -“-
  3.             -“-
  4. Pour off the supernatant, remove as much of the residual media as possible and resuspend in 82 µl human T cell nucleofector solution, 18 µl supplement 1 (Lonza; both at room temperatue) plus 10 µl DNA solution
  5. Transfer cell-nucleofector solution-DNA mixture to a 2 mm gap cuvette and apply nucleofection programme X-001
  6. As per Protocol 1
  7.             -“-