Transfection of Wild-Type Amoeba via Electroporation with Bacterial Feeding Support

Samenvatting

Source: Paschke, P., et al. Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria. J. Vis. Exp. (2019).

This video demonstrates the procedure for introducing a plasmid carrying a target gene into amoeba cells via electroporation, followed by recovery in a bacterial supplement to enable efficient gene delivery in wild-type amoeba.

Protocol

1. Setting up Dictyostelium cells for transfection
   1. Grow Klebsiella aerogenes to confluence in SM (Skim milk) medium (nutrient-rich medium) overnight at RT (Room temperature). Cultures can be stored for up to 2 weeks at 4 °C.
   2. Add about 400 µL of this bacterial suspension onto a SM agar plate (peptone 10 g/L; yeast extract 1 g/L; glucose 10 g/L; KH₂PO₄ 1.9 g/L; K₂HPO₄ x 3 H₂O, 1.3 g/L; MgSO₄ anhydrous 0.49 g/L; 1.7% agar) and spread evenly. Take a sterile loop and inoculate with Dictyostelium cells. Spread the cells at one edge of the plate.
   3. Incubate the plate at 22 °C for 2 days to ensure sufficiently large growth zones for transfection.
      NOTE: For Dictyostelium strains that do not make large growth zones (e.g., Ax3, DH1, or JH10), or for inexperienced experimenters, use clearing plates instead.
   4. Take a sterile loop and inoculate with Dictyostelium cells (approximately 2-4 x 10⁵ cells). Transfer the cells to 800 µL of a dense K. aerogenes suspension in SM. Mix cells by pipetting up and down.
   5. Transfer 400 µL, 200 µL, 100 µL, and 50 µL on fresh SM agar plates. Add to every plate 400 µL of additional SM K. aerogenes suspension, spread evenly, and dry.
   6. Incubate the plates at 22 °C for about 2 days until the plates become translucent.
      NOTE: Due to their faster growth rate, the bacteria initially produce a confluent lawn, and the amoebae subsequently "clear" the plate of the bacteria. The time required for this process can differ depending on the strain background and the ability of mutant cells to grow on bacteria.

2. Transfection of Dictyostelium Cells Based on Bacterial Selection

1. To prepare plates with K. aerogenes suspension, add 10 mL of SorMC buffer containing K. aerogenes bacteria to a density of OD (Optical density)₆₀₀ = 2 (add 200 µL of the prepared OD₆₀₀ = 100 K. aerogenes stock solution for the desired bacteria concentration) into a 10 cm tissue culture-treated petri dish.
   NOTE: This plate is later needed to cultivate the transfected D. discoideum cells. Alternatively, a 6-well tissue-culture plate can be used. In the case of transfection of the extrachromosomal plasmids, the 6-well tissue-culture plate is more resource-efficient. Use 2 mL of K. aerogenes SorMC (OD₆₀₀ = 2) suspension per well.
2. Preparation of Dictyostelium cells
   1. Using a 10 µL disposable inoculation loop, scrape cells from the growth zones (approximately 3 cm) of the culture plate (edge of the cleared area) or clearing plate. Transfer cells into a 1.5 mL tube containing 1 mL of ice-cold H40 buffer.
      NOTE: The timing of harvesting cells from clearing plates is crucial. Harvesting too early yields too little an amount of cells, while harvesting to late increases the risk of yielding partially developed cells.
   2. Wash the cells by spinning down for 2 min at 1,000 x g or flash-spinning for 2 s at 10,000 x g. Discard the supernatant and resuspend cells in H40 buffer to a final density of 2-4 x 10⁷ cells/mL. Keep the cells cold during the whole transfection procedure. Use an ice-water slurry to ensure direct contact of the tubes and ice.
3. Electroporation
   1. Add 100 µL of cells to a tube with 1-2 µg of DNA (Deoxyribonucleic acid). Mix carefully by pipetting up and down.
   2. Transfer the cell/DNA mixture into a pre-chilled electroporation cuvette (2 mm gap).
   3. Pulse the cells using the following square-wave settings: 350 V, 8 ms, 2 pulses, and 1 s pulse interval.
      NOTE: Do not add more than 2 µg of DNA. Higher amounts are toxic to cells and decrease transfection efficiency. The total added DNA volume should not exceed 5 µL.
   4. Transfer cells immediately to the earlier prepared 10 cm Petri dish with K. aerogenes SorMC and allow the cells to recover for 5 h.
      NOTE: Check the cells in the Petri dish under an inverted microscope. Cells will appear round directly after electroporation but will return to their amoeboid shape after about 30 min, when they have had enough time to attach properly to the surface.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Eppendorf Microcentrifuges 5424/5424R	ThermoScientific	05-400-002
Eppendorf 5702R Centrifuges with A-4-38 Model Rotor	ThermoScientific	12823252
Safety Cabinet Foruna - SCANLAF	Labogene
BioPhotometer Plus	Eppendorf
CLSM 710	Zeiss
Purified Agar	Oxoid	LP0028
SM broth	Formedium	SMB0102
2x LB broth	Formedium	LBD0102
HEPES free acid	Sigma Aldrich/Merck	391340 EMD
KH₂PO₄	VWR	P/4800/53
Na₂HPO₄ 2H₂O	VWR	10028-24-7
MgCl₂ 6H₂O	Sigma Aldrich/Merck	5982 EMD
CaCl₂ 2H₂O	Sigma Aldrich	442909
Folic Acid	Sigma Aldrich	F7876
KOH	VWR	26668.263
100 x 20 mm style Tissue Culture Dish, Tissue Culture Treated	Corning Incorporated	353003
2mm gap electroporation cuvettes, long electrode	Geneflow Limited	E6-0062
10 µl Inoculating Loop, Blue 10 Micro L	ThermoScientific	129399
Spreader, L-shaped, sterile	greiner bio-one	730190