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Embryonic stem cells, mouse

Multiporator Transfection Protocol Protocol No. 4308 915.039 12/1999 Cell line Embryonic stem cells (ES cells), mouse, R1. Passage 15 Transfection with Linearized targeting vector (12 kb) with neoR-selection marker Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium DMEM / 15% FCS, 2 mM glutamine, 1 mM pyruvate, 1 x MEM, 1 x nucleoside mix, 1 x Pen/Strep, 1000 U LIF Cuvette Eppendorf, 4 mm gap width, 800 l Temperature RT (20-25 C) Reference Dr. Michael Bsl ZMNH Transgene Technologie Martinistr. 42 D-20246 Hamburg
Phone +49 40 42803 6663 Fax +49 40 42803 6659 e-mail: boesl@plexus.uke.uni-hamburg.de
  1. Harvest the cells 36-40 hours after the last passage, determine the number of cells and centrifuge the cells (5 minutes, 200 x g, at room temperature).
  2. Wash the cells in Hypoosmolar Electroporation Buffer: Resuspend the cells in 10 ml Hypoosmolar Buffer and collect them by centrifugation (for 5-10 minutes, 200 x g, at room temperature). Remove supernatant.

    Note: The overall incubation time in the Eppendorf Electroporation Buffer must not exceed 30 minutes to guarantee a successful electroporation!

  3. Resuspend the cells at a concentration of 1 x 107 cells/ml in Hypoosmolar Electroporation Buffer.
  4. Mix 750 l cell suspension with 50 l DNA solution (1 g/l in Isoosmolar Electroporation Buffer).
  5. Transfer 800 l cell/DNA solution into electroporation cuvettes (4 mm gap width). The cell suspension must be free of air bubbles.
  6. Electroporation:

    Mode Eukaryotes Voltage (V) 300 V Time constant (T) 100 s No. of pulses (n) 1
  7. After the pulse, let the cells shortly recover for 5 minutes at room temperature and then carefully transfer the cells to 10 ml ES-medium.
  8. Plate the cells onto 100 mm culture dishes prepared with inactivated mouse embryonic fibroblasts feeder layers at a density of 1-1.5 x 106 cells per plate.
  9. The next day change medium and start selection 24 hours after the electroporation with 250 g/ml medium of G418. Change selection medium every day.
  10. 7-8 days after the electroporation pick the surviving individual ES-cell colonies under a stereo microscope, culture and expand them individually. Take only colonies with sharp borders and proper morphology.
  11. After expansion freeze down at least one aliquot of each clone and use another for DNA preparation to identify correctly transfected clones originating from homologous recombination by PCR or Southern Blot analysis.

Detection methods for transfection:
Selection with G418 to identify G418 resistant, stably transfected colonies carrying the neoR marker of the linearized targeting vector.


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