PRODUCTION OF COMPLETELY ES CELL-DERIVED FETUSES BY AGGREGATION WITH TETRAPLOID

The technique described here is a slight modification (March 1997) of methods presented in: Nagy A., J. Rossant. 1993. Production of completely ES cell-derived fetuses. In : Gene Targeting: A Practical Approach. (ed. A. Joyner). IRL Press at Oxford University Press.

Recovery of 2-cell stage embryos

Recovery of 2-cell stage embryos is very similar to that of the 8-cell stage embryos and it must be done on the day 1.5 dpc. It is safer to collect late 2-cells stage embryos to avoid the so-called two-cell-stage block. The presence of 10-15% 3-4-cell stage embryos among 2-cell indicates the appropriate timing. The flushing 46 + hours after hCG injection is recommended.

Materials:

Dissecting microscope;       Flushing needle (The sharp tip of No. 30 G 1/2 needle is cut off and then rounded using sharpening stone);       1 ml syringe;       Dissecting instruments (fine-pointed scissors, fine forceps);       No. 5 forceps (Dumont);       Mouth pipette (aspirator mouth piece, latex tubing, blue tip) alternatively:       Finger controlled pipette (Manostat tubing, yellow tip, scalpel blade);       9'' Pasteur pipettes;       70% Ethanol;       Alcohol burner or Bunsen burner;       Sterile plastic Petri dishes (100 x 15 mm);       Sterile Organ Culture dishes (60 x 15 mm, Falcon, 3037);       M2 and KSOM media.

Method:       The oviducts with the upper part of the uterus attached are removed from 2.5 days post-coitum (dpc) superovulated CD-1 females and placed into a drop of M2.       Under dissecting microscope the oviducts are flushed by inserting the flushing needle attached to a 1 ml syringe of M2 into the infundibulum.       The embryos are collected using mouth or finger controlled pipette and washed through several drops of M2 medium to remove any debris.       The embryos are washed in KSOM medium and cultured in organ culture dish in KSOM at 37o C, 5% CO2.

Production of tetraploid embryos

Fusion of the blastomeres of 2-cell stage embryos occurs when a square pulse is applied perpendicular to the plane of contact of the two cells. The pulse parameters varry depending on the electrodes and pulse generator. We use Cell-fusion instrument, CF-150B available from BLS Ltd., Hungary with following parameters (for non-electrolyte fusion):

Voltage -30 V; Duration - 35 microsec; Number of pulses - 2 ;      Adjustable AC field is applied to allow the correct orientation of embryos (enable, 1 or 2 V on the display). Too high an AC field can cause lysis.

Materials:

CF-150B cell-fusion instrument with an electrode chamber ( The company manufacture three kinds of electrode chambers: GSS-250, GSS-500 or GSS-1000, which differ in the gap distance. All work well for tetraploid production. The choice is based on personal preference.)

two dissecting microscopes;       M2 and KSOM media;       0.3 M Mannitol (Sigma M4125) (dissolved in ultra pure water with added 0.3% BSA (Sigma A4378) and filtered through 0.22micron Millipore filter, stored in aliquots at -20 C);       tissue culture dish with KSOM microdrops covered with oil (Sigma M8410);       mouth or finger controlled pipette;

Method: 

1. Turn on the CF-150B pulse generator. Make sure you set the switch on the backside of the mashine to "Non-electrolyte".Do NOT use the "Normal" position!

2. Put a 100 mm Petri dish containing the electrode - chamber under a dissecting microscope, connect the cables to the pulse generator and adjust all param eters by setting the button to Voltage and turning the appropriate dial on the left side, then setting the to duration and turning the appropriate dial until the desired number is displayed.

Typical settings for GSS-250: 40 V, 30 microsec, 2 repeats, for GSS-500: 75 V, 35 microsec and for GSS-1000: 137 V, 26 microsec.The parameters however could depend on local conditions and embryos used. They should be optimized empirically.On the front panel, in the lower right corner, you have a two buttons and a dial. Push the first button, which is called "HF SINUS" - "ENABLE" until the diod lits up and it is so enabled. Push the button on the other side of the dial until also this is enabled. This button is called "ATTENUATOR" - "AMP/10. Now turn the dial until 1.0-2.0 is shown on the display. Which strength you should choose depends on how quickly you can work, and the sensitivity of your embryos. The higher this value, the faster will the embryos align in the right position, but a too high value for more then 20-30 seconds seconds will harm them. This you should optimize empirically.