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 37^o 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.

3. Place two large drops of M2 medium and two drops of mannitol solution in the second 100 mm Petri dish under other dissecting microscope. Place large drop of mannitol over electrodes.

4. Place 50 - 100 embryos in one drop of M2. The number of embryos depends on the speed since the drop of mannitol over the electrode chamber can not be used for longer that 10 - 15 minutes (it evaporates and the fusion becomes less efficient) and should be changed to fresh one after that time.

Pass the group of 20-25 embryos through the drop of mannitol and place them between electrodes one by one, leaving distance between them. Most embryos will properly orient. Pick up those which are not oriented and place them right manually.

When all the embryos are properly oriented push the trigger to apply the pulse.

Transfer the embryos into the new M2 drop.

Repeat steps 5-7 with new groups of embryos.

Rinse embryos through KSOM drops and place them into microdrops under the oil in the incubator.

Repeat steps 4-9 with the rest of embryos.

Fusion of blastomeres should be completed in 20-40 minutes. Since embryos are recovered at the late 2-cell stage, the second mitotic division is expected soon after fusion. Therefore it is important to select for the perfectly fused tetraploid embryos 20 - 60 min after application of the pulse. It is safest to transfer the tetraploids into a new culture dish or new microdrop. Under optimal conditions the rate of unfused and lysed embryos does not exceed 5 %. The tetraploid embryos are cultured overnight.
Majority of them form the 2-cell stage. By noon of the next day most of them have cleaved once more and reached the 4-cell stage. This stage is equivalent to the diploid 8-cell stage and should be used for aggregation with ES-cells, tetraploid embryos start compacting at this stage. Some embryos are still at the 2-cell stage the next stage, they are delayed. Depending on the total number of 4-cell stage embryos and number of recipients, 2-cell stage might still be used for aggregations but with limited success.

Preparation of aggregation plate Materials: 
      Dissecting microscope; 
      Sterile tissue culture dishes (Easy Grip 35 x 10 mm, Falcon 3001-3); 
      1 ml syringe with 26 G 1/2 needle of KSOM medium; 
      light mineral oil (EMBRYO TESTED) (e.g. Sigma: M8410); 
      aggregation (darning) needle (DN-09, BLS Ltd., Hungary,) 
      70% ethanol.

Method:

Place few rows of KSOM microdrops (roughly 3 mm in diameter) into tissue culture dish using syringe (e.g. 3 drops in the first and fourth and 4-5 in the second and third rows), cover with oil.

Sterilize aggregation needle by washing in ethanol.

Make six or more depressions in each microdrop (leaving a few drops intact for ES cell selection.

Keep the plate at 37^o C, 5% CO₂.

Removal of Zona Pellucida Materials: 
      Dissecting microscope; 
      Acid Tyrode's (e.g. Sigma: T1788); 
      Sterile plastic Petri dishes (100 x 15 mm); 
      M2 and KSOM medium in 1 ml syringes; 
      9'' Pasteur pipettes; 
      Mouth or Finger controlled pipette.

Method:

Place a few drops of M2, KSOM and acid Tyrode's in the Petri dish.

Wash the group of embryos with as little medium as possible through one drop of Acid Tyrode's, then transfer to a fresh drop of the same solution.

Observe zona dissolution.

Immediately transfer the embryos into a drop of M2 medium as soon as the dissolution is completed.

Wash the embryos at least twice in KSOM drops before putting them in the aggregation plate.

Transfer embryos into aggregation plate by placing them one by one beside each depression as well as inside each depression (two embryos will be used to form "sandwich" with ES-cells clump). It is possible to use only one tetraploid embryo to aggregate with ES-cells but such aggregates will need an additional night of culture to form blastocysts.

Prepare ES-cells for aggregation by that time.

ES cells/ tetraploid embryo "SANDWICH" aggregation Materials: 
      Dissecting microscope; 
      Prepared aggregation plate with depressions and embryos with removed zona; 
      Trypsinized ES-cells; 
      Mouth or Finger controlled pipette; 
      9'' Pasteur pipette.

Method: 

Choose clumps of loosely connected ES cells and transfer them into microdrops (not containing embryos) of aggregation plate for final selection.

Select few clumps of ES cells (8 -15 cells each); place each in a depression next to an embryo.

Pick up the corresponding embryo outside the depression and place it on the free side of the ES cell clump, forming "sandwich".
Assemble all aggregates in this manner, check the plate, and culture overnight at 37^o C, 5% CO₂ .

The following afternoon, the majority of aggregates should have formed blastocysts. At this time they should be transferred into the uterus

of 2.5 dpc pseudopregnant females. We transfer a maximum of 8-10 embryos into each uterine horn of a pseudopregnant recipient. Mature CD-1 females are used as pseudopregnant foster mothers and ordered at a weight of 30⁺ g.

In the event of a recipient shortage, it is possible:
      to transfer up to 24-26 embryos per recipient; 
      to culture the aggregates (preferably morulae) for one more day and transfer them into 2.5 day pseudopregnant females; 
      to use 3.5 day pseudopregnant females.

Transfer of embryos Materials: 
      2.5 dpc pseudopregnant females; 
      Instruments: 
      Scissors; 
      Semken forceps (straight or curved with serrated tips); 
      Forceps with 1 x 2 teeth; 
      Dumont ss/mc or No. 5 forceps; 
      Serrefine (e.g. Fine Scientific Tools: 1 8050-28 or 8051-28); 
      26G1/2 or 30G1/2 needle; 
      Autoclip applier (Clay Adams B-D 763007); 
      Autoclips (Clay Adams B-D 7631); 
      Mouth controlled pipette; 
      M2 medium; 
      2.5% Avertin: 
      2,2,2,-Tribromethanol 2.5 g (Morre-Tec Industries #1693); 
      Tert-amyl alcohol 5.0 ml (Fisher: A-730-1).

Dissolve tribromethanol in Tert-amyl alcohol, then add to 200 ml distilled water. Place on a magnetic stirrer until solution is in one phase. Store in brown bottle and keep refrigerated until use. Should be warmed and shaken before use. Dosage is 0.2 ml/10 g body weight.

Method:

The embryo transfer procedure is described in details in many publications, such as the following:

1. Hogan, B., F. Constantini, E. Lacy. 1986. Manipulating the Mouse Embryo. Cold Spring Harbor, New York.

2. Bradley, A. 1987. Production and analysis of chimeric mice in Teratocarcinomas and Embryonic Stem cells: a Practical Approach (ed. E.J.Robertson) IRL Press, Oxford, Washington, D.C.

3. Pappaioannou, V., R. Johnson. 1993. Production of chimeras and genetically defined offspring from targeted ES cells. In Gene Targeting: A Practical Approach (ed. A.Joyner) IRL Press at Oxford University Press

4. Stewart C.L. 1993. Production of Chimeras between Embryonic Stem Cells and Embryos. in Methods in Enzymology. vol.225. Academic Press Inc.