PRODUCTION OF COMPLETELY ES CELL-DERIVED FETUSES BY AGGREGATION WITH TETRAPLOID EMBRYOS
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Recovery of 2-cell stage embryos
Production of tetraploid embryos
Preparation of aggregation plate
Removal of Zona Pellucida
ES cells/ tetraploid embryo "SANDWICH" aggregation
Transfer of embryos
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 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.
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.
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 parameters by setting the <mode> button to Voltage and turning the appropriate dial on the left side, then setting the <mode> 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.
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.
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:
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.
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.
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