This procedure describes how to retrieve mouse oocytes and/or embryos, clean and process them for ChIP or RNASeq experiments
Collection and handling of mouse oocytes/embryos
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posted 27 Jan, 2020
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M2 medium (Merck Millipore MR-015-D)
KSOM Embryomax medium (Merck Millipore MR-106-D)
M2 medium with hylarunidase (MR-051-F)
Acid Tyrodes solution (Sigma T-1788)
Leica brightfield dissection microscope
Basic mouse dissection tools with forceps
Pulled glass capillaries
Mouth pipette with filter for embryo handling
1. For collection of MII oocytes, four to eight-week-old donor female mice were injected with 5 units of pregnant mare serum gonadotropin (PMSG) at 18.00 (100 µl of 50 I.U/ml solution)
2. this is followed by 5 units of human chorionic gonadotropin (hCG) at 17.00 (100 µl of 50 I.U/ml solution) the following day.
3. Donor mice were sacrificed by cervical dislocation 14-15 hours post hCG injection.
4. Oviducts are transferred to a clean petridish dish with M2 (Sigma) medium.
5. Cumuli were released from the ampulla in M2 medium and disaggregated in M2 medium containing 0.3 mg/ml hyaluronidase
6. This is followed by four washes in M2 medium.
7. For zona removal, oocytes/ preimplantation embryos are transferred in a 50µl drop of Tyrode’s solution (Sigma) to remove the zona pellucida.
8. this is followed by three washes in M2 medium droplets before downstream processing for Chip or RNA Seq.
9a. For Chip Seq, oocytes were given a fourth final wash in M2 before crosslinking and fixation as described in a separate protocol.
9b. For RNASeq, the oocytes and embryos were washed three times in sterile PBS before a final wash in nuclease free water supplemented with RNAse inhibitor, prior to tubing single oocytes/embryos in single 0.25mL units of a sterile 8-tube PCR strip for lysis and downstream cDNA amplification.
1 day from time of harvest of oocytes/embryos
Javier Martin Gonzalez, Transgenic Core Facility, University of Copenhagen