Method for physically removing an oocyte zona pellucida
An oocyte and zona pellucida technology, applied in the biological field, can solve the problems of difficult to maintain oocyte activity, difficult to master the operation method, low efficiency of removing the zona pellucida, etc., and achieves less mechanical damage, fast removal of the zona pellucida, and maintaining activity. Effect
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Embodiment 1
[0029] Main equipment: microinjector (Eppendorf, Germany, FemtoJet 4i); Nikon stereo microscope (Japan); pneumatic manual microinjector (brand: Eppendorf, model: CellTram Air, place of origin: Germany); injection needle (MIC -35-35, ORIGIO, USA); egg-holding needle (MPH-MED-35, ORIGIO, USA); Petri dishes and Pasteur pipettes were purchased from Falcon, USA; pipettes were purchased from Eppendorf, Germany.
[0030] The oocytes of female cynomolgus monkeys were collected according to the conventional method of ovulation induction, and the zona pellucida was physically removed from the oocytes of cynomolgus monkeys. The specific steps were as follows:
[0031] (1) Prepare microdroplets of micromanipulation solution: add 50uL serum albumin (BSA, Sigma, USA) to 10mL TL-hepes solution (Caisson, USA), then add 50uL double antibody (Gibco, USA), mix well Obtain the micromanipulation solution; place the prepared micromanipulation solution in the operation tray, and divide the dish into...
Embodiment 2
[0038] The oocytes of female C57 mice were collected according to conventional methods of ovulation induction, and the zona pellucida was physically removed from the oocytes of C57 mice. The specific steps were as follows:
[0039] (1) Same as embodiment 1;
[0040] (2) Same as embodiment 1;
[0041] (3) Place the operating disk on the micromanipulator table of the micromanipulator, use a pipette gun to draw 5-10uL sterile PBS solution into the inner cavity of the tip of the injection needle, and install the egg-holding needle and the injection needle on the micromanipulator. On the fixed rod of the manipulator, adjust the screw, and observe in the field of view of the inverted microscope, so that the oocyte, egg-holding needle and injection needle are at the same horizontal position;
[0042] (4) The preset injection pressure Pi of the microinjector in cleaning mode is 120hPa, and the balance pressure Pc is 220hPa. Connect the egg holding needle to the pneumatic manual micro...
Embodiment 3
[0048] The oocytes of female buffalo are collected according to the conventional method of ovulation induction, and the zona pellucida is removed from the buffalo oocytes by physical methods. The specific steps are as follows:
[0049] (1) Same as embodiment 1;
[0050] (2) Same as embodiment 1;
[0051] (3) Place the operating disk on the micromanipulator table of the micromanipulator, use a pipette gun to draw 5-10uL sterile PBS solution into the inner cavity of the tip of the injection needle, and install the egg-holding needle and the injection needle on the micromanipulator. On the fixed rod of the manipulator, adjust the screw, and observe in the field of view of the inverted microscope, so that the oocyte, egg-holding needle and injection needle are at the same horizontal position;
[0052] (4) The preset injection pressure Pi of the microinjector in cleaning mode is 200hPa, and the balance pressure Pc is 250hPa. Connect the egg holding needle to the pneumatic manual m...
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