细胞核移植 z-bo 2020-10-14 318

　　(1) Improved NCSU-23 culture medium

　　CSU-23 culture medium is a phosphate buffer without NaHCO 3, and the physiological pH phosphate buffer is composed of dihydrogen phosphate anions and dihydrogen phosphate anions with a molar ratio of 3:1. These ion exchanges cause constant changes in the Na + and K + concentrations, so that the NaCl and KCl concentrations can be adjusted to maintain the osmotic pressure and the Na +/K + ratio.

　　(2) Excessive ovulation of donor sows, collection of oocytes and fertilized eggs

　　For hybrid young sows weighing 125 to 145 kg, mix 18 to 20 mg of tofu with feed and germinate at the same time. I will. Depending on the duration of the sexual cycle, the sows are fed for 5-14 days, and 250 μg of estrogen (Bayer) is injected intramuscularly on the last day of lesbian treatment. 15 to 17 hours after the last leguminous feeding, 1500 IU of PMSG was injected intramuscularly to induce superovulation in sows. 82 hours after PMSG injection, 1000 IU HCG was injected intramuscularly. 46-54 hours after HCG injection, the oviduct was washed back with Dalbecco phosphate buffer containing 4 g/L bovine fetal serum and preheated to collect oocytes. 24 to 36 hours after HCG injection, fertilized eggs are collected by artificial seeding or natural mating. 52-54 hours after HCG injection, the eggs were washed with PBS containing 4 g/L fetal bovine serum.

　　(3) Separation and culture of porcine granulosa cells 28-51 hours after HCG injection. Granulosa cells are collected by aspirating the follicular fluid of hybrid sows with 2-8 mm ovary and centrifuging for 10 minutes. DMEM (10% bovine fetal serum, 0.01 mM non-essential amino acids, 2g/ml basic fibroblast growth factor (bFGF), 6μl/ml gentamicin) continue to grow for several days, and then cryopreserve. Before nuclear transfer, put (1-5) x 104 granular cells into each 35 mm petri dish, which contains 0.1 mM non-essential amino acids, 2g/ml bFGIF and 10% bovine serum in DMEM solution . The mixed culture completes the merger. These cells were cultured in DMEM containing 0.59% fetal bovine serum for 48-72 hours under serum starvation. It was then digested with membrane protease and collected, placed in a modified NCSU-23 phosphate medium, and then used as a nuclear donor, suspended and stored at 38.5°C for 20-120 minutes.

　　(4) Activation of oocytes

　　Place the egg cell in the activation medium and stimulate it with 1.0 kV/cm DC. The electric shock every 5 seconds is 60 μs, a total of 2 electric shocks. The activation medium is a D-sorbitol solution containing 0.1 mM DDL4, 0.05 mM CaCl2 and 0.3 M.

　　(5) First nuclear transfer embryo reconstruction

　　The collected egg cells were washed several times with PBS containing 4 g/LBSA (38°C), and then transplanted into NCSU-23 phosphate buffer without calcium at 38°C. done. Return the liquid to the laboratory. In order to remove the cell nucleus, a small amount of cytoplasm under the first pole was aspirated with a glass needle with a diameter of 18 mm, and the aspirated nucleus was exposed to ultraviolet light to confirm the presence of the metaphase plate. Incubate the egg cells for 20 minutes at 38°C in decalcified phosphate buffer NCSU-23, which contains 5 pg/ml cytokeratin B and 7.5 μg/ml Hoechet-33342, each granuloma The cells are placed under the shingles of each enucleated oocyte. The chimera is transferred to the fusion device. The fusion medium is 700μl of deionized water, which contains 0.3M mannitol, 0.1mM DDL4 and 0.1mM CaCl2, and the osmotic pressure is 280mosm. After 5V AC electric shock for 5 minutes, two 1.5kV/cM DC electric shocks (60μs") to fuse and activate cells. The equipment used is ECM2001 electric stimulation cell manipulator. (BTXIncSan-Diego): Use dibasic carbonic acid Wash the chimeras with salt solution NCSU-23 several times, and incubate them in a humid atmosphere at 38.5°C containing 5% CO 2 for 0.5 to 1 hour, and expand the fused chimera by 300 times. Observe under an inverted microscope with 1.2 The cells were stimulated again with kV/cM DC, and two consecutive electric shocks were performed, each shock was 60μs. After treatment, they were cultured in NSCU-23 medium overnight.

　　(6) Reconstruction of the second embryo transfer The fertilized eggs were centrifuged at 14900g for 15 minutes in a Biofuge-13 centrifuge, and centrifuged at 38° in phosphate buffer NCSU-23 containing 5.0 μg/ml cytokeratin B. Put it in C for 20 minutes, use the fertilized egg and 2 pronuclei, 25.? A 35μm glass tube sucks the membrane containing 2 pronuclei and connects to the membrane. Take out (if the second polar body is excreted, also take it out) From that day on, the first pseudo-nucleus transplantation began to prepare the nuclear body containing the pseudo-nucleus. Except for the use of a 45μm glass needle for enucleation, the rest is the same as the method for nucleation. Place a single nucleus in the space between the egg circumference of a single enucleated fertilized egg. After using the alternating current 5V for 5 seconds, then 1.2 2 kV/cM direct current. Each time 60μs, the fusion chimera was washed several times with NSCU-23 buffer at 38°C, and incubated in a humid environment (containing 5% CO2) for 30-60 minutes.

　　(7) PMSG and HCG can be used in combination to maintain pregnancy when sows receive treatment. On the 10th day, the intramuscular injection was 3 to 3.5 days, and 10,000 IU PMSG was injected, and then on the 13th day of the estrus cycle, the HCG was injected intramuscularly.