Method for producing clened cows
A technology of male cattle and oocytes, applied in the field of production of cloned cattle, can solve the problems of unsatisfactory, low embryo output, low productivity of cloned animals, etc.
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Embodiment 1
[0039] The present invention is further illustrated with the following examples, which are not intended to limit the scope of the present invention. Example 1: Preparation of Donor Cells and Recipient Oocytes
[0040] In order to prepare donor cells, tissues were collected from the inner skin of Korean bull ears, washed with PBS (phosphate-buffered saline, Gibco's BRL, Life Technology, USA), and crushed to 100 mesh. Then at 39°C, 5% CO 2 Incubate the tissue for 1 hour in PBS containing 0.25% trypsin, 1 mM EDTA and 1 mg / ml type II collagenase, and after the tissue is digested by the enzyme, centrifuge at 1500 rpm for two minutes, then Suspended in DMEM (Dulbecco's Modified Eagle's Medium, Gibco's BRL, Life Technology, USA) supplemented with 10% FBS, 1% NEAA (non-essential amino acids), and 1% penicillin-streptomycin. Transfer the suspension into a cell culture dish and store at 39°C, 5% CO 2 ambient incubation to obtain somatic cell lines. Thereafter, the cells were treated...
Embodiment 3
[0049] Replace the cutting tube mounted on the micromanipulator with an injection tube. Enucleated oocytes were washed three times with TCM199 wash medium and then pipetted into injection droplets. The donor cells are drawn into the syringe and then transferred into the injection droplet. Figure 4 Indicates the process of transferring a somatic cell into an enucleated oocyte. Such as Figure 4 As shown, enucleated oocytes were placed with their cleft orientation at 1 o'clock, held with a holding tube, and then injected into donor cells through the cleft using a syringe and hydraulic pressure to obtain a reconstructed embryo. Embryos were washed three times with TCM199 wash medium and then incubated in TCM199 wash medium. Example 3: Electrofusion and activation
[0050] Electrofusion and activation Reconstructed embryos were electrofused using an electrocytomanipulator (USA, BTX, ECM2001) and then reactivated. Add 15 microliters containing 0.28M mannitol, 0.5M mhepes (pH7...
Embodiment 4
[0051] For activation, embryos were incubated for 4 minutes in the dark in a solution of ionomycin (Sigma Chemical Co., USA), a TCM199 wash solution containing 5 [mu]M ionomycin and 1% BSA. The ionomycin stock solution was prepared by dissolving 1 mg of ionomycin in 1.34 ml of DMSO. Activated embryos were incubated for 5 minutes in 35 mm dishes containing TCM199 wash medium supplemented with 10% FBS to wash ionomycin from the embryos. Example 4: Post-activation and in vitro culture of electrofused embryos
[0052] The activated embryos were activated for 4 hours in 25 microliters of cycloheximide (Sigma Chemical Co., USA) solution, which was obtained by adding actinomycetes in the culture medium mTALP for in vitro culture. Ketone stock solution (10 mg / ml in ethanol) was prepared by diluting to 10 μg / ml. Embryos were then screened, and the selected embryos were incubated at a temperature of 39°C in 5% CO 2 Incubate for 7 days under ambient conditions.
[0053] In applying t...
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