Method for isolating pig embryonic stem cells

  Pig embryo isolation and culture system will change the expression of pluripotent genes in nuclear transfer embryos, parthenogenetic embryos and in vivo embryos, and will also affect the efficiency and quality of pig pluripotent stem cell formation. Isolation of porcine embryonic stem cells is essential, as follows:

  Cell culture: Porcine fetal fibroblasts for nuclear transfer donors were isolated from the ears of newborn piglets. The cell culture medium was DMEM, 15% fetal bovine serum, and the culture temperature was 37 ° C, 5% CO 2 . The cells were cryopreserved, digested with 0.25% (w/v) trypsin, neutralized and centrifuged at 200 x g for 5 min. The cells were resuspended and an equal amount of cryopreservation solution (40% DMEM, 40% FBS and 20% DMSO) was added and stored in liquid nitrogen.

  Oocyte collection and preparation: Porcine ovaries were collected from the slaughterhouse and stored in physiological saline containing cyan/streptomycin at a transport temperature of 32-38 ° C and returned to the laboratory within 3 h. Ovarian oocytes were harvested by aspiration. The oocytes in the 3~6mm follicles were taken out in a 15mL centrifuge tube and allowed to stand in a 37°C water bath for several minutes. After the oocytes were settled, wash them with the egg washing solution twice, and pick up the granule cell layer under the microscope for 3 layers. The above cumulus oocyte complexes were then transferred to a U-shaped dish for 42-44 hours, and 500 μL of mature medium was added to each well, and 50-70 pieces were placed in each well.

  Parthenogenetic activation and embryo culture: Mature oocytes were electrically activated by electrofusion. After rinsing, the oocytes were transferred to 2 mmol/L 6D for 2.5 h. The oocytes were transferred to embryo culture medium G1, NCSU23 and porcine fertilized egg medium respectively, and the cleavage rate was recorded 24 hours later. After 3 days, the embryos developed in the medium G1 were transferred to the blastocyst medium G2. After culturing for another 3 to 4 days, the number of blastocysts was counted and collected.

  Somatic cell nuclear transfer Embryo culture: The first polar body of the mature oocyte is removed and subsequently injected into the donor cell. After micromanipulation, the cells were transferred to M199 medium containing 10% FBS, incubated at 38.5 ° C, 5% CO 2 for 0.5 h, activated by electrofusion, transferred to 6D for 2.5 h, and then reconstituted in NCSU23 medium. The medium was cultured for 3 days, and then the NC-SU23 was replaced and cultured for 3 to 5 days to observe the development of the embryo.

  In vivo embryo collection: The 7th to 8th blastocysts were collected from artificially fertilized sows, all methods are referred to Stokes et al. The estrus of the sow was monitored daily, the estrus date was taken as 0d, and the artificial insemination was performed at 6, 12 and 18 hours after the estrus was identified. A pre-warmed 10% FBS phosphate buffered saline solution was used to flush the embryos from the uterus.

  Isolation of embryonic pluripotent stem cells from pig embryos: The blastocysts from which the zona pellucida was removed were inoculated onto the mitomycin C-treated mouse embryonic fibroblast feeder layer for 2 to 4 days, and the blastocyst inner cell mass appeared after 3 to 5 days. . When the ICM size is moderate, the cells are mechanically separated, re-plated onto a new feeder layer, and different porcine embryonic stem cell culture media are used. Embryonic pluripotent stem cell clones are mechanically passaged every 5-7 days.

  Immunofluorescence assay: embryos were fixed in 4% paraformaldehyde for 10 min, washed twice with PBS, then added with 0.25% Triton-X for 30 min at room temperature, washed three times with PBS, 1% BSA was blocked at room temperature for 1 h, and the primary antibody was incubated at 4 °C for 24 h (1:1) 200, CDX2; 1:200 NANOG). Rinse 3 times with PBS and incubate the fluorescently labeled secondary antibody (1:1000) for 1 h at room temperature. After rinsing twice with PBS, DAPI staining was performed for 2 min, and PBS was washed twice, and photographing was observed under a fluorescence microscope.

  qRT-PCR: After removing the zona pellucida of pig embryos with 0.5% pronase A, immediately add 5 μL of lysis buffer (5 mmol/L dithiothreitol (DTT), 1% NP-40, 1 U/uL RNA Enzyme inhibitor), incubated on ice for 30 min. The lysate was reverse transcribed into cDNA using a reverse transcription kit. qRT-PCR system: cDNA 2 μL, SYBRgreenmix 12.5 μL, upstream and downstream primers each 0.3 μmol / L 0.5 μL, ddH2O supplemented to 25 μL. Reaction procedure: 95 ° C for 30 s; 95 ° C for 5 s, 60 ° C for 30 s for a total of 40 cycles.

 

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