Nuclear transplantation is a technical process in which the nucleus of an embryonic cell or adult cell is transferred into a denucleated oocyte using microsurgical methods to construct a recombinant embryo, which is then cultured for a period of time and transplanted into a surrogate to produce an offspring of the same genotype as that of the donor cell, which is also known as animal cloning technology.
Operation method
Preparation of transgenic pigs using somatic cell nuclear transfer technology
Materials and Instruments
Equipment: Move The basic process of preparing transgenic pigs using somatic cell nuclear transfer technology can be divided into the following steps: 1. Acquisition of porcine fetal tissues (1) Anesthetize the pregnant sows of 35 to 40 days of pregnancy. (2) Surgically cut open the abdominal cavity and remove the entire uterus. (3) Remove the fetus wrapped in fetal membranes and place it in a petri dish. Remove the membranes and expose the fetus on an ultra-clean bench. (4) The fetus was rinsed thoroughly with DPBS 3 times. (5) Remove the head and internal organs (intestines, liver, heart, etc.) of the fetus. (6) Fetal tissue is cut into 1 mm3 pieces in DPBS using sterilized ophthalmic scissors. (7) Collect the cut pieces of tissue into a 15 mL centrifuge tube, settle and discard the supernatant. 2. Porcine fetal tissue digestion (1) Transfer the tissue pieces to a 50 mL centrifuge tube. (2) Add 10 mL of trypsin solution (Trypsin-EDTA). (3) Oscillate at 39 ℃ for 30 minutes. (4) Disperse the tissue block by blowing with a gun tip. (5) Leave for a few moments to remove the residual tissue mass at the bottom. (6) Centrifuge the cell suspension at 500 g for 5 minutes and discard the supernatant. (7) Resuspend the cells with cell culture medium in 10 cm dishes and incubate at 39 ℃ in 5% CO2 incubator. 3. Porcine fetal fibroblasts subculture After the cells have grown to confluence, it is necessary to subculture the cells for expansion. (1) Aspirate the cell culture medium. (2) Wash the cells with DPBS. (3) Add appropriate amount of 0.05% trypsin to cover the cell surface and incubate at 39 ℃ for 5 minutes. (4) Add an equal volume of serum-containing cell culture medium, blow the bottom of the dish, and collect the cells into a 15 mL centrifuge tube. (5) Allow to stand for a few moments to remove any residual tissue mass from the bottom. (6) Centrifuge at 500 g for 5 minutes to collect cells. (7) Discard supernatant and resuspend cells in cell culture medium. (8) Cultivate the cells in the ratio of 1:3. 4. Cell freezing (1) Digest and centrifuge the cells according to the method in step 3. (2) Resuspend the cells with cell freezing solution, count the cells, and adjust the cell suspension to 107 cells/mL with freezing solution according to the counting result. (3) Dispense cells into freezing tubes (100 μl per tube). (4) Place the freezing tubes in a programmed freezer box and place in a refrigerator at -80 ℃ overnight. (5) Remove the frozen tubes from the -80 ℃ refrigerator and put them into liquid nitrogen for long-term storage. 5. Cell thawing (1) Take out the cell freezing tube from liquid nitrogen and put it into 37 ℃ water bath for thawing. (2) Transfer the cells to a 15 mL centrifuge tube filled with cell culture medium and mix well, centrifuge at 500 g for 5 minutes, discard the supernatant and collect the cells. (3) Resuspend the cells with cell culture medium, spread them in 10 cm Petri dishes, and incubate them at 39 ℃, 5% CO2 incubator, and pass them on every 2-3 days. 1. Collection of oocytes
① syringe
② sterile surgical instruments
③Cell culture dish
④ Egg transfer needle
⑤37 ℃, 5% CO
2
⑤37 ℃, 5% CO2 incubator
⑥ Inverted microscope, stereoscope
⑦PCR instrument, electrotransfer apparatus, centrifuge
Reagents:
①Materials: Pigs
② Pregnant horse serum gonadotropin, human chorionic gonadotropin
③DPBS
④Hyaluronidase
⑤ Pentobarbital sodium solution
⑥70% ethanol
⑦ Trypsin solution (Trypsin-EDTA)
⑧ Freezing solution, operating solution, DMEM medium, PZM-3 liquid, NCSU23-BSA liquid
⑨PVA-TL-Hepes
⑩ Colchicine (colchicine, Sigma, C9754), bisbenzamide (Sigma, B1155), Thimerosal (Sigma, T2295), DTT (Sigma, 45777-9)
(1) Collect the ovaries from the slaughterhouse in sterilized saline, keep them warm at 30-35 ℃, and send them back to the laboratory within 4 hours.
(2) Rinse the ovaries with 37 ℃ saline 3 to 4 times to remove blood stains.
(3) Use a 10 mL syringe with an 18-gauge needle to aspirate follicular contents of 3-6 mm in diameter and place them in a centrifuge tube held at 37 ℃.
(4) The follicular fluid was allowed to rest for 5 minutes, the supernatant was discarded, and the follicular fluid was washed three times with PVA-TL-Hepes.
(5) Under the stereomicroscope, the complexes (COCs) with homogeneous cytoplasm and multilayered outer layer of oocytes were picked out, and washed in PVA-TL-Hepes for two times, and then washed in maturation solution for three times.
(6) The COCs were placed in a 4-well plate for maturation culture, with 40-70 pieces per well, and cultured in an incubator with saturated humidity, 39 ℃, and 5% CO2 for 42-44 h. The COCs were then washed in PVA-TL-Hepes solution for 2 times, and then washed in maturation solution for 3 times.
2. Treatment of mature oocytes
(1) Place the mature oocytes in a 1.5 mL centrifuge tube containing 1 mL of hyaluronidase (1 mg/mL) after 42-44 hours of culture.
(2) Shake vigorously for 4 to 5 minutes and transfer to a 35 mm dish containing operating solution.
(3) Transfer the oocytes with intact plasma membrane, regular shape, and obvious perivitelline gap to the new operating solution for use.
(iii) Handling of donor cells1. Acquisition of cycling cells
(1) Resuscitate the cells and pass them on to a 24-well plate or a 4-well plate.
(2) After 2-3 days of culture, collect the cells by confluent digestion.
(3) Centrifuge the collected cells and resuspend them with 200 μl of the operating solution to be used as donor cells for nuclear transplantation. 2.
Acquisition of G-phase cells (serum starvation)
(1) Thaw the cells and spread them in a 24-well plate or 4-well plate.
(2) After 12-24 hours, when the cells grow to confluence, aspirate the medium, add 500 μl of DMEM medium containing 0.5% FBS, and incubate for 5 days.
(3) Replace the liquid, add 500 μl of DMEM medium containing 0.1% FBS, and continue to incubate for 3 days.
(4) Digest and collect the cells by centrifugation.
(5) Resuspend the cells with embryo manipulation solution and use as donor cells. 3.
3. Acquisition of G2/M phase cells
(1) Thaw the cells and spread them in 24-well plates or 4-well plates.
(2) After 12-24 hours, when the cells grow to confluence, aspirate and discard the medium, add 500 μl of cell culture medium containing 1.0 μmol/L colchicine (Sigma, C9754), and incubate for 24 hours.
(3) Digest and collect the cells by centrifugation.
(4) Resuspend the cells with embryo manipulation solution and use as donor cells.
4. Preparation of thawed cells
(1) Thaw the cells in a water bath at 37 ℃ and add 200 μl of DPBS.
(2) Leave at room temperature for 30 minutes.
(3) Add 800 μl of cell culture medium, centrifuge at 500 g for 5 minutes and discard the supernatant.
(4) Resuspend the cells with 50-100 μl of operating solution and use as donor cells for nuclear transplantation (Figure 5-1-27).
(iv) Nuclear transplantation1. Denucleation of mature oocytes
(1) Mature oocytes are stained with 5 μg/mL bisbenzamide (Sigma, B1155) for 30 minutes.
(2) The oocytes were then transferred to a mineral oil-covered enucleation droplet and left for 5 min.
(3) Under a micromanipulator, part of the cytoplasm containing the mid-stage nucleus in the vicinity of the first polar body and its vicinity was aspirated with a 25-30 μm diameter enucleation tube.
(4) Confirm that the aspirated cytoplasm contains an intermediate nuclear chromosome under ultraviolet light.
2. Importation of nuclei from donor cells
(1) Direct injection method
(1) Place the oocytes in the embryo culture medium after nucleation is completed in batches, and return them to the incubator to recover for 30 minutes.
(2) Replace the needle with a 10 μm diameter (G/G stage cells) or 15 μm diameter (G2/M stage cells) with a pointed needle, and place the enucleated oocytes and donor cells in a mineral oil-covered microscopic drop.
3) The donor cells were repeatedly blown with the syringe needle to rupture their cell membranes.
(4) Inhale the nucleus of the donor cell together with the residual cytoplasmic components into the injection needle, enter the denuded oocyte through the notch in the zona pellucida left during denudation, penetrate the plasma membrane, and inject the donor nucleus and cytoplasmic components into the denuded oocyte.
(2) Fusion method
1) The nucleus and polar body of the aspirated oocyte are spat out after the completion of the denucleation of each oocyte in the denucleation operation drop.
2) Aspirate a donor cell with a denuding tube, enter through the gap in the zona pellucida formed during denudation, place the donor cell under the zona pellucida, and squeeze the zona pellucida with a denuding needle to ensure that the donor cell is in close contact with the cytoplasm of the recipient oocyte.
3）After the completion of denucleation and nuclear injection, the oocyte-somatic cell pair was placed in NCSU23-BSA liquid, put back to the incubator and wait for the fusion operation.
(4) Adjust the parameters of the electrofusion instrument: 1.20 kV/cm, 30 µs DC pulse, 2 times, 1 second interval.
(5) The oocyte-somatic cell pairs to be fused were washed in the fusion solution for 3 times and placed between the fusion electrodes covered with the fusion solution, and the glass microneedle was pivoted to make the contact surface of the oocyte-somatic cells parallel to the direction of the electrodes, and the electrode buttons were triggered to execute the electric pulses for cell fusion.
(6) After electrical stimulation, place the cell pairs in the culture medium for 30 minutes, check the fusion situation under the body microscope, and place them in the incubator for further cultivation.
3. Activation
(1) Electrical activation: Mature porcine oocytes can be easily activated by electrical stimulation, and the activation of reconstructed embryos can be realized by adopting the above parameters of electrical fusion. If the method of electric fusion is used in nuclear transfer, there is no need to further activate the reconstructed embryo, and the embryo has already been activated at the same time of fusion; while if the method of direct injection is used, it is necessary to activate the reconstructed embryo, at this time, if electric activation is used, the reconstructed embryo only needs to be electrically stimulated by electric pulse using the same parameters as those of electric fusion.
(2) Chemical activation: Porcine oocytes can also be activated under the action of chemical activators, and the co-treatment of Thimerosal and DTT is commonly used for the chemical activation of porcine oocytes or reconstituted embryos.
(1) Oocytes or reconstituted embryos were treated with 200 μmol/L Thimerosal (Sigma, T2295) for 10 min.
2) Oocytes or reconstituted embryos were washed once in the operating solution.
3) Oocytes or reconstituted embryos were treated in 8 mmol/L DTT (Sigma, 45777-9) for 30 min.
(4) Oocytes or reconstituted embryos were washed once in operating solution.
(5) The oocytes or reconstructed embryos were washed twice in the embryo culture medium and cultured in an incubator.
(E) In vitro culture of embryoAfter activation, the reconstructed embryo is placed in a four-well plate containing 500 μl of embryo culture medium, covered with mineral oil, and cultured in an incubator with saturated humidity, 39 ℃, and 5% CO2. The formation of prokaryotic nucleus can be observed 8-15 hours after activation, egg cleavage can be observed 24-36 hours later, and blastocyst can be formed in 6-7 days.
(VI) Embryo Transfer1. Preparation of embryo transfer recipient pigs Natural estrus recipient pigs: conduct estrus detection for a certain number of sows twice a day, and select healthy sows in estrus on the day before or on the day of embryo transfer as embryo transfer recipients. Simultaneous estrus in recipient pigs can also be induced by drugs, and the experimental methods are:
(1) According to the estrus condition of the recipient pigs and the transfer plan, 18-22 mg of Regu-Mate was mixed into the daily feed of a certain number of recipient pigs.
(2) Stop feeding Regu-Mate and inject HCG 1000U intramuscularly 105 hours later.
(3) Embryo transfer was performed 22 to 26 hours after HCG injection.
2. Surgical operation of embryo transfer
(1) Anesthesia was induced by tachyzoin in the recipient pig, anesthesia mask was put on, the anesthesia machine was turned on, and anesthesia was maintained by isoflurane.
(2) Conventional surgical methods were used to cut 7-10 cm at the penultimate pair of papillae in the abdominal midline, traction out one side of the uterine horn, and take out the ovaries along the uterine horn and oviducts on that side, and then rinse the uterus and ovaries with 37 ℃ physiological saline to maintain the temperature and humidity of the reproductive tract, and observe the ovulation situation.
(3) The embryos were removed from the incubator and placed in the operating fluid, and transported to the site of the transfer procedure in a 39 ℃ incubator.
(4) The embryos were aspirated into the embryo transfer tubes with a 1 mL syringe under a somatoscope (Tomcat catheter, Sherwood Medical, St. Louis,MO).
(5) The transfer tube containing the embryos was inserted into the fallopian tube, delivered as deep as possible into the fallopian tube, and withdrawn from the transfer tube after slowly pushing out the embryos.
(6) The fallopian tube and uterus were reset, penicillin and streptomycin were applied to the incision, and it was sutured according to the three layers of peritoneum, subcutaneous fascia, and skin.
II. Preparation of transgenic pigs based on cloning technology of freehand operation (freehand cloning)1. Acquisition of porcine mature oocytes in vitro-Collect cultured oocytes according to the method before this book.
2. Preparation of porcine donor cells - Cultivate and prepare donor cells according to the method in this book.
3. Cutting and denucleation of oocytes
4. Reconstruction of cloned embryos
(1) Fusion and activation
(1) First electrofusion: The parameters of the first fusion are 200 V/cm, 9 microseconds. Take half of the cut half eggs (cytoplasm) to be the fusion receptor, i.e. the fusion of cytoplasm and somatic cells, each time, take 2~3 half eggs into PHA (1 μg/mL) for about 3 seconds, transfer and paste the digested somatic cells preserved in the T2 droplet, one half egg corresponds to one somatic cell, paste them well, put them into fusion solution for equilibrium, and repeat the above work. Subsequently, electrofusion is performed in the fusion tank as outlined in Figure 5-1-35. After electrocution, the egg-somatic cells are moved into the T11 droplet until all are complete.
2) Second Fusion and Electroactivation: The parameters for the second fusion and electroactivation are 86 V/cm for 80 µs. Eggs with somatic cells already fused as described above were fused one by one with half of the retained half-eggs. The eggs were incubated in a T2 droplet for 15-20 minutes until the two halves were fused into a single reconstructed embryo.
(3) Chemical activation and in vitro culture: The reconstructed embryos were then transferred into PZM-3 liquid containing 5 μg/mL CB and 10 μg/mL CX for chemical activation. 3 hours later, the chemically activated reconstructed embryos were washed 2-3 times in PZM-3 liquid without CB and CX, and then transferred into prepared wells for in vitro cultivation in PZM-3 liquid in an incubator containing 5% O2, 5% CO2, and 5% CX. The incubator conditions were 5% O2, 5% CO2, 90% N2, 38.5 ℃, and 100% humidity. Every 1 day, the eggs were taken out and observed for splitting and recorded (be careful when taking out the eggs, otherwise the eggs will probably be shaken out from the small holes).
5. Embryo transfer
(1) Selection of embryos for transfer: Since the cloned embryos are without zona pellucida, the cloned embryos that have developed to the level of mulberry embryo and blastocyst are selected on the 5th and 6th day after cloning (the day of cloning is day 0) (Fig. 5-1-38). The embryos selected for transfer are then transported to the farm where the surgical transfer is performed using a holding tank.
(2) Embryo transfer: Sow recipients are prepared in the farm. The estrus period of the sow recipients should be basically the same as the developmental period of the embryos, i.e., sows on the 4th to 5th day of estrus are used as the recipients for the embryo transfer, and the oviducts and uterine horns of the recipients are found by surgical methods. The embryos are aspirated into the tube to which they are attached using a 1 mL syringe, and the embryos are aspirated in a sequence of liquid-bubble-liquid-bubble-embryo and liquid, and transferred into the surgically prepared sow recipient (Fig. 5-1-39), followed by surgical suturing. Pregnancy is tested weekly from 20 days onwards.
(3) Cloned piglets: Observe the pregnancy of pregnant recipient sows on day 114-117, and be ready to deliver cloned piglets.
Gene transfection and screening of donor cells1. In accordance with the experimental steps described earlier in this section, thaw and subcultivate the cryopreserved porcine fetal fibroblasts to 70%-80% confluence. 2.
2. Aspirate the culture medium, digest, centrifuge and collect the cells according to the procedure described earlier in this section, and wash them with PBS once or twice.
3. Suspend the collected cells in antibiotic-free PBS and count them, adjusting the cell density to 5x10° cells/mL. 4.
4. Add 200 μl of cell suspension to the shock cup at a time, then add 4 μg of purified linear DNA, gently tap the bottom of the cup to mix, and place in the shock bath.
5. According to the specification of the electroshocking cup used or the model of the equipment, set the corresponding parameter combinations for electroshocking (reference parameters: U: 230 V; TC: 5 ms; No: 1; Cuvettes: 0.4 cm), and then let it stand for 2 minutes at room temperature after electroshocking, and add it to the six-well cell culture plate containing culture medium and put it into the incubator for culture.
6. According to the design scheme of the transgenic vector, screening will be carried out; if there is a screening marker gene (e.g. neomycin resistance gene) in the vector, the transfected cells will be screened by adding the culture medium containing the screening drug (G418) after 48 hours of cultivation, the culture medium will be changed every two days, and the number of cell clones formed in each well will be observed after two weeks; if the vector does not contain a screening marker gene, the single clones will be separated by gradient dilution in If the vector does not contain the screening marker gene, it can be isolated by gradient dilution of the culture, single clones, and then identified one by one.
7. Transgenerate the cell clones for transgene identification.
8. Expand the correctly identified positive cell clones for subsequent nuclear transplantation or cryopreservation.For more product details, please visit Aladdin Scientific website.