nuclear transplantation

Summary

Nuclear transplantation is based on the theory that all nuclei from the same embryo or cell line contain exactly the same genetic information as the fertilized egg, and should have the same full potential to guide individual development in terms of genetic composition as the fertilized egg. When a donor nucleus is transplanted into the cytoplasm of a recipient, the donor nucleus undergoes reprogramming by cytoplasmic factors.

(When the donor nucleus is transplanted into the cytoplasm of the recipient, the donor nucleus undergoes reprogramming by cytoplasmic factors, thus returning to the state of a syncytial nucleus and guiding the development of the recombinant embryo into a complete individual. The success of mammalian nuclear transplantation demonstrates that the nucleus of highly differentiated somatic cells still possesses totipotency, i.e., it is capable of reprogramming and guiding the development of early embryonic cells into new individuals.

Author: Xuedao Pei, this experiment is from "Stem Cell Guide".

Operation method

nuclear transplantation

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Major equipment and reagents required for nuclear transplantation operations Materials and Equipment Required for Nuclear Transplantation

Microprocessor (with IOx and 20x objectives, including heating and cooling elements, glass holder, and stereomicroscope combination), platinum-iridium filament, needle puller (with variations in the choice of glass filament and control), glass capillary tubes [for holding needles (1x90m m ), for denuding or injecting needles (1.2m m OD, 0.90m m ID), and for puncture needles (10 ul), respectively], Micromanipulators, inverted microscopes, electrofusion systems, microscope heating/cooling plates, CO2 incubators, piezoelectric micropipettes to drive the micropipettes, and other equipment.
Piezo micropipette drive unit, operating chamber, transfer pipette, 2 7-gauge needles, and 30 m m petri dish.

Culture media and reagents

Operational media: M 2 medium, medium for in vitro culture, M 16 medium.
Hyaluronidase, M 2 medium at a final concentration of 300 units/m2.

Medium for microinjection: For microinjection, HEPES-buffered medium, e.g., modified BMOC-3 plus HEPES) supplemented with cytochalasin B (5 ^ig/ml), may be used. If the egg membrane is dragged into the cytoplasm during injection, the egg will probably lyse quickly. Cytochalasin B hardens the membrane during injection and prevents egg lysis. Another method with similar results is to use 7% (W/V) ethanol. Using a platform cooling device to lower the temperature to IoC also hardens the egg membranes. As long as the eggs are brought back up to 37.5°C: within about 45 m i n , egg survival will not be altered. The cooling effect takes up to lOmin to harden the egg membranes significantly, so the simpler method of cytochalasin B is preferred.

Cytoskeletal inhibitors: cytochalasin B, nocodazole; stock solution (IOOOx) prepared in dimethyl sulfoxide and stored at -20 °C or dissolved and immediately chilled to 4 °C.

0.25% Trypsin: Prepared with phosphate containing 0.2% EDTA. 0.25% Trypsin: Prepared in saline (PBS) containing 0.2% EDTA in phosphate buffer (PBS) for digestion of sphere and cells.

Inactivated Sendai virus, paraffin oil, Hoechst 33342 [storage solution (lmg/ml) prepared in double-distilled water], Zimmermann cell fusion medium, O.3mol/L mannitol, aphidicolineX storage solution (lmg/ml commissioned in DMSO, stored at -20°C or immediately cooled after dissolution), P V P (polyvinylpyrrolidine), and PVP (polyvinylpyrrolidone). (polyvinylpyrrolidone), P V A (polyvinyl alcohol, used instead of B S A to prevent cytoplasmic inclusions from sticking to the donor cells when administering direct injections), strontium-supplemented medium (Appendix 3), complement [for isolation of the inner cell mass (ICM)], Cell Proliferation Kit (cell cycle kit for the detection of synchronized embryos), hormones for ovulation monitoring, PMSG (Pregnant Horse Serum) and Gonadotropin. (Pregnant Horse Serum Gonadotropin) and H C G (Human Chorionic Gonadotropin).

The main instruments necessary for the nuclear transfer procedure are

The main instruments include a fluorescence inverted microscope and a micromanipulator. Microscopes of different configurations should have good differential interference contrast (DIC). Typically, 180 to 400 times magnification is used, and DIC is usually required for microprocessing at a magnification of 200 x. In addition, there are cell fusion instruments, needle pullers, needle forgers, and needle grinders. The devices are described below. (1) Needle holder. Micromanipulator manufacturers offer a number of needle holders or peripheral collar kits. For example, Leica's needle holder is a peripheral collar kit that secures two brass fittings with adapter washers to form a tight connection. The assembly is capped with a metal threaded cap that adjusts to the encircled syringe needle to form a leak-proof washer. The diameter of the washer should match the outside diameter of the injection needle and needs to be replaced frequently to make the tubing used effective. (ii) Adjustment devices for micromanipulators. The micromanipulator has a number of adjustable dials to adjust the sensitivity and tension of the joystick. The various adjustments made on the micromanipulator should be demonstrated by a qualified technician. ③Shock Resistance
Table. When the microinjection needle is inserted into the egg, the slightest vibration can damage the egg, causing it to cleave (immediate or delayed) and making localization of the pronucleus difficult. To avoid such errors, vibration-free tables must be used. For example, a vibration-resistant base supports a stainless steel-covered lead frame (or composite or steel frame) on four nitrogen-driven pistons. Otherwise, marble tables are good and inexpensive in most cases. In certain cases, the location and environment of the laboratory will also play a role.

There are three types of glass tools needed for nuclear transplantation: a holding needle, a nucleation or injection needle, and a fine glass needle. Holding needles are used to hold the oocyte during microsurgery, denuding needles are used to remove chromosomes or nuclei from the oocyte or syncytiotrophoblast, injection needles are used to inject donor cells or nuclei into the peritrophic space or ooplasm, and fine-glass needles are used to cut the zona pellucida for nuclear transplantation. In terms of the quality of the material used for the instruments: the holding needles are to be made from thick glass tubes. The thinned part or center of the capillary is heated by hand over a flame and then quickly stretched. When used for holding eggs, the most suitable outer diameter is equal to or slightly larger than the diameter of the egg (80 to 100 um). The tip of the needle can be examined under a microscope to see if it is flat or has the proper outer diameter. Microscopy helps in observation and shaping of the needle by means of a needle manipulator equipped with a platinum-iridium wire (IOOum). The glass capillary is held perpendicular to the platinum-iridium wire on the needle former and heated slightly. Due to the melting of the glass, the end of the glass capillary becomes smaller and the end results in a slight bend of 30°. Fine glass needles can be made by heating the thinned part or the center of the capillary over a flame and then quickly stretching it by hand. To sharpen the needle, hold the glass capillary perpendicular to the platinum-iridium wire and pull the tip of the capillary downwards while the platinum-iridium wire is heated at a lower temperature. Repeat this step 2 to 3 times to make the capillary tip very thin.

All microtools can be slightly curved (about 1 mm from the tip) with the end slightly in contact with the heated wire, depending on the requirements of the microtome used, and more curved ones can be done by hand over a flame. Nucleating needles Nucleating syringes are made from thin-walled glass tubes. The glass capillary is first pulled with a needle puller and finally shaped with a needle forger. The glass capillary and platinum iridium wire into a horizontal direction fixed, the outer diameter of 1 〇 ~ 20p m of the end of the capillary tube gently contact the pin Yi silk end on the glass beads, slowly heated, the end of the capillary tube began to melt, the formation of a very thin glass needle. The appropriate outer diameter of the syringe needle should be similar to or slightly smaller than the diameter of the donor cell.

The main steps of the nuclear transplantation technique

Nuclear transplantation technique mainly includes micromanipulation technique and cell fusion technique, which includes a series of complex procedures: preparation of recipient cells, denucleation, preparation of donor cells, cell nuclear transplantation, fusion and activation, in vitro culture of reconstructed embryos and embryo transfer. Successful nuclear transfer relies on a variety of factors, including the type of recipient cell, the source of the recipient cell, the method of reconstitution, activation, the culture of the embryo, the type of donor cell, and the selection of donor and recipient cell cycles. Each of these is described below according to the procedure.

Preparation of recipient cells

To date, the main types of nuclear recipients used in mammalian nuclear transplantation are enucleated mature oocytes and prokaryotic fertilized eggs. Denucleated oocytes are the most commonly used. Nowadays, in vitro mature oocytes (M stage II) are increasingly used as receptor cytoplasm.

Mature oocytes as receptor cytoplasm

Stage M II oocytes are the most commonly used recipients for nuclear transfer, although the timing of activation varies from laboratory to laboratory (pre-nucleus transfer, nucleus transfer, delayed activation after nucleus transfer), and both early embryonic cleavage spheroids, embryonic stem cells (ESCs), and somatic cells can be transplanted into these recipients to successfully obtain progeny.

Mature oocytes are obtained in two ways: in vitro and in vivo. In vivo maturation is obtained by superovulation. While in vitro maturation culture is convenient and favorable for obtaining a large number of oocytes, there are several sources of oocytes for in vitro culture: (1) directly extracted or stripped from luminal follicles on the ovary; (2) obtained by pre-luminal follicle culture; and (3) oocytes preserved in cryopreservation. Currently, the former is widely used, while the latter two provide a broader source of oocytes.

Ovary collection and oocyte isolation

At present, the ovaries of animals mainly come from large slaughterhouses. Before collecting the ovaries, sterile saline should be heated to a temperature slightly higher than the predetermined temperature (e.g., 38--39 ℃), and then poured into pre-sterilized thermos flasks with a certain concentration of double antibiotic (penicillin 100U/ml; streptomycin 100U/ml) before departure. The ovaries were collected to minimize contamination of the ovaries, moving quickly and rinsing with warm saline before placing in the thermos flask. After removing the ovarian tissues, the ovaries should be preserved in saline with double antibiotic, and it was proved that the ovaries were best preserved at 30--38℃ for 4 h or less. The temperature and duration of ovarian preservation affected the development of blastocysts in IVF (in vitro fertilization) to a different extent, and the preservation of ovaries at 33-35 ℃ for 8 h did not reduce the ability of oocytes to mature in vitro. There are two methods to isolate the cumulus oocyte complex (COC): stripping and extraction. The extraction method is simple and rapid, and is currently used more often.

The oocytes are brought back to the laboratory, rinsed with sterilized warm saline or PBS, and then isolated on an ultraclean table. For aspiration, use a sterilized 12-gauge needle and aspirate some oocyte washings into a syringe prior to aspiration, then inject the aspirate into a clean, sterile centrifuge tube. Keep warm throughout the procedure to minimize temperature changes. Ovary collection and oocyte isolation are the most vulnerable to contamination, so it is important to maximize the sterility of the procedure.

Obtaining oocytes by superovulation

In mice, for example, nucleus transfer recipient chorionic cells are collected from inbred mice (C57BL/6xCBA or C3 H), and mature females (6 weeks) are superovulated by injections of 5U of pregnant mare serum gonadotropin (P M S G ) and 5U of human chorionic gonadotropin (H C G ) at 48-h intervals. gonadotrophin (H C G ) 5U for superovulation.
The oviducts were collected after mating with male rats of the same species. Twenty to twenty-two hours after the injection of HCG, the conidia were collected from the cut oviducts with a 27-gauge needle. The collected COC was treated with hyaluronidase (300N F units/m 2 ) at 37 ℃ for l to 3 min to dissociate the eggs from the zygotic cells. Naked 1-cell eggs were aspirated with an egg transfer needle and washed three times with M2 medium. Selected 1-cell eggs with two pronuclei and dipolar body were incubated with M16 medium at 5% CO2 in an incubator.

Forty-two to forty-four hours after superovulation with HCG, the oviducts were flushed with M2 medium and 2-cell stage embryos were collected from the oviducts of female rats. 4- and 8-cell embryos were collected from 2-cell embryos cultured in vitro, the latter being flushed from the oviducts and uterine horns 65 h after H C G injection. Tightly bundled mulberry stage embryos were obtained from 8-cell stage embryos after 24 h of in vitro culture. The collected embryos were warmed in M2 medium for use.

Oocyte maturation culture

The extracted fluid was poured into a dish to examine the eggs, and the oocytes were aspirated from 26 m m follicles on the surface of the ovary, and those with intact oocytes and homogeneous cytoplasm were selected and washed several times with well-balanced culture medium, and then put into the maturation culture medium. In vitro maturation culture was carried out in a CO2 incubator at a concentration of 5 % CO2 at 38.5 p.m. for 18 to 24 h to check the maturation rate. The maturation medium was composed of 10% FBS, 0.2 mmol/L sodium pyruvate, 0.1 m m ○ l /L glutamine, 5ug/ml
FSH, 0.3 IU/ml LH (luteinizing hormone), 1 ug/m l estradiol and 25 ug/m l gentamicin in T CM 199-H C 0 3 (with Earle's salt). The incubation conditions were 38.5 °C and 5 % CO2 relative humidity.

A sign of oocyte maturity was the radial dispersion of the oocytes, which was removed by blowing on the oocytes with a pipette, followed by microscopic observation of polar body excretion. The mature oocytes were put into a 0.1% hyaluronidase tube and shaken for 2~3 min, and then blown gently with a glass tube to completely detach the oocyte from the oocyte, and the oocyte with intact morphology and homogeneous cytoplasm, which had discharged the first polar body, was selected as the somatic cell nuclear receptor.

Freezing and preservation of oocytes

Oocytes are equilibrated with cryoprotectant, cooled down to -5~-7℃ at the rate of l ℃/min, planted with ice, and then cooled down to -30-40℃ at the rate of l ℃/m i n , and put into liquid nitrogen. Thaw at a rate of 25℃/m i n or more, thawing is generally carried out at room temperature or 37℃.

Processing of nucleated cells for cell cycle synchronization

When M-stage oocytes are used as recipient cytoplasm, the cell cycle of the donor embryo must be synchronized at G1, G2, or M stage because of the high activity of the maturationpromotion factor (M P F ) in the cytoplasm of M II stage oocytes. When the donor cell fuses with the MII oocyte, chromosome condensation occurs early due to the activity of MPF in the cytoplasm of the oocyte. When the donor nucleus in the oocyte is activated during chromosome condensation, the nuclear membrane is initially formed again in the G1 phase of the cell cycle and begins to form a prokaryotic structure. If the donor cell that fuses with the M II oocyte is in S phase, DNA will be abnormal due to chromosome condensation and subsequent activation. Therefore, the donor cell
Therefore, the donor cell must be in G1, G2 or M phase. After synchronization to G1 phase, the medium must be supplemented with bacteriocin until fusion with the oocyte cytoplasm occurs. If the donor cells are in G2 phase, exclusion of polar bodies after activation is necessary.

1 ) 4 Cells to Mulberry Stage Embryos

Synchronization of the cell cycle of mouse 2-cell stage preimplantation embryos to the M stage is well established and has no adverse effects. However, to synchronize the embryo cell cycle to the 4-cell stage, the 2-cell embryo is incubated in M 16 medium supplemented with nocodazole (3ug/ml) for I2 to 14 h, and then washed and cultured in M 16 medium supplemented with bacteriophage (5ug g/ml_ M 16 medium) in order to inhibit the synthesis of DNA. The division of both dividing spheres was synchronized at about 55 min after nocodazole was withdrawn. 8-cell stage embryos were treated with nocodazole-containing M 16 medium for 6 h, followed by incubation in medium supplemented with colistin when the mulberry stage embryos needed to be synchronized at the G 1 stage. When nuclei were used as donors at the 4-cell stage, nucleoplasts were removed by inserting a nucleolytic needle into the perivitelline space of each dividing sphere. When the dividing spheres are small and difficult to remove the nucleoplasm, such as in mulberry stage embryos, the zona pellucida is removed and the dividing spheres are disassembled into individual spheres with a needle in trypsin-EDTA solution. The disassembled sphere is kept at room temperature in medium supplemented with bacteriophage for use.

2) Inner cell mass (ICM) and luminal wall trophoblast cells of saccular embryos
Blastocysts can be recovered from female mice after 4 days of natural mating or from mulberry-stage embryos cultured for 24 h. In vitro culture was performed with M 16 medium supplemented with nocodazole (3ug/ml). The blastocysts are synchronized to the MII phase of the cell cycle by 12 h of in vitro culture in M16 medium supplemented with nocodazole (3ug/ml). After synchronization to the MII phase, the blastocysts were cultured in M16 medium without nocodazole but supplemented with bacteriophage (5ug/ml) for Ih to inhibit DNA synthesis. The zona pellucida of the blastocysts were removed by Tyrode acetate solution or protease, except for those at G1 stage.

The ICM of blastocysts was isolated by immunosurgery: blastocysts were treated with appropriate concentrations of polyclonal antibodies against mouse liver and kidney for 30 min at 37 ℃, then washed three times with M2 medium, and transferred to medium supplemented with complement (low-toxicity guinea-pig complement) (1:7) for 30 min at 37 ℃. By this time, the trophoblast ectoderm (TE) was removed from the culture. TE)
ICM cells were isolated by breaking the trophectoderm (TE).

In the preparation of mouse T cells, G1 blastocysts without transparent zones were further cultured in M16 medium supplemented with bacteriophage (5 ug/ml) until the blastocyst lumen was recovered. Mouse T cells were carefully isolated by immunosurgery. Isolated ICM and mouse T cells were treated with PBS containing trypsin (0.01%) and EDTA (0.02%) or collagenase (200 U) and DNA enzyme (lug/ml) for 30 min at 4 ℃ to disassemble into individual cells.

3 ) Determination of cell cycle

To determine whether the donor dividing sphere is in the G 1 phase of the cell cycle, the thymine analog 5-bromo-Z-deoxypyrimidine purine (BrdU) can be doped. If BrdU is doped into the nucleus, the nucleus has entered S phase. Therefore, the presence of BrdU in the nucleus indicates whether the cell has stopped in G1 phase. Embryos that are likely to be in M phase are cultured for 4 h in M16 medium with germacrene (5 ug/ml) and B rd U. At the end of the culture period, the embryos are incubated with acidophilus (5 ug/ml). At the end of the culture period, the embryos were fixed in acidic ethanol and incubated for lh at room temperature with a monoclonal antibody against B rd U. The embryos were washed and treated with peroxidase anti-mouse _IgG2a for 30 min at room temperature, treated with substrates and enhancers, and observed in the presence of diaminobenzidine. As a positive control, embryos that might be in M stage were cultured in medium containing B r d U without supplementation with bacteriophage. For counterstaining, 0.5% eosin was used for 2--3 min, and BDRU positive cells in the S stage were stained blue-black.

Denucleation

Oocyte denucleation

After oocyte maturation, the original cell nucleus should be removed or its function should be lost, and there are two methods: denucleation and inactivation, and at present, most of them use microscopic denucleation. Nucleation of mature oocytes is an important part of nuclear transplantation technology, which requires clean removal and minimizes damage to the cytoplasm. Oocyte denucleation is mainly based on microsurgical operation. Mainly include: ① blind suction method: blind suction in the first polar body with a fine glass tube, suction out the first polar body and in the middle of the division of chromosomes and some of the surrounding cytoplasm. ② Negative pressure aspiration method: in the differential interference difference inverted microscope under the high-resolution field of view to make the oocyte slightly rotating, when the spindle and the focal plane perpendicular to the refractive index of the nuclear region and the cytoplasm of the slight difference, can be accurately suctioned out of the cell nucleus and a small amount of cytoplasm, the rate of denuclearization can be up to 100%. Squeezing method: make a cut in the zona pellucida near the first polar body, and squeeze the oocyte with a nuclear transfer tube, so that the first polar body and 1/3 of the cytoplasm in the vicinity can be squeezed out of the zona pellucida. This method is related to the cell division phase and must be carried out at the right time, otherwise the chromatin cannot be removed cleanly. Half egg method: cut the zona pellucida, suck out half of the cytoplasm and move it into another empty zona pellucida, when observing the naked oocytes under an inverted microscope with an IOx eyepiece and a 20x objective lens, you can see a small swelling area containing M II chromosomes, the collected M II oocytes are put into M2 medium without cytoskeletal inhibitors at 37 ℃, then cut the zona pellucida next to the swelling area, after cutting the zona pellucida, the oocytes are in the cytosol containing cytosolic phase, and then the zona pellucida is cut. After the zona pellucida was cut, the oocytes were incubated in M2 medium containing cytochalasin B (5u=g/ml). After treatment with cytochalasin B, the swollen zone became difficult to recognize. The oocyte was held in position opposite the zona pellucida cutting slit, and a denuding needle was inserted through the slit into the perivitelline space. The translucent region containing the M II chromosome moves as the needle touches the egg mass, and the region is then aspirated into the needle while the presence of chromatin is determined by the naked eye. After all the oocytes were denucleated with M2
medium, these eggs were washed and incubated at room temperature or 37°C for use.

If it is difficult to find the area containing M2 chromosome, it can be stained with Hoechst 33342 first, and then visualized by fluorescence microscope during enucleation. Oocytes cultured for 20~24 h were put into M2 solution containing 600IU/m l hyaluronidase and Ca2+ and Mg2+ free, and the oocytes with the first polar body discharged (MII, middle stage) were selected for denucleation by blowing the oocytes with a glass microneedle. Oocytes were stained with 0.5 |ig/ml DNA fluorescent dye (Hoechst 3342), 7.5ug/m l cytochalasin B in Ca2+ and Mg2+ free M2 solution for 15-20 min at room temperature, and then washed three times with M2 medium without Hoechst 33342. The cells were then washed 3 times with Hoechst 33342-free M2 medium. M2 chromosomes were removed under a fluorescence microscope with a denuding needle (blue-violet, blue-violet, and blue-violet, blue-violet, blue-violet).
UV-excited filters 400 to 450 nm). The duration of UV irradiation is critical for oocyte viability and should not exceed 15s.

In addition, there is functional denucleation, that is, the recipient oocyte is placed in Hoechest specific stain, with UV or laser irradiation, so that the nucleus is not functional, but the oocyte in the UV irradiation time should not be more than 30s, otherwise its viability will be seriously impaired.

Nucleation of syncytia

Prior to microsurgery, collected conidia were held in M2 medium supplemented with a cytoskeleton inhibitor (cytochalasin B, nocodazole). For microsurgery, syncytia were manipulated in a drop of flattened M2 medium in an operating chamber using a micromanipulator and an inverted microscope. The zona pellucida was cut with a fine glass needle. The haptic is held in place by applying negative pressure to the hyaline strip with a holding needle. The tip of the needle is then carefully advanced toward the periplasm so that the egg plasma membrane is not damaged. The haptic is released from the holding pin by forking the fine glass needle and repeated pressure is applied against the wall of the holding pin until an incision is created. After the zona pellucida is cut, the hapten is held in place with the holding pin on the opposite side of the zona pellucida incision. A denuding needle or syringe needle was inserted through the incision slit into the perivitelline gap.
The tip of the needle is then inserted into the perivitelline space at a point near one of the two anterior nuclei. One pronucleus with a small amount of cytoplasm is aspirated into the syringe needle, and this step is repeated for the other pronucleus, where cytoskeletal inhibitors are necessary to avoid penetration of the egg plasma membrane. After the enucleation of the syncytium, it is washed with M2 medium and kept at room temperature.

Preparation of nucleocytes

Body cells as nucleocytes

At present, there have been reports of successful cloning of a variety of somatic cells as nuclear donors, including sheep mammary epithelial cells, porcine ovarian thalamus cells, bovine oviductal epithelial cells, and bovine ear fibroblasts. The cell cycle synchronization of donor and recipient cells is an important factor affecting the success of nuclear transplantation, and most of them use G() phase cells. There are two main ways to obtain G() phase cells: one is by limiting a certain component of the cell culture medium so that most of the cells remain in a certain phase of the cell cycle: e.g., starvation with decreasing serum concentration to make the cultured cells temporarily withdraw from the proliferative cycle; wilmut et al. used starvation with decreasing serum concentration, i.e., they cultured mammary epithelial cells in a culture with 10% fetal calf serum (FCS), then cultured the cells with 10% FCS, then cultured the cells with 10% FCS, then cultured the cells with 10% FCS. wilmut et al. utilized a decreasing serum concentration starvation method, in which mammary epithelial cells were first cultured in 10% fetal calf serum (FCS) and then transferred to medium containing only 0.5% FCS for 5 days, thereby temporarily withdrawing the cultured cells from the proliferative cycle. Alternatively, G0-phase cells can be obtained by selective cell type selection, e.g., more than 90% of the surrounding oocytes of a newly discharged oocyte are in the G0 phase. Recently, however, it has been found that nuclear transplantation of G1 or G2 or even M-phase donor cells without dormancy induction has also been successful.

Donor cells were obtained by chopping the tissue blocks and digesting them with D-Hank buffer containing 0.25% trypsin and 0.02% EDTA at 30 T for 30 min, with stirring several times in between. The digested tissue was rinsed several times with D-Hank buffer, centrifuged at IOOg for 5 min, and the supernatant was rinsed several times with D-Hank buffer, and then centrifuged at 700g for IOmin to obtain the cell mass. The cell pellet was suspended in DMEM with 10% FC S and cultured in a humidified incubator at 37℃ with 5% CO2. After 100-14 days, confluent monolayer fibroblasts were obtained for passaging, and thereafter, cells were passaged every 24 h. After a sufficient number of cells were passaged, the cells were frozen. Thereafter, the cells were passaged every 24 h. After the number of passaged cells was sufficient, the cells were frozen and set aside. 3--10 days before nuclear transplantation, the fibroblasts were cultured in TCM199 containing 0.5% serum by serum starvation, so as to make them regress.
Fibroblasts were serum-starved in TCM199 with 0.5% serum in order to exit the cell cycle and enter the G0/G1 phase. Cells were digested with 0.25% trypsin and 0.02% EDTA for a short time, then washed with TCM199 and put into the operating solution.

Embryonic stem cells as nucleus donor cells

Embryonic stem (ES) cells are a type of totipotent cells selected from early embryonic cells or blastocystic ICM cells in culture after inhibition of differentiation. These cells have a normal diploid karyotype, and can either proliferate indefinitely without differentiation or differentiate into a variety of cells and tissues, including the germ line, under specific conditions. E S cells are totipotent for in vitro development and can be passed on indefinitely in vitro, making them ideal donor cells for animal cloning. To date, mouse and human ESC cell lines have been established, and ESC cells have been utilized for the following purposes
The main research direction of nuclear transplantation is to utilize these cells to produce transgenic animals. The method of preparing ESCs as donor cells for nuclear transplantation is similar to that of somatic cells, and cell cycle regulation is also required.

The process of obtaining E S cells from oocytes: the mucin layer and zona pellucida on the outside of the oocytes were first digested with trypsin, then the embryos were digested in PBS buffer containing 0.25% trypsin, and the cells were dispersed by blowing them with a fine glass tube. If we want to get single cells, first cut the tissue into pieces, and then digest them with PBS buffer containing 0.25% trypsin plus 0.02% EDTA at 30 ℃ for 30 min with occasional stirring, and then centrifuge the digested solution at IOOg for 5 min to take the supernatant, and then put the embryos in the buffer at 30 ℃.
Centrifuge the supernatant at IOOg for 5 min, and then centrifuge the supernatant at 700 g for IOmin to obtain the granular precipitate, dilute the precipitate with a-MEM +10% FCS, and incubate the sample at 37 ℃ under 5% CO2, and then set aside.

Basic operation procedure for embryonic stem cell maintenance:
The ES cells are passaged from a four-well plate to a 35 mm dish, and then labeled as 〇代. Generally, ES cells are passaged every 2 to 3 days at a ratio of 1:3. It is preferable to treat the dishes for culturing ES cells with 0.1% gelatin before inoculating cells in the feeder layer.

(1) Remove the original medium and wash once with 1.5 ml of PBS.

(2) Add 1.5 ml of 0.25% trypsin-EDTA solution. (2) Add 1.5 ml of 0.25% trypsin-EDTA solution, digest for 5 m i

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