Rabbit somatic cell nuclear transplantation

Summary

Rabbits share similar biochemical and physiological processes with humans and are ideal model animals for the study of human reproduction, cardiovascular disease and regenerative biology. In the 1980s and 1990s, it was possible to produce cloned rabbits by transplanting nuclei from embryonic cells into enucleated oocytes. It was found that using differentiated somatic cells as the donor cells for nuclear transplantation, rabbits were more difficult to produce cloned animals relative to other species, probably due to the shorter cell cycle of the preimplantation embryo and other unknown mechanisms of nuclear reprogramming in rabbits, and that cloned embryos from rabbits exhibited a higher rate of miscarriage during embryo implantation and gestation. To date, only a few cases of successful acquisition of somatic cell nuclear transplanted rabbits have been reported.

Principle

The principle of somatic cell nuclear transfer is that all the nuclei of cells originating from the same embryo contain exactly the same genetic information as the fertilized egg, and have the same full potential to guide the development of the individual in terms of genetic composition as the fertilized egg.

However, as the embryo develops and the cells differentiate, some of the genomes of the nuclei change, resulting in these genomes not being able to revert to their former state, and thus losing their nuclear totipotency. However, they contain all the genetic information of the organism and, under the regulation of specific environmental factors, can be restored to the fertilized egg-like state and develop from scratch into a complete biological individual.

Operation method

Rabbit somatic cell nuclear transplantation

Principle

The principle of somatic cell nuclear transfer is that all the nuclei of cells originating from the same embryo contain exactly the same genetic information as the fertilized egg, and have the same full potential to guide the development of the individual in terms of genetic composition as the fertilized egg. However, as the embryo develops and the cells differentiate, some of the genomes of the nuclei change, resulting in these genomes not being able to revert to their former state, and thus losing their nuclear totipotency. However, they contain all the genetic information of the organism and, under the regulation of specific environmental factors, can be restored to the fertilized egg-like state and develop from scratch into a complete biological individual.

Materials and Instruments

Equipment:
① syringe
② glass microneedle
③ Optical microscope
④ sterile surgical instruments
⑤ Incubator
⑥ light conditions for 8: 16 (light: dark) hours of the environment clean-grade animal breeding room
Reagents:
①Materials: Rabbit
② Protease
③ 0.1% EDTA without Ca, Mg
2
Hanks solution
④ hCG, follicle stimulating hormone (FSH), GnRH
⑤ Egg flushing solution
⑥PBS
⑦Hyaluronidase
⑧PVP-40
⑧PVP-40 ⑨EDTA
⑩DMEM
○11pmnaseE
○12Hoechest 33342

Move

The basic process of the rabbit somatic cell nuclear transfer technique can be divided into the following steps:

I. Supernumerary ovulation and oocyte collection

1. Sexually mature female New Zealand white rabbits (6 to 18 months of age) were used as embryo donors and kept in an environmentally clean animal room with a light condition of 16:8 (light:dark) hours.

2. Donor rabbits were treated with supernumerary ovulation by intramuscular injection of 0.3 mg, 0.4 mg, and 0.5 mg of follicle stimulating hormone (FSH) twice daily for 3 consecutive days. Twelve hours after FSH treatment, 200 U of human chorionic gonadotropin (hCG) was injected intramuscularly.

3. Supernumerary ovulation-treated female rabbits were not mated with male rabbits and were used as donors of oocytes for nucleus transfer; supernumerary ovulation-treated female rabbits were mated with male rabbits in shared cages and were used as nucleus donors for embryos.

Dutch Belted female rabbits were injected with GnRH (15 μg each) for oestrus synchronization (22 h later than the donor) and served as recipient rabbits for reconstituted embryo transfer. After embryo transfer, the recipient rabbits were housed in an environmentally clean animal room with light conditions of 8:16 (light:dark) hours.

4. 10-12 hours after hCG injection, donor rabbits were opened from the mid-abdominal line, the oviducts and ovaries were surgically clipped, and oocytes were flushed from the juxtapical portion of the oviducts to the flail with 5 ml of egg-flushing fluid.

5. The complex of oocyte-egg (COC) was treated with PBS + 0.5 mg/ml of hyaluronidase for 1 min. The oocyte was gently blown away from the oocyte zona pellucida with a mouth pipette, and the oocyte was examined under a light microscope for the discharge of the oocyte polar body to determine whether the oocyte was in stage MII.

6. Oocytes can also be released from the follicles of the ovary by picking them with the tip of a fine needle. COCs collected on the ovary are removed from the oocytes in the same way as tubal oocytes. Oocytes identified as showing polar bodies can be used for nucleus transplantation experiments.

II. Preparation of nucleus donor cells

(1) Granulosa cells as nucleus donor cells: Oviductal cells blown off from COC can be directly used as nucleus donor cells for nucleus transplantation. The collected granulosa cells were first washed in D-PBS + 10% PVP-40 without _Ca2 and _Mg2.

The addition of PVP-40 helped to remove membrane-damaged granulosa cells, and the cells were collected by centrifugation at 1000 r/min. The cells were digested with 0.05% trypsin + 0.5 mmol/L EDTA for 3 min at 37 ℃, and the treated oocytes were suspended in DMEM + 10% FBS and stored at 37 ℃ to be used for nucleus transplantation.

(2) rhES as nucleus donor cells: rhES were recovered as described above, washed once in DMEM, suspended in PBS + 15% FBS, and stored at 37 ℃ for nucleus transplantation.

(3) Embryonic cells as nucleus donor cells: After the fertilized eggs were cultured in vitro and developed into 8～16 cell stage or 32～64 cell stage, the zona pellucida was removed by D-PBS at pH 2.3, and then the naked embryos were dispersed into single embryonic cells in pmnaseE solution, suspended in PBS＋15% FBS. 37 ℃ was stored and used for nucleus transplantation.

III. Nuclear transplantation 1. Under the inverted microscope, a small opening was made in the zona pellucida of the oocyte with a nucleating glass needle, and pressure was exerted outside the zona pellucida with a nucleating needle, so that the polar body and the nearby cytoplasm were discharged and pressed out from the opening.

2. The treated oocytes were stained with 10 μg/ml of Hoechest 33342 and examined for successful denucleation under ultraviolet light.

3. Nucleus removal was performed by direct injection: nucleus removal needles (8 μm inner diameter) cleaned with 10% PVP-360 were used to mechanically lyse the nucleus donor cells. The donor cells with partially disrupted cell membranes were injected into the cytoplasm of enucleated oocytes using the Poezo-Drill system, and the success of the transfer was examined under 400× magnification.

Nucleus transfer by cell fusion: Donor cells with a diameter of approximately 20 μm were transferred under the zona pellucida of the denuded oocytes with a nucleus transfer needle (25 μm inner diameter) (Fig. 4-7-1e). The "nucleus-plasmid" is incubated in cell fusion solution for 3 min.

The cells are then transferred into a fusion chamber containing the fusion solution, and 3 DC pulses are applied with a cell fusion apparatus (BTX 200) at a pulse intensity of 3.2 kV/cm. After shocking, the "nucelles-plasmids" are incubated at 38.5°C for at least 15 minutes, and the cells are examined for fusion under a microscope.

4. Injected or fused oocytes were incubated in M199 + 10% FBS for 1 hr and then subjected to orphan activation. Clonally reconstituted embryos were activated by direct current stimulation (the method was the same as that for cell fusion), and the activated embryos were incubated in M199 + 10% FBS + 2 mmol/L 6-dimethylaminopurine + 5 μg/ml cyeloheximide for 1 hour.

IV. In vitro development of rabbit cloned embryos 1. Reconstructed embryos were cultured in B22.5% FBS, the cleavage ratio was recorded after 14-18 hours of incubation, and embryos that had developed to 4-8 cells were embryo transferred.

2. Cloned embryos can also continue to be cultured in vitro for 4 days before developing into blastocysts, and the number of cells in the developing embryo is examined.

The bES cells are trypsinized into single cells and transferred into enucleated rabbit oocytes, which undergo cell fusion to form reconstituted embryos. The reconstructed embryos were electrochemically activated and cultured in vitro for 5 days to develop to the blastocyst stage.

V. In vivo development of rabbit cloned embryo 1. After the cloned embryo was cultured in vitro, embryo transfer was carried out. The recipient rabbit was opened in the mid-abdominal line, and 10-20 embryos were loaded with 5pl quantitative capillary tubes and transplanted into one side of the oviduct, and the embryo recipients were sent to the environmentally clean animal room under the light condition of 8:16 hrs (light:darkness) for rearing after embryo transfer.

2. Pregnancy was checked by abdominal palpation 14-16 days after transfer. Recipients' pregnancy expired on 31-33 days of gestation, and cloned dead fetuses were obtained by natural delivery or cloned live pups were obtained by cesarean section surgery.

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