Nuclear transfer, also known as microinjection, is a core component of cloning in mammalian technology and has been highly regarded by the scientific community since the day of its success. The birth of Dolly, the world's first adult somatic cell cloned animal, was a historic breakthrough in animal genetics and animal embryology, and drew great attention from scientists to animal cloning technology.
Principle
The principle of nuclear transplantation is to transplant the nucleus of a cell into a denucleated oocyte or a denucleated egg by microscopic manipulation to form a new recombinant body, so that the nucleus of the donor cell undergoes reprogramming in the cytoplasm of the oocyte, and then is transplanted into the recipient animal, where it develops into a new individual. Serum promotes the growth of the cells, and the medium with very low serum concentration only maintains the cells in a certain state without proliferation. In nuclear transplantation studies, this treatment coordinates the cycles of donor and recipient cells to achieve synchronization, which facilitates the remodeling of the donor nucleus in recombinant embryos and thus promotes the development of the nuclear transplanted embryo.
Operation method
Microinjection techniques
Principle
The principle of nuclear transplantation is to transplant the nucleus of a cell into a denucleated oocyte or a denucleated egg by microscopic manipulation to form a new recombinant body, so that the nucleus of the donor cell undergoes reprogramming in the cytoplasm of the oocyte, and is then transplanted into the recipient animal, where it develops into a new individual. Serum promotes cell growth, and medium with very low serum concentrations only maintains the cells in a certain state without proliferation. In nuclear transplantation studies, this treatment coordinates the cycles of donor and recipient cells to achieve synchronization, which facilitates the remodeling of the donor nucleus in recombinant embryos and thus promotes the development of the nuclear transplanted embryo.
Materials and Instruments
Equipment. Move The basic process of nuclear transplantation can be divided into the following steps: (3) Under a body-view microscope, in culture medium containing 0.1% hyaluronidase, the dilated tubal jugal was broken open with a sharp forceps or a needle. The mass of the oviduct-oocyte complex will be released into the culture medium and slowly separated under the action of the enzyme. 1800 V/cm Voltage, 30 ms pulses were used to activate the recombinant embry Caveat (1) Prepare synchronized rat fibroblasts in advance.(2) Prepare egg donor mice in advance.(3) Careful operation to improve the efficiency of enucleation. For more product details, please visit Aladdin Scientific website.
①Stereomicroscope
② CO
2
Incubator
③Electrofusion meter
④ Ultra-clean bench
⑤ Microscope
Reagents
① Materials: synchronized rat fibroblasts, egg donor mice, concave slides, ophthalmic instruments, egg holders, egg transfer tubes, enucleation needles, enucleation needles
② Mineral oil
Mineral oil ③ Calf serum
④ Fetal calf serum
⑤ 0.25% trypsin
⑥ PBS
⑦ M16 culture medium
⑧ Cell relaxin B (CCB)
⑨ 0.1% hyaluronidase
⑩ Anhydrous ethanol
1 . Collection of oocytes
(1) The female egg donor mice were killed by cervical dislocation method, soaked in 75% ethanol for 5 min, fixed on the mouse board in ventral position, and the skin was cut horizontally from the lumbar posterior dorsal side, and then torn toward the head with large forceps.
(2) Cut open the abdominal cavity on both sides of the back, move the intestines to one side, and pinch the ovaries by the fat pad. The ovary was cut at the uterine horn, and the ovary and fallopian tube were transferred to a flat dish and washed with PBS to remove blood and debris.
2 . Blind aspiration of oocytes for denucleation
(1) Incubate the oocytes with obvious first polar body into the M16 culture medium containing 10 μg/ml of cytosolic relaxin B for 15 min, and then transfer them to the micromanipulator. (1) The oocytes were incubated in M16 culture medium containing 10 μg/ml cytochalasin B for 15 min.
(2) Fix the oocyte on the opposite side of the first polar body with an egg-holding needle, adjust the beveled mouth of the nucleation needle in the fixation tube close to the first polar body, puncture the zona pellucida, enter the perivitelline space, and aspirate the cytoplasm of the first polar body and its vicinity, and aspirate 1/4~1/3 of the total amount of cytoplasm.
(3) Cultivate the denucleated oocyte in a 37 ℃ 5% CO2 incubator for 30 min, and incubate the oocyte in an incubator for 30 min, in which all oocytes with complete cell structure and undissociated cytoplasm could be incubated. Any oocyte with complete cell structure and undiscrete cytoplasm can be judged as successful denucleation.
3 . Nuclear transplantation
(1) Synchronized rat fibroblasts were routinely digested with 0.25% trypsin and suspended in M16 culture medium.
(2) Transfer to concave slides with an oocyte pipette, oocytes were transferred into the same liquid and covered with mineral oil to prevent evaporation.
(3) On the microscope operating table, hold the oocyte tube to fix the opposite side of the denucleation, and use the nuclear injection needle to repeatedly aspirate the donor cells in the liquid, so as to break the membrane of the donor cells, and then inject the nucleus of the donor cells into the cytoplasm of the recipient cells by the denucleation port.
4 . Recombinant Embryo Activation
The activated recombinant embryos for nuclear transfer were transferred into M16 culture medium and cultured at 37 ℃ in a 5% CO2 incubator.
6 . Experimental results and analyses
(1) The oviposition of the recombinant embryos was observed at 48 h after culture.
(2) At 72 h after culture, the recombinant embryos were observed to continue to develop, and some of them could develop into mulberry embryos.