The so-called nuclear transfer technology, is to remove the donor nucleus into the oocyte nucleus, so that the latter without the process of sperm penetration and other sexual or asexual reproduction can be activated and split the concurrent incubation of new individuals, making the nuclear donor gene is fully copied. To the source of donor nuclei can be divided into embryonic cell nuclear transfer and somatic cell nuclear transfer two.
【Research Progress in Mammalian Nuclear Transfer】
Mammalian nuclear transfer is an exogenous nucleus with an enucleated oocyte binding, resulting in genetically homogeneous animal technology. It is in animal breeding, preparation of transgenic animals, gene therapy and organ transplantation has a major role.
Specific methods
『1』 donor nuclei obtained
Donor nuclei can be early embryonic cells, embryonic stem cells and somatic cells. Early zygotic nucleus are used to 64-cell stage embryo blastomeres nuclei plasmid as donor nuclei, 80 years, with embryonic stem cells (embryo stem cells, es) of the building and its research, the people of embryonic stem cells The prospect of nuclear transplantation have high hopes, but es build system too difficult to be successful only in mice. teruhiko wakayama and other Department of cloned mice using es, resulting in 29% of the reconstructed embryos can develop in vitro to the blastocyst stage, transplanted to pseudopregnant mice embryo implantation of 8% and the birth of mice. 1997 dolly sheep there, and began somatic cell nuclear transfer, and develop rapidly.
1.1 embryonic cells
1.2 cells
『2 receptor cells and to obtain nuclear』
The receptor cell nuclear transfer, as there are three categories: to the nucleus of oocytes, zygotes and 2 - cell embryos, of which the most widely used oocytes. Recently, someone with two to go nuclear oocyte cytoplasm as a fusion receptor generated by nuclear transfer generations in order to increase the number of nuclear transfer embryos and cell transplantation to improve the efficiency of subculture. Studies have shown that in vitro maturation of oocytes than the success rate of transplantation in vivo mature oocytes, the reason may be that oocyte maturation in vitro, synthesis of some proteins need to complete the first meiotic division, a number of in vitro maturation oocytes are likely to be restrained in its activities.
2.1 enucleated oocyte method
2.1.1 Blind suction method using fine glass tubes under the blind in the first polar body suction, suction in the first polar body and metaphase chromosomes and the surrounding part of the cytoplasm, but the method of the low success rate. To improve the enucleation rate, the use of hoechst33342 the specificity of dye staining on the role of chromatin in the nucleus after fluorescence microscope to determine down to the core is complete, go nuclear accuracy greatly improved. stice, etc. with hoechst33342 (1μg/ml) staining under UV light exposure time control in less than 10s, not observed negative effects on embryonic development.
2.1.2 Semi egg method using micro-needles in a transparent glass tube to do to bring all the mouth, with a tiny glass tube to absorb half of the chromatin to another empty zona pellucida, the oocytes will be divided into two halves, and then stained with hoechst33342 to determine the non-receptor cytoplasmic half of the chromosome. Its operation as follows, to 35mm into oocytes with mpbsa (phosphate buffer, of which d-glucose 1ooomg / l, pyruvate 36mg / l, 0.4% bovine serum albumin, 1% penicillin and streptomycin 10000μg/ml ) in a Petri dish for micromanipulation, zona pellucida first split in two steps, that is, first cut in a transparent bring a small mouth, and then expand the incision with the other cutting needle, which split the zona pellucida. Zona pellucida was removed and transferred to the containing mpbsa +5 μg / ml cytochalasin b, 35mm Petri dish for the role of 3 to 5min, and then use the fixed needle (inner diameter of the zona pellucida of the l / 5 of a 1 / 3, OD close to the zona pellucida diameter) fixed, split pin into the mouth of the zona pellucida of the gap and the needle fixed in place rely on the zona pellucida, oocytes slowly absorb fluid, when the split needle to draw fluid half of oocytes, this gap when the needle removed from the yolk, and relying on cutting edge with a slightly transparent grazed in order to achieve complete division, its needles in the oocyte fluid into the space ready for the zona pellucida, and use hoechst33342 staining is not observed under fluorescence microscope as a fluorescent receptor oocytes.
2.1.3 centrifugation to enucleated tatham other 150oog, 2min centrifugation of bovine oocytes with pronase to remove oocyte zona pellucida (zp), by the osmotic pressure gradient centrifugation, m Ⅱ of spindle cells from the majority of oocytes the separation of the non-transparent cytoplasm with enucleated for nuclear transfer for the transfer, together with the split by electric fusion into a ball of nuclear transfer embryos, and finally off into the zona pellucida in alginate, in vitro cleavage and development, but its effect has yet to be studied further.
2.1.4 the end of stage Ⅱ and put forward to the nucleus bordignon to oocyte activation so that in the end of the first stage Ⅱ, the second polar body in the discharge of the second polar body and suck out a small amount of surrounding cytoplasm, so as to achieve to the nucleus. This method avoids the use of dna dyes and exposure to ultraviolet light to locate the chromosome, and the removal of the cytoplasm is relatively small. Way to go nuclear to nuclear than m Ⅱ of the success rate has significantly increased, but the cytoplasm easy to aging, whether the full weight of the genome sequencing, the successful completion of post-development, be studied.
2.2 The time to go nuclear to nuclear success rate
2.3 persons cytoplasmic volume potential and the development of reconstructed embryos
It was in inverse proportion with the nuclear relationship between the development of reconstructed embryos, the results show that the oocyte quality and the expected removal of the cell size for a considerable time the nuclear volume, cell cycle interactions can create the best conditions for the removal of too little or too much is not satisfactory. Based on the amount of removal and the cytoplasm is closely related to the nucleation rate, therefore, to ensure a high nuclear case. Removal should be minimal cytoplasm.
Nuclear reorganization 』『 3 eggs
Under the control of the micromanipulation device, with a diameter close to the transplantation of large blastomeres isolated needle draw a complete blastomere, into the enucleated oocyte receptor for the gap, according to the site transferred into different taken off the transplant and can be divided into the cytoplasm injection. Blastomere separation of those embryos can be difficult to add a calcium and magnesium - free phosphate buffered saline solution and incubated for 30-6omin mpbsa the cell division stopped. But the incubation for more than 6omin, cell membrane integrity will be destroyed, the process of melting rate in the cells will increase. After removal of the transplant needle on the top of the transparent blastomere transplantation with pressure the blastomeres and half-oocyte contact.
『4 cell fusion / activation』
4.1 Fusion and activation
Because micro-pipette destroyed part of the egg membrane and the cytoplasm, if the direct transfer, the success rate is very low, it takes on the integration of reconstructed embryos, and its methods used Sendai virus, electric fusion method, which is more commonly used. The _select_ion of fusion power and pulse frequency of the voltage range of integration depends on the distance between two electrodes in the box. The distance from the fusion box 2ooμm, to a few millimeters, so a multiple of the pulse is 200μsec and can transfer lkv / cm DC basically meet the requirements. Mice, rabbits and other animals found in embryonic nuclear transfer Shihai, receptor oocytes reconstructed embryos were activated when the power state of fusion and embryo development rate of repurchase is closely related to the pulse intensity is 2.0-3.6k / cm, fusion time is appropriate for the 60-2ooμsec range. If the pulse is too strong, the integration time is too long, the further development of reconstructed embryos is extremely unfavorable. kono and put forward for different periods of the donor nucleus to activate the procedures are different and good results: ① for g2 and m of the donor nucleus of the donor dna 4 times, can be used in the fusion does not cause oocyte activation or intracytoplasmic injection of Sendai virus and then given active treatment, so that the polar body half way dna discharge, followed by the formation of 2 times the original core and normal cells, should this method has produced the normal mice; ② For g0, g1 and s of the donor nucleus, activation and fusion before fusion can be activated in two ways, in order to prevent the formation of donor nuclei caused metaphase plate of chromosomes is not normal. szollosi with thymocytes for nuclear transplantation experiments found that only mature enucleated oocytes to be activated before or after activation 3omin fused into the donor nucleus before degradation, and re-aggregate to form a new nuclear membrane, if more than 3omin then touch together, though only into the degradation of the donor nucleus, which may cause the donor and recipient cytoplasm chromatin effective interaction of various factors inhibited, so that hindered the process of reprogramming. wakayama, who use chemical receptors in oocytes and reconstructed embryos activated chemical fusion method, also in mouse somatic cell cloning research, cloning and then achieved the desired results.
Rabbit eggs after electrofusion of oocytes activated by electrical stimulation is also due to start a new programming and development, but mice, rats and cattle need to be further developed to get the full activation. The methods of chemical and electrical activation of two types of chemical agents, 7% ethanol shock, ionomycin (ionomycin), calcium ionophore a23187, after activation by ionomycin treatment with 6-dmap 3h, can be avoided pcc (premature chromosome condensation ), and increase the development rate of reconstructed embryos sheep and the birth rate.
And cell fusion rate of 4.2
prather et al (1989) results show that the fusion rate and no significant difference in cell stage, indicating that the volume of smaller blastomere fusion rate of no significant impact, Zhang Yong (1992) study in mice has drawn similar results. robal, also believes that the fusion rate of oocytes with the receptor when the age related, and also in contact with the nuclear donor area and the close degree of contact with, and into the cleavage and at what time the ball has nothing to do, but the reconstructed embryos development rate of the donor embryo cell stage are related.
4.3 temperature, time of oocyte maturation of oocytes activated
As the oocyte maturation time in vitro, and then aging, mature promoting factor (mpf) down, easy activation. Aging in vitro maturation of bovine oocytes induced activation of a high degree of prosperity to the temperature sense, in a certain temperature induced activation, the chromatin of oocytes Condensation, "Automatic to go nuclear," the frequency. Immature oocytes are not easily activated at room temperature, even if the activation is not stable, may be reversed to the medium term.
『5 reconstructed embryo culture and transplantation』
Reconstructed embryos develop to be some time before transfer to the receptor, rabbit and pig embryos reconstructed within 24h in vitro, to be passed by non-surgical transplantation, while sheep and cattle embryos from a longer incubation time required for the general development to the morula or blastocyst stage when the transplant. Culture methods are available to electric fusion of reconstructed embryos to enlarge 1o% fcs rd droplets within the culture can also be micro-manipulation of embryos into the oviducts of homogeneous or heterogeneous in cultured oviduct flushing after a few days, recovery reconstructed embryos. The latter experiment is as follows, first made of agar and 0.9% naci 1.0% and 1.2% agarose, 1.0% agar into the Petri dish 35mm, when the temperature is 37 ℃, the embryo into agar in. Agar cutting needle draw a bubble and mpbsa +20% newborn calf serum, and then draw the embryo containing agar, the needle at room temperature for a few seconds after it into mpbsa +20% neonatal calf serum and cultured skin's dish. 1.2% agar when the temperature reaches 37 ℃, the method used above in the wash several times in the agarose, and then double-agar composition and use of surgically implanted into the oviduct. In vitro culture of reconstructed embryos, _select_ the appropriate medium is very important. Reconstructed embryo transfer and embryo transfer method is basically the same as that of animals, according to the different receptors can be divided into surgical and non-transplant surgery transplantation.
』『 6 Conclusion
So far, although the rapid development of nuclear transfer, but the technology still has many problems, such as nuclear transfer success rates were generally low, low rate of reconstructed embryos, a high rate of abnormal embryos. Prolonged in vitro culture medium composition or transfer embryos may lead to abortion and the death soon after birth of the newborn animal. Recombinant programming mechanism is not clear which mpf, nebd (nuclear envelope breakdown) and pcc restated in the genome need to clarify the role of the process. Genomic imprinting of nuclear transfer and reprogramming of gene imprinting and the success of animal cloning and the lack of any relationship is unclear. |
