CN101580828B - Nuclear transfer method - Google Patents
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Abstract
本发明公开了一种细胞核移植方法，包括以下步骤：构建卵母细胞；采用浓度为0.05～20ug/ml的去核试剂，将所述卵母细胞处理0.5～26小时，获得细胞质体；构建具有所需遗传特性的供体细胞或细胞核，将细胞质体与所述供体细胞或细胞核融合，得到重建的胚胎。本发明通过使用去核试剂灭活卵母细胞来去除卵母细胞的细胞核，取代现有技术的细胞核移植技术中需要机械操作去除卵母细胞细胞核的步骤，简化了该步骤的操作，可广泛应用于多种物种的细胞核移植操作中。
The invention discloses a cell nucleus transplantation method, comprising the following steps: constructing oocytes; using an enucleation reagent with a concentration of 0.05-20ug/ml, treating the oocytes for 0.5-26 hours to obtain cytoplastids; constructing oocytes with A donor cell or nucleus of desired genetic characteristics is fused with a cytoplastid to obtain a reconstructed embryo. The present invention removes the nucleus of the oocyte by using an enucleating agent to inactivate the oocyte to replace the step of removing the oocyte nucleus by mechanical operation in the prior art nuclear transfer technology, simplifies the operation of this step, and can be widely used in nuclear transfer procedures in a variety of species.
Description
Technical field
The present invention relates to the fetology field, relate in particular to a kind of nuclear transfer method.
Background technology
" clone " is English " clone " transliteration, but is meant the offspring that an animal via vegetative propagation parthenogenesis produces, and all offsprings' of clone genetic composition is identical, claims clone again.
Animal cloning is meant by an animal and directly obtains to have identical genetic homogeneity offspring's process with the parent without amphigenetic mode, comprises lonely female activation reproduction, enzygotic twins, the embryo is cut apart and nuclear transplantation etc.Animal cloning is at present mainly cut apart two kinds of methods through nuclear transplantation and embryo and is obtained, and normal development is the new individuality with identical genetic material.
Nuclear transplantation is through micrurgy, laboratory means such as electricity fusion, and with the nuclear donor of growing certain phase, like embryonic cell or somatocyte, the nuclear receptor that reaches the corresponding etap carries out external reconstruct, like the protokaryon embryo or the mature oocyte of stoning.Through the embryo transfer of reconstructed embryo, reach the mammiferous a kind of biotechnology of mass production heredity homogeneity.Utilization nuclear transplantation method; Cloned animal of successful acquisition; Need accomplish following process effectively: the acquisition of the selection of donorcells and processing, acceptor ovocyte and processing, under the micrurgy appearance, get nuclear or with the stoning of blade excision method, inject in vitro culture and the embryo transfer and the body of cell, caryoplasm fusion and activation of oocytes, reconstructed embryo and grow with microneedle is micro-.
In the above scheme, the stoning step has adopted with the micro-nuclear of getting of microneedle in the nuclear transplantation process, or with the stoning of blade excision method; This operation steps is not only loaded down with trivial details; And need manual carrying out, and cause misoperation easily, can not well guarantee the success ratio of nuclear transplantation.
Therefore, prior art is still waiting to improve and improve.
Summary of the invention
The objective of the invention is to above-mentioned shortcoming, provide a kind of and need not to adopt micrurgy appearance mechanical process to prior art, or blade excision method removal acceptor ovocyte karyon, the nuclear transfer method of easy handling and stoning quick and precisely.
Technical scheme of the present invention is following:
A kind of nuclear transfer method may further comprise the steps:
A, structure ovocyte;
B, employing concentration are the stoning reagent of 0.05～20ug/ml, and said ovocyte was handled 0.5～26 hour, obtain cytosome;
Donorcells or nucleus that C, structure have required hereditary property are with cytosome and said donorcells or nucleus fusion, the embryo who obtains rebuilding.
Method of the present invention, wherein, the ovocyte of said ovocyte for needing zona pellucida to adhere to.
Method of the present invention, wherein, said ovocyte is the ovocyte of zona pellucida.
Method of the present invention, wherein, among the said step B, the concentration of said stoning reagent is 0.1～10ug/ml, the time of handling said ovocyte is 0.5～20 hour.
Method of the present invention, wherein, said stoning reagent is one or more in defence line rhzomorph, Zorubicin, daunorubicin, Plicamycin, Plicamycin, amanitine and the dichloro benzimidazole furan type riboside.
Method of the present invention, wherein, said ovocyte derives from Mammals.
Method of the present invention, wherein, said Mammals is a pig.
The present invention removes the nucleus of ovocyte through using stoning reagent deactivation ovocyte; Need power operation to remove the step of ovocyte karyon in the nuclear transplantation technology of replacement prior art; Simplified the operation of this step, can be widely used in the nuclear transplantation operation of multiple species.
Description of drawings
Fig. 1 is the nuclear transfer method schema of the embodiment of the invention.
Embodiment
Below in conjunction with accompanying drawing, preferred embodiment of the present invention is specified.
Nuclear transfer method schema provided by the invention is as shown in Figure 1, specifically may further comprise the steps:
S101, structure ovocyte;
Among all embodiment of the inventive method, ovocyte can be the ovocyte of any species.Gather ripe mammal ovocyte through live body, or after the ovary extraction of exsomatizing, obtain through maturation in vitro.In the specific embodiment of the present invention, Mammals is preferably pig.Gather ovum from the ovary that exsomatizes, comprise the collection of ovary, the collection of ovum and the maturation of ovum are cultivated.
S102, the constructed ovocyte of employing stoning agent treated obtain cytosome;
Remove the reagent that nucleus adopted of recipient oocyte among the present invention; In comprising following examples the cited reagent; Also comprise the whole replaceable reagent of bringing into play similar functions; Comprise most of antitumor drugs; As Zorubicin (Doxorubicin), defence line rhzomorph A/D (Actinomycin A/D), daunorubicin (daunorubicin), Plicamycin (plicamycin), Plicamycin (mithramycin), amanitine (a-amanitin) and dichloro benzimidazole furan type riboside (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) etc.The reagent that this removal recipient cell karyon is adopted below all is called stoning reagent, and its concentration of treatment to ovocyte is 0.05～20ug/ml, and be 0.5～26 hour action time, is preferably 0.1～10ug/ml, is preferably 0.5～20 hour action time.
Donorcells or nucleus that S103, structure have required hereditary property are with cytosome and donorcells or nucleus fusion, the embryo who obtains rebuilding.
10 groups of specific embodiments below are provided, so that the best implementation method of nuclear transplantation provided by the present invention to be described.Wherein, embodiment 1,2,3,4 and 5 carries out the operation of nuclear transplantation to the ovocyte that needs zona pellucida to adhere to, and embodiment 6,7,8,9 and 10 is to going the ovocyte of zona pellucida to carry out the operation of nuclear transplantation.
Embodiment 1
The operation one that the ovocyte that adheres to the need zona pellucida is cloned:
(1) with 0.1ug/ml defence line rhzomorph as stoning reagent, handle through the sophisticated porcine oocytes of vitro culture 20 hours, the deactivation ovocyte makes it to carry out rna transcription and synthetic proteins;
(2) ovocyte-ovarian cumulus complex body after the taking-up maturation with mucinase enzymic digestion cumulus cell, forms ova nuda, is convenient to carry out micrurgy;
(3) ova nuda being put into the operation of micrurgy dish drips; From the fixing ovocyte of polar body offside, draw donor cell with fixed tube, directly inject donor cell in the ovocyte matter through the stoning pipe of point; During the withdraw of the needle; Take advantage of a situation first polar body is removed in the lump, promptly remove the genetic material of polar body, and arrangement and recovery egg membrane otch.If adopt ovum week crack injection method, for helping merging, draw small amounts of cells matter earlier with the stoning pipe, again tenuigenin is injected ovum week crack together with donorcells, donorcells and egg membrane are close together;
(4) set electric fusion parameters according to different experiments, the cell after the nuclear transplantation is merged, the calcium ion fluctuation during the simulation insemination; Make donorcells fully incorporate in the ovocyte; Reconstituted embryo after electricity swashs moves in the nutrient solution, in constant incubator, cultivates 40 minutes;
(5) according to the different experiments setup parameter, with the chemical treatment of carrying out of nuclear transplantation reconstituted embryo, suppress karyomit(e) and discharge, guarantee reconstructed embryo times type, the reconstituted embryo after the processing is put into nutrient solution, at 38.5 ℃, 5% CO
2Cultivated 5 days in the incubator, the developmental state of observing blastaea is good.
Embodiment 2
The operation two that the ovocyte that adheres to the need zona pellucida is cloned:
(1) with the 10ug/ml Zorubicin as stoning reagent, handle through the sophisticated porcine oocytes of vitro culture 0.5 hour, the deactivation ovocyte makes it to carry out rna transcription and synthetic proteins;
(2) ovocyte-ovarian cumulus complex body after the taking-up maturation with mucinase enzymic digestion cumulus cell, forms ova nuda, is convenient to carry out micrurgy;
(3) ova nuda being put into the operation of micrurgy dish drips; From the fixing ovocyte of polar body offside, draw donor cell with fixed tube, directly inject donor cell in the ovocyte matter through the stoning pipe of point; During the withdraw of the needle; Take advantage of a situation first polar body is removed in the lump, promptly remove the genetic material of polar body, and arrangement and recovery egg membrane otch; If adopt ovum week crack injection method, for helping merging, draw small amounts of cells matter earlier with the stoning pipe, again tenuigenin is injected ovum week crack together with donorcells, donorcells and egg membrane are close together;
(4) set electric fusion parameters according to different experiments, the cell after the nuclear transplantation is merged, the calcium ion fluctuation during the simulation insemination; Make donorcells fully incorporate in the ovocyte; Reconstituted embryo after electricity swashs moves in the nutrient solution, in constant incubator, cultivates 30 minutes;
(5) according to the different experiments setup parameter, with the chemical treatment of carrying out of nuclear transplantation reconstituted embryo, suppress karyomit(e) and discharge, guarantee reconstructed embryo times type, the reconstituted embryo after the processing is put into nutrient solution, at 38.5 ℃, 5% CO
2Cultivated 7 days in the incubator, the developmental state of observing blastaea is good.
Embodiment 3
The operation three that the ovocyte that adheres to the need zona pellucida is cloned:
(1) with the 5ug/ml daunorubicin as stoning reagent, handle through the sophisticated porcine oocytes of vitro culture 10 hours, the deactivation ovocyte makes it to carry out rna transcription and synthetic proteins;
(2) ovocyte-ovarian cumulus complex body after the taking-up maturation with mucinase enzymic digestion cumulus cell, forms ova nuda, is convenient to carry out micrurgy;
(3) ova nuda being put into the operation of micrurgy dish drips; From the fixing ovocyte of polar body offside, draw donor cell with fixed tube, directly inject donor cell in the ovocyte matter through the stoning pipe of point; During the withdraw of the needle; Take advantage of a situation first polar body is removed in the lump, promptly remove the genetic material of polar body, and arrangement and recovery egg membrane otch; If adopt ovum week crack injection method, for helping merging, draw small amounts of cells matter earlier with the stoning pipe, again tenuigenin is injected ovum week crack together with donorcells, donorcells and egg membrane are close together;
(4) set electric fusion parameters according to different experiments, the cell after the nuclear transplantation is merged, the calcium ion fluctuation during the simulation insemination; Make donorcells fully incorporate in the ovocyte; Reconstituted embryo after electricity swashs moves in the nutrient solution, in constant incubator, cultivates 50 minutes;
(5) according to the different experiments setup parameter, with the chemical treatment of carrying out of nuclear transplantation reconstituted embryo, suppress karyomit(e) and discharge, guarantee reconstructed embryo times type, the reconstituted embryo after the processing is put into nutrient solution, in 38.5 ℃, 5% CO
2Cultivated 6 days in the incubator, the developmental state of observing blastaea is good.
Embodiment 4,
The operation four that the ovocyte that adheres to the need zona pellucida is cloned:
(1) with 0.05ug/ml defence line rhzomorph as stoning reagent, handle through the sophisticated porcine oocytes of vitro culture 26 hours, the deactivation ovocyte makes it to carry out rna transcription and synthetic proteins;
(2) ovocyte-ovarian cumulus complex body after the taking-up maturation with mucinase enzymic digestion cumulus cell, forms ova nuda, is convenient to carry out micrurgy;
(3) ova nuda being put into the operation of micrurgy dish drips; From the fixing ovocyte of polar body offside, draw donor cell with fixed tube, directly inject donor cell in the ovocyte matter through the stoning pipe of point; During the withdraw of the needle; Take advantage of a situation first polar body is removed in the lump, promptly remove the genetic material of polar body, and arrangement and recovery egg membrane otch; If adopt ovum week crack injection method, for helping merging, draw small amounts of cells matter earlier with the stoning pipe, again tenuigenin is injected ovum week crack together with donorcells, donorcells and egg membrane are close together;
(4) set electric fusion parameters according to different experiments, the cell after the nuclear transplantation is merged, the calcium ion fluctuation during the simulation insemination; Make donorcells fully incorporate in the ovocyte; Reconstituted embryo after electricity swashs moves in the nutrient solution, in constant incubator, cultivates 30-60 minute;
(5) according to the different experiments setup parameter, with the chemical treatment of carrying out of nuclear transplantation reconstituted embryo, suppress karyomit(e) and discharge, guarantee reconstructed embryo times type, the reconstituted embryo after the processing is put into nutrient solution, in 38.5 ℃, 5% CO
2Cultivated 5 days in the incubator, the developmental state of observing blastaea is good.
Embodiment 5
The operation five that the ovocyte that adheres to the need zona pellucida is cloned:
(1) with 20ug/ml defence line rhzomorph as stoning reagent, handle through the sophisticated porcine oocytes of vitro culture 10 hours, the deactivation ovocyte makes it to carry out rna transcription and synthetic proteins;
(2) ovocyte-ovarian cumulus complex body after the taking-up maturation with mucinase enzymic digestion cumulus cell, forms ova nuda, is convenient to carry out micrurgy;
(3) ova nuda being put into the operation of micrurgy dish drips; From the fixing ovocyte of polar body offside, draw donor cell with fixed tube, directly inject donor cell in the ovocyte matter through the stoning pipe of point; During the withdraw of the needle; Take advantage of a situation first polar body is removed in the lump, promptly remove the genetic material of polar body, and arrangement and recovery egg membrane otch; If adopt ovum week crack injection method, for helping merging, draw small amounts of cells matter earlier with the stoning pipe, again tenuigenin is injected ovum week crack together with donorcells, donorcells and egg membrane are close together;
(4) set electric fusion parameters according to different experiments, the cell after the nuclear transplantation is merged, the calcium ion fluctuation during the simulation insemination; Make donorcells fully incorporate in the ovocyte; Reconstituted embryo after electricity swashs moves in the nutrient solution, in constant incubator, cultivates 60 minutes;
(5) according to the different experiments setup parameter, with the chemical treatment of carrying out of nuclear transplantation reconstituted embryo, suppress karyomit(e) and discharge, guarantee reconstructed embryo times type, the reconstituted embryo after the processing is put into nutrient solution, in 38.5 ℃, 5%CO
2Cultivated 5 days in the incubator, the developmental state of observing blastaea is good.
Embodiment 6
The operation one of cloning to the ovocyte that removes zona pellucida:
(1) with the 0.1ug/ml Plicamycin as stoning reagent, handle through the sophisticated porcine oocytes of vitro culture 26 hours, the deactivation ovocyte makes it to carry out rna transcription and synthetic proteins;
(2) ovocyte-ovarian cumulus complex body after the taking-up maturation with mucinase enzymic digestion cumulus cell, forms ova nuda, is convenient to follow-up clone operations;
(3) ova nuda that will slough cumulus cell is placed in the pronase and digests, and removes zona pellucida;
(4) blow and beat ovocyte repeatedly with a mouthful suction pipe and remove the genetic material that its surperficial first polar body is promptly removed polar body;
The ovocyte that (5) will remove first polar body is handled with phytohemagglutinin, and making it surface viscosity increases, and binds a donorcells in the cytogamy appearance, under the electric shock effect of 2.0KV/cm, makes the two fusion;
(6) embryo after will merging was placed in the nutrient solution 60 minutes, made donorcells fully incorporate in the ovocyte;
(7) embryo after will merging is placed on and activates in the liquid, activates the calcium fluctuation when promptly simulating insemination with the electric shock of 0.85KV/cm;
(8) reconstructed embryo is placed contain 5ug/ml cytochalasin and 10ug/ml cycloheximide blended nutrient solution and handled 4 hours, suppress karyomit(e) and discharge, guarantee reconstructed embryo times type;
After (9) 4 hours, reconstructed embryo is placed in the culture system of little cave (well of the well), in embryo medium, cultivated 5 days, it is all right to observe fetal development.
Embodiment 7
The operation two of cloning to the ovocyte that removes zona pellucida:
(1) with 10ug/ml defence line rhzomorph as stoning reagent, handle through the sophisticated porcine oocytes of vitro culture 0.5 hour, the deactivation ovocyte makes it to carry out rna transcription and synthetic proteins;
(2) ovocyte-ovarian cumulus complex body after the taking-up maturation with mucinase enzymic digestion cumulus cell, forms ova nuda, is convenient to follow-up clone operations;
(3) ova nuda that will slough cumulus cell is placed in the pronase and digests, and removes zona pellucida;
(4) blow and beat ovocyte repeatedly with a mouthful suction pipe and remove the genetic material that its surperficial first polar body is promptly removed polar body;
The ovocyte that (5) will remove first polar body is handled with phytohemagglutinin, and making it surface viscosity increases, and binds a donorcells in the cytogamy appearance, under the electric shock effect of 2.0KV/cm, makes the two fusion;
(6) embryo after will merging was placed in the nutrient solution 60 minutes, made donorcells fully incorporate in the ovocyte;
(7) embryo after will merging is placed on and activates in the liquid, activates the calcium fluctuation when promptly simulating insemination with the electric shock of 0.85KV/cm;
(8) reconstructed embryo is placed contain 5ug/ml cytochalasin and 10ug/ml cycloheximide blended nutrient solution and handled 5 hours, suppress karyomit(e) and discharge, guarantee reconstructed embryo times type;
After (9) 4 hours, reconstructed embryo is placed in the culture system of little cave (well of the well), in embryo medium, cultivated 7 days, it is all right to observe fetal development.
Embodiment 8
The operation three of cloning to the ovocyte that removes zona pellucida:
(1) with 5ug/ml defence line rhzomorph as stoning reagent, handle through the sophisticated porcine oocytes of vitro culture 5 hours, the deactivation ovocyte makes it to carry out rna transcription and synthetic proteins;
(2) ovocyte-ovarian cumulus complex body after the taking-up maturation with mucinase enzymic digestion cumulus cell, forms ova nuda, is convenient to follow-up clone operations;
(3) ova nuda that will slough cumulus cell is placed in the pronase and digests, and removes zona pellucida;
(4) blow and beat ovocyte repeatedly with a mouthful suction pipe and remove the genetic material that its surperficial first polar body is promptly removed polar body;
The ovocyte that (5) will remove first polar body is handled with phytohemagglutinin, and making it surface viscosity increases, and binds a donorcells in the cytogamy appearance, under the electric shock effect of 2.0KV/cm, makes the two fusion;
(6) embryo after will merging was placed in the nutrient solution 60 minutes, made donorcells fully incorporate in the ovocyte;
(7) embryo after will merging is placed on and activates in the liquid, activates the calcium fluctuation when promptly simulating insemination with the electric shock of 0.85KV/cm;
(8) reconstructed embryo is placed contain 5ug/ml cytochalasin and 10ug/ml cycloheximide blended nutrient solution and handled 4 hours, suppress karyomit(e) and discharge, guarantee reconstructed embryo times type;
After (9) 4 hours, reconstructed embryo is placed in the culture system of little cave (well of the well), in embryo medium, cultivated 7 days, it is all right to observe fetal development.
Embodiment 9
The operation four of cloning to the ovocyte that removes zona pellucida:
(1) with 0.05ug/ml defence line rhzomorph as stoning reagent, handle through the sophisticated porcine oocytes of vitro culture 20 hours, the deactivation ovocyte makes it to carry out rna transcription and synthetic proteins;
(2) ovocyte-ovarian cumulus complex body after the taking-up maturation with mucinase enzymic digestion cumulus cell, forms ova nuda, is convenient to follow-up clone operations;
(3) ova nuda that will slough cumulus cell is placed in the pronase and digests, and removes zona pellucida;
(4) blow and beat ovocyte repeatedly with a mouthful suction pipe and remove the genetic material that its surperficial first polar body is promptly removed polar body;
The ovocyte that (5) will remove first polar body is handled with phytohemagglutinin, and making it surface viscosity increases, and binds a donorcells in the cytogamy appearance, under the electric shock effect of 2.0KV/cm, makes the two fusion;
(6) embryo after will merging was placed in the nutrient solution 60 minutes, made donorcells fully incorporate in the ovocyte;
(7) embryo after will merging is placed on and activates in the liquid, activates the calcium fluctuation when promptly simulating insemination with the electric shock of 0.85KV/cm;
(8) reconstructed embryo is placed contain 5ug/ml cytochalasin and 10ug/ml cycloheximide blended nutrient solution and handled 4 hours, suppress karyomit(e) and discharge, guarantee reconstructed embryo times type;
After (9) 4 hours, reconstructed embryo is placed in the culture system of little cave (well of the well), in embryo medium, cultivated 5 days, it is all right to observe fetal development.
Embodiment 10
The operation five of cloning to the ovocyte that removes zona pellucida:
(1) with 20ug/ml defence line rhzomorph as stoning reagent, handle through the sophisticated porcine oocytes of vitro culture 5 hours, the deactivation ovocyte makes it to carry out rna transcription and synthetic proteins;
(2) ovocyte-ovarian cumulus complex body after the taking-up maturation with mucinase enzymic digestion cumulus cell, forms ova nuda, is convenient to follow-up clone operations;
(3) ova nuda that will slough cumulus cell is placed in the pronase and digests, and removes zona pellucida;
(4) blow and beat ovocyte repeatedly with a mouthful suction pipe and remove the genetic material that its surperficial first polar body is promptly removed polar body;
The ovocyte that (5) will remove first polar body is handled with phytohemagglutinin, and making it surface viscosity increases, and binds a donorcells in the cytogamy appearance, under the electric shock effect of 2.0KV/cm, makes the two fusion;
(6) embryo after will merging was placed in the nutrient solution 60 minutes, made donorcells fully incorporate in the ovocyte;
(7) embryo after will merging is placed on and activates in the liquid, activates the calcium fluctuation when promptly simulating insemination with the electric shock of 0.85KV/cm;
(8) reconstructed embryo is placed contain 5ug/ml cytochalasin and 10ug/ml cycloheximide blended nutrient solution and handled 4 hours, suppress karyomit(e) and discharge, guarantee reconstructed embryo times type;
After (9) 4 hours, reconstructed embryo is placed in the culture system of little cave (well of the well), in embryo medium, cultivated 7 days, it is all right to observe fetal development.
Through measuring, the nuclear transfer method among the above embodiment all can obtain the sophisticated embryo of ability normal development.
In sum; The present invention removes the nucleus of ovocyte through using stoning reagent deactivation ovocyte; Need power operation to remove the step of ovocyte karyon in the nuclear transplantation technology of replacement prior art; Simplified the operation of this step, can be widely used in the nuclear transplantation operation of multiple species.
Should be understood that, concerning those of ordinary skills, can improve or conversion, and these improvement and conversion all should belong to the protection domain of accompanying claims of the present invention according to above-mentioned explanation.
Claims (2)
1. nuclear transfer method may further comprise the steps:
With the 0.1ug/ml Plicamycin as stoning reagent; Processing was through the sophisticated porcine oocytes of vitro culture 26 hours; Or with 0.05ug/ml defence line rhzomorph as stoning reagent; Processing was through the sophisticated porcine oocytes of vitro culture 20 hours, and the deactivation ovocyte makes it to carry out rna transcription and synthetic proteins; Ovocyte-ovarian cumulus complex body after the taking-up maturation with mucinase enzymic digestion cumulus cell, forms ova nuda, is convenient to follow-up clone operations; Ova nuda is placed in the pronase digests, remove zona pellucida;
Blow and beat ovocyte repeatedly with a mouthful suction pipe and remove the genetic material that its surperficial first polar body is promptly removed polar body; To remove the ovocyte of first polar body handles with phytohemagglutinin; Making it surface viscosity increases; Bind a donorcells in the cytogamy appearance, under the electric shock effect of 2.0KV/cm, make the two fusion; Embryo after merging was placed in the nutrient solution 60 minutes, donorcells is fully incorporated in the ovocyte, the embryo after merging is placed on activates in the liquid, activate the calcium fluctuation when promptly simulating insemination with the electric shock of 0.85KV/cm;
Reconstructed embryo placed contain 5ug/ml cytochalasin and 10ug/ml cycloheximide blended nutrient solution and handled 4 hours, suppress karyomit(e) and discharge, guarantee reconstructed embryo times type; After 4 hours, reconstructed embryo is placed in the culture system of little cave, in embryo medium, cultivated 5 days.
2. nuclear transfer method may further comprise the steps:
, handle through the sophisticated porcine oocytes of vitro culture 5 hours as stoning reagent with 5ug/ml or 20ug/ml defence line rhzomorph, the deactivation ovocyte makes it to carry out rna transcription and synthetic proteins; Ovocyte-ovarian cumulus complex body after the taking-up maturation with mucinase enzymic digestion cumulus cell, forms ova nuda, is convenient to follow-up clone operations; Ova nuda is placed in the pronase digests, remove zona pellucida;
Blow and beat ovocyte repeatedly with a mouthful suction pipe and remove the genetic material that its surperficial first polar body is promptly removed polar body; To remove the ovocyte of first polar body handles with phytohemagglutinin; Making it surface viscosity increases; Bind a donorcells in the cytogamy appearance, under the electric shock effect of 2.0KV/cm, make the two fusion; Embryo after merging was placed in the nutrient solution 60 minutes, donorcells is fully incorporated in the ovocyte, the embryo after merging is placed on activates in the liquid, activate the calcium fluctuation when promptly simulating insemination with the electric shock of 0.85KV/cm;
Reconstructed embryo placed contain 5ug/ml cytochalasin and 10ug/ml cycloheximide blended nutrient solution and handled 4 hours, suppress karyomit(e) and discharge, guarantee reconstructed embryo times type;
After 4 hours, reconstructed embryo is placed in the culture system of little cave, in embryo medium, cultivated 7 days.
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| CN2009101072665A CN101580828B (en) | 2009-05-06 | 2009-05-06 | Nuclear transfer method |
| PCT/CN2010/000631 WO2010127559A1 (en) | 2009-05-06 | 2010-05-05 | Nuclear transfer method |
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| CN101580828B (en) * | 2009-05-06 | 2012-06-20 | 深圳华大基因科技有限公司 | Nuclear transfer method |
| CN102367454B (en) * | 2011-10-11 | 2015-04-01 | 深圳华大基因研究院 | Transgenic animal and preparation method thereof |
| CN106754348B (en) * | 2016-12-23 | 2024-02-09 | 南京农业大学 | Electroactivated equipment and RNA interference method of oocyte or embryo using same |
| CN119331864A (en) * | 2024-11-02 | 2025-01-21 | 广州市中誉仪器科技有限公司 | A method for electrofusion microinsemination |
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| Marcelo Tigre Moura et al.Analysis of actinomycin D treated cattle oocytes and their use for somatic cell nuclear transfer.《Anim Reprod Sci》.2007,第109卷(第1-4期),40-49. * |
| 刘吉.猪体外成熟卵母细胞孤雌激活及胚胎体外发育的研究.《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》.2007,全文. * |
| 孙兴参.猪体细胞核移植及几个相关问题的研究.《中国优秀博硕士学位论文全文数据库（博士）农业科技辑》.2002, * |
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