WO2010127559A1 - Procédé de transfert nucléaire - Google Patents

Procédé de transfert nucléaire Download PDF

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Publication number
WO2010127559A1
WO2010127559A1 PCT/CN2010/000631 CN2010000631W WO2010127559A1 WO 2010127559 A1 WO2010127559 A1 WO 2010127559A1 CN 2010000631 W CN2010000631 W CN 2010000631W WO 2010127559 A1 WO2010127559 A1 WO 2010127559A1
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WO
WIPO (PCT)
Prior art keywords
oocyte
hours
oocytes
agent
nucleus
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Ceased
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PCT/CN2010/000631
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English (en)
Chinese (zh)
Inventor
杜玉涛
王俊
汪建
杨焕明
林琳
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication of WO2010127559A1 publication Critical patent/WO2010127559A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877—Techniques for producing new mammalian cloned embryos

Definitions

  • the present invention relates to the field of embryology, and in particular to a method of nuclear transfer. Background technique
  • “Clone” is an English “clone” transliteration, which refers to the offspring of an animal that can be parthenogenetically propagated. The genetic composition of all cloned offspring is identical, also known as the asexual reproduction cell line.
  • Animal cloning refers to the process of obtaining the same genetically homologous offspring from an animal without sexual reproduction, including parthenogenetic reproduction, identical twins, embryo division, and nuclear transfer. At present, animal cloning is mainly obtained by nuclear transfer and embryo division, and normally develops into a new individual with the same genetic material.
  • Nuclear transfer is a method of micromanipulation, electrofusion, etc., to reconstitute a nuclear donor at a certain stage of development, such as embryonic cells or somatic cells, and nuclear receptors at corresponding developmental stages, such as enucleated pronuclear embryos. Or mature oocytes. Through the embryo transfer of reconstructed embryos, a bioengineering technique for mass production of genetically homogenous mammals is achieved.
  • the enucleation step in the process of nuclear transfer is performed by microscopically taking a microneedle with a microneedle, or by a blade resection method.
  • This operation step is not only cumbersome, but also needs to be performed manually, which may easily lead to operational errors, and cannot be very Good to ensure cell nuclear transfer The success rate of planting.
  • the object of the present invention is to provide a nuclear transfer which is easy to operate and can quickly and accurately remove nuclei without using a micromanipulator mechanical method or a blade excision method to remove a receptor oocyte nuclei without using the micromanipulator mechanical method or the blade excision method. method.
  • the method of the present invention, the method of the invention, the method of the present invention 5 ⁇ 26 ⁇ ,, preferably, 0. 5 - 26 hours, more preferably 0. 05 - 20 ug / ml, more preferably 0. 1 - 10 ug / ml of the denucleating agent for 10 minutes to 48 hours, preferably 20 minutes to 30 hours, more preferably 0. 5 ⁇ 26 hours, More preferably, it is 0.5 to 20 hours, preferably 5 to 10 hours, to obtain a cytoplast.
  • the invention also relates to a method of nuclear transfer, comprising the following steps
  • A constructing an oocyte or obtaining or providing an oocyte
  • oocyte is treated for 10 minutes to 48 hours, preferably 20 minutes to 30 hours, more preferably 0.5 to 26 hours, more preferably 0.5 to 20 hours, preferably 5 to 10 hours, to obtain a cytoplast;
  • the oocyte is preferably a mature oocyte.
  • the oocyte can be from a mammal, such as a scorpion animal or a livestock animal or a human.
  • the oocyte is not a human oocyte (ie, from a non-human mammal).
  • the obtained oocyte may be obtained by collecting mature mammalian oocytes in vivo or by in vitro maturation after being extracted from an isolated ovary.
  • the in vitro maturation of oocytes is well known to those of ordinary skill in the art, and for example, mature oocytes can be obtained in vitro according to the method described in CN101313072A (published on Nov. 26, 2008).
  • the oocyte is an oocyte to which a zona pellucida is attached, or an oocyte to which a zona pellucida is attached.
  • the oocyte is an oocyte that is opaque.
  • the concentration of the enucleating agent is 0.1 to 10 ug/ml, and the time for processing the oocyte is 0. 5 to 20 hours.
  • the final concentration of the denuclear reagent is 0. 001-100 ug/ml, preferably 0, in the step of the method of the present invention.
  • the appropriate temperature for the treatment, contact or placement can be determined by one skilled in the art, and the temperature can be from room temperature to 39 degrees Celsius, such as room temperature - 37 degrees Celsius or 37-39 degrees Celsius.
  • the enucleating agent can be an agent capable of functional enucleation/inactivation of oocytes.
  • the enucleating agent can be an agent that inactivates the oocyte, rendering it incapable of RNA transcription and synthesis of the protein.
  • the enucleating agent can be an anti-tumor drug or an analog thereof.
  • the enucleating agent may be actinomycin, doxorubicin, daunorubicin, pucamycin, phleomycin, muscimol and dichlorobenzimidazole furan riboside One or several of them (such as two, three, or four).
  • a preferred enucleating agent is actinomycin, doxorubicin, daunorubicin, or phosfomycin or a combination of one or more thereof.
  • the oocyte is derived from a mammal.
  • the mammal is a pig.
  • the donor cell or the donor cell or the donor cell nucleus may be derived from an autologous cell or a stem cell.
  • the donor cell or donor cell or donor cell nucleus may be from a mammal, such as an experimental animal or a livestock animal.
  • the donor cell or donor cell or donor cell nucleus is not human, e.g., from a non-human mammal.
  • the present invention removes the nucleus of an oocyte by inactivating an oocyte by using an enucleating reagent, making it impossible to perform RNA transcription and synthesizing a protein, and replacing the step of mechanically removing the oocyte nucleus in the prior art nuclear transfer technique. , simplifies the operation of this step, and can be widely applied to nuclear transfer operations of various species.
  • the invention also provides a composition for removing a cell nucleus of a cell comprising an enucleating agent.
  • the enucleating agent may be capable of functional enucleation or inactivation of oocytes Reagents.
  • the enucleating agent can be an agent that inactivates the oocyte, rendering it incapable of RNA transcription and protein synthesis.
  • the enucleating agent can be an anti-tumor drug or an analog thereof.
  • the enucleating agent may be actinomycin, doxorubicin, daunorubicin, pucamycin, phleomycin, muscimol and dichlorobenzimidazole furan riboside One or several of them (such as two, three, or four).
  • the cells are preferably oocytes, in particular mature oocytes.
  • the composition can be used to remove the nuclei of oocytes to provide enucleated oocytes for nuclear transfer or other uses.
  • the invention also relates to an agent capable of functionally enucleating or inactivating cells for removing the nucleus of a cell.
  • the invention further relates to the use of an agent capable of functional enucleation or inactivation of a cell in the preparation of an enucleating agent for removing the nucleus of a cell.
  • the invention also relates to the use of an agent capable of functional enucleation or inactivation of cells for removing the nucleus of a cell.
  • the cell is preferably an oocyte, in particular a mature oocyte.
  • the agent capable of functionally enucleating or inactivating the oocyte may be an agent that inactivates the oocyte and makes it impossible to perform RNA transcription and synthesis of a protein.
  • the agent can be an anti-tumor drug or an analog thereof.
  • the agent may be in the actinomycin, doxorubicin, daunorubicin, pucamycin, phleomycin, muscimol, and dichlorobenzimidazole furan riboside. One or several (such as two, three, or four).
  • FIG. 1 is a flow chart of a method for nuclear transfer of an embodiment of the present invention.
  • Figure 2 is a typical photograph of the embryo obtained in the step (5) of Example 1-5 and the embryo obtained in the step (9) of Example 6-10, showing that the obtained embryo was well developed. detailed description
  • FIG. 1 The flow chart of the nuclear transfer method provided by the present invention is shown in FIG. 1 , and specifically includes the following steps:
  • the oocyte can be an oocyte of any species.
  • the oocyte can be obtained by collecting the mature mammalian oocyte by living body, or extracting it from the isolated ovary, and obtaining it by in vitro maturation.
  • the mammal is preferably a pig.
  • Eggs are collected from isolated ovaries, including the collection of ovaries, the collection of eggs, and the maturation of eggs.
  • the reagent for removing the nucleus of the recipient egg cell in the present invention includes, in addition to the reagents listed in the following examples, an inactivated egg All alternative reagents for mother cells, including most anti-tumor drugs, such as Doxorubicin, Actinomycin A/D, daunorubicin, Pakamycin Pl icamyc in , mi thramycin , a -amani t in and dichlorobenzimidazole furan riboside ( 5 , 6-dichloro-l- ⁇ -Dr ibofuranosylbenz imidazole ⁇ 5 ⁇ 26 ⁇ , ⁇
  • the inoculation of the nucleus of the nucleus of the nucleus of the nucleus of the nucleus of the nucleus is 0.
  • the action time is 0. 5 ⁇ 26 hours, preferably 0. 1 ⁇ 10ug / ml, the action time is preferably 0. 5 ⁇ 20 hours.
  • the treatment temperature can be from room temperature to 39 degrees Celsius, such as room temperature - 37 degrees Celsius or 37-39 degrees Celsius.
  • Examples 1, 2, 3, 4, and 5 are for the need for a transparent belt
  • Examples 6, 7, 8, 9, and 10 are operations for performing nuclear transfer on oocytes that have gone to the zona pellucida.
  • the culture solution in the following examples was Porcine zygote medium - 3 (PZM-3).
  • composition of the fusion medium used in the following embodiment is: 0. 3 M mannitol, 0.01% polyvinyl alcohol.
  • composition of the activating liquid in the following examples is: 0.3 M mannitol, 0.01% polyvinyl alcohol,
  • the electrofusion parameters are set according to different experiments, and the cells after nuclear transplantation are fused (in this embodiment, the enucleated oocyte and the donor cell are fused under the electric shock of 2. OKV/cm). ), simulating calcium ion fluctuations when sperm enters the egg (in this case) In the example, this is achieved by activation with an electric shock of 0.85 KV/cm, so that the donor cells are fully integrated into the oocyte, and the electrogenerated embryos are transferred into the culture medium in a constant temperature incubator (38. 5 ), cultivating for 40 minutes;
  • Chemically treating the nuclear transfer recombinant embryo according to different experimental setting parameters (in the present embodiment, this is carried out by: placing the recombinant constructor in a medium containing 5 ug/ml cytochalasin and 10 ug/ml The mixture of the cytosine mixed culture solution for 4 hours to 6 hours) inhibits chromosome excretion, ensures the reconstructed embryo ploidy, and the treated recombinant embryo is placed in the culture medium at 38.5, 5% C0 2 incubator The culture was carried out for 5 days, and the development of the embryo was observed to be good.
  • the electrofusion parameters are set according to different actual conditions, and the cells after nuclear transplantation are fused (in this embodiment, both the enucleated oocyte and the donor cell are subjected to an electric shock of 2. OKV/cm. Fusion), simulating calcium ion fluctuations when sperm enters the egg (in this case) In the example, this is achieved by activation with an electric shock of 0.85 KV/cm, and the donor cells are fully integrated into the oocyte, and the electrogenerated embryos are transferred into the culture medium in a constant temperature incubator (38. 5 ⁇ ) , culture for 30 minutes;
  • Chemically treating the nuclear transfer recombinant embryo according to different experimental setting parameters (in the present embodiment, this is carried out by: placing the recombinant constructor in a medium containing 5 ug/ml cytochalasin and 10 ug/ml The mixture of the cytosine mixed culture solution for 4 hours to 6 hours) inhibits chromosome excretion, ensures the reconstructed embryo ploidy, and the treated recombinant embryo is placed in the culture solution at 38.5, 5% C0 2 incubator The medium was cultured for 7 days, and the development of the embryo was observed to be good.
  • the electrofusion parameters are set according to different experiments, and the cells after nuclear transplantation are fused (in this embodiment, the enucleated oocyte and the donor cell are fused under the electric shock of 2. OKV/cm). ), simulating calcium ion fluctuations when sperm enters the egg (in this case) In the example, this is achieved by activation with an electric shock of 0.85 KV/cm, so that the donor cells are fully integrated into the oocyte, and the electrogenerated embryos are transferred into the culture medium in a constant temperature incubator (38. 5 V), culture for 50 minutes;
  • the electrofusion parameters are set according to different experiments, and the cells after nuclear transplantation are fused (in this embodiment, the enucleated oocyte and the donor cell are fused under the electric shock of 2. OKV/cm). ), simulating calcium ion fluctuations when sperm enters the egg (in this case) In the example, this is achieved by activation with an electric shock of 0.85 KV/cm, and the donor cells are fully integrated into the oocyte, and the electrogenerated embryos are transferred into the culture medium in a constant temperature incubator (38. 5 ⁇ ) , culture for 30-60 minutes;
  • Chemically treating the nuclear transfer recombinant embryo according to different experimental setting parameters (in this embodiment, this is carried out by: placing the recombinant constructor in a medium containing 5 ug/ml cytochalasin and 10 ug/ml The mixture of the cytosine mixed culture solution for 4 hours to 6 hours) inhibits chromosome excretion, ensures the reconstructed embryo ploidy, and the treated recombinant embryo is placed in the culture solution at 38. 5 ⁇ €, 5% of C0 2 The culture was carried out for 5 days in the incubator, and the development of the embryos was observed to be good.
  • the electrofusion parameters are set according to different experiments, and the cells after nuclear transplantation are fused (in this embodiment, the enucleated oocyte and the donor cell are fused under the electric shock of 2. OKV/cm). ), simulating calcium ion fluctuations when sperm enters the egg (in this case) In the example, this is achieved by activation with an electric shock of 0.85 KV/cm), the donor cells are fully integrated into the oocyte, and the electrogenerated recombinant embryos are transferred into the culture medium in a constant temperature incubator (38.5), cultured. 60 minutes;
  • the oocyte from which the first polar body is removed is treated with the plant lectin to increase the surface viscosity, and a donor cell is bonded in the cell fusion instrument, and the two are fused under the electric shock of 2. OKV/cm. ;
  • the fused embryo was placed in an activating solution and activated by an electric shock of 0.85 KV/cm, that is, a calcium fluctuation when simulating sperm entry;
  • the reconstructed embryos were treated in a medium containing 5 ug/ml cytochalasin and lOug/ml cycloheximide for 4 hours to inhibit chromosome excretion and ensure the reconstructed embryo ploidy;
  • the oocyte from which the first polar body is removed is treated with the plant lectin to increase the surface viscosity, and a donor cell is bonded in the cell fusion instrument, and the two are fused under the electric shock of 2. OKV/cm. ;
  • the reconstructed embryos were treated in a medium containing 5 ug/ml cytochalasin and 10 ug/ml cycloheximide for 5 hours to inhibit chromosome excretion and ensure the reconstructed embryo ploidy;
  • the oocyte from which the first polar body is removed is treated with the plant lectin to increase the surface viscosity, and a donor cell is bonded in the cell fusion instrument, and the two are fused under the electric shock of 2. OKV/cm. ;
  • the reconstructed embryos were treated in a medium containing 5 ug/ml of cytochalasin and 10 ug/ml of cycloheximide for 4 hours to inhibit chromosome excretion and ensure the reconstructed embryo ploidy;
  • the oocyte from which the first polar body is removed is treated with the plant lectin to increase the surface viscosity, and a donor cell is bonded in the cell fusion instrument, and the two are fused under the electric shock of 2. OKV/cm. ;
  • the reconstructed embryos were treated in a medium containing 5 ug/ml of cytochalasin and 10 ug/ml of cycloheximide for 4 hours to inhibit chromosome excretion and ensure the reconstructed embryo ploidy;
  • the oocyte from which the first polar body is removed is treated with the plant lectin to increase the surface viscosity, and a donor cell is bonded in the cell fusion instrument, and the two are fused under the electric shock of 2. OKV/cm. ;
  • the reconstructed embryos were treated in a medium containing 5 ug/ml cytochalasin and 10 ug/ml cycloheximide for 4 hours to inhibit chromosome excretion and ensure the reconstructed embryo ploidy;
  • the present invention removes the nucleus of an oocyte by inactivating an oocyte by using a denucleating agent, and replaces the step of mechanically removing the nucleus of the oocyte in the prior art nuclear transfer technique, which simplifies the step.
  • the operation can be widely applied to nuclear transfer operations of various species.

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Abstract

La présente invention porte sur un procédé de transfert nucléaire et le procédé comprend les étapes suivantes : la construction d'un ovocyte; l'utilisation d'un agent de dénucléation à une concentration de 0,05-20 µg/ml pour traiter l'ovocyte pendant 0,5-26 heures pour obtenir un cytoplaste; la construction d'une cellule donneuse ou d'un noyau doté de propriétés génétiques requises et la fusion du cytoplaste avec la cellule donneuse ou le noyau pour obtenir un embryon reconstitué. La présente invention remplace l'étape qui nécessite une opération mécanique pour enlever le noyau de l'ovocyte dans la technologie de transfert nucléaire de l'art antérieur, par l'utilisation d'un agent de dénucléation pour inactiver l'ovocyte pour enlever le noyau de l'ovocyte, ce qui simplifie de cette manière la mise en œuvre de ladite étape et permet son application large dans la mise en œuvre d'un transfert nucléaire d'un grand nombre d'espèces.
PCT/CN2010/000631 2009-05-06 2010-05-05 Procédé de transfert nucléaire Ceased WO2010127559A1 (fr)

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CN200910107266.5 2009-05-06
CN2009101072665A CN101580828B (zh) 2009-05-06 2009-05-06 一种细胞核移植方法

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Cited By (1)

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CN119331864A (zh) * 2024-11-02 2025-01-21 广州市中誉仪器科技有限公司 一种电融合显微授精方法

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CN101580828B (zh) * 2009-05-06 2012-06-20 深圳华大基因科技有限公司 一种细胞核移植方法
CN102367454B (zh) * 2011-10-11 2015-04-01 深圳华大基因研究院 转基因动物及其制备方法
CN106754348B (zh) * 2016-12-23 2024-02-09 南京农业大学 一种电激活设备及采用其的卵母细胞或者胚胎的rna干扰方法

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WO2004010771A2 (fr) * 2002-07-29 2004-02-05 Trustees Of Tufts College Procede de formation d'embryon de transfert nucleaire
CN101580828A (zh) * 2009-05-06 2009-11-18 深圳华大基因科技有限公司 一种细胞核移植方法

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AUPR451401A0 (en) * 2001-04-20 2001-05-24 Monash University A method of nuclear transfer

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WO2004010771A2 (fr) * 2002-07-29 2004-02-05 Trustees Of Tufts College Procede de formation d'embryon de transfert nucleaire
CN101580828A (zh) * 2009-05-06 2009-11-18 深圳华大基因科技有限公司 一种细胞核移植方法

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MOURA, M.T. ET AL.: "Analysis of actinomycin D treated cattle oocytes and their use for somatic cell nuclear transfer.", ANIMAL REPRODUCTION SCIENCE., vol. 109, 26 December 2007 (2007-12-26), pages 40 - 49 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119331864A (zh) * 2024-11-02 2025-01-21 广州市中誉仪器科技有限公司 一种电融合显微授精方法

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