CN108504691B - Method for editing female gene - Google Patents
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- CN108504691B CN108504691B CN201810237058.6A CN201810237058A CN108504691B CN 108504691 B CN108504691 B CN 108504691B CN 201810237058 A CN201810237058 A CN 201810237058A CN 108504691 B CN108504691 B CN 108504691B
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- 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
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- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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Abstract
The invention discloses an editing method aiming at female genes, which comprises the following steps: obtaining parthenogenetic fertilized eggs, editing parthenogenetic fertilized egg genes, separating parthenogenetic blastomeres of the parthenogenetic embryos after editing, reconstructing parthenogenetic embryos, and identifying editing results. The method can indirectly realize the function of accurately editing the mammalian ovum, can ensure the final gene editing effect, and is beneficial to the transformation of the gene editing technology to clinical medicine.
Description
Technical Field
The invention relates to a method for editing female genes, in particular to a method for editing female genes by combining a gene editing technology with a cell nucleus transplantation technology.
Background
Gene editing techniques are currently gaining more and more progress in the research field and have been extensively tried in human cell samples. However, the main risks of the current gene editing technology are still the problems of editing efficiency and off-target, which is particularly critical to clinical popularization and application, and the gene editing technology can be safely popularized to the clinic only by overcoming the related risks. At present, aiming at embryo gene editing, the efficiency of editing cannot be clearly determined before all cells of an embryo are not detected, so that before detection, a user cannot predict which cell is successfully edited and which cell is failed to be edited. But once molecularly detected, the cell loses its value of utilization, which is fatal to the embryo itself. On the other hand, under the existing technical conditions, the relevant gene editing can not be carried out on the female chromosome of the mammal, which further limits the future application range of the gene editing technology.
Disclosure of Invention
In order to effectively edit the female gene 100% successfully, the invention provides a gene editing method combining a gene editing technology and a nuclear transfer technology, which can confirm and utilize the editing effect of the female gene.
In order to solve the above technical problems, the present invention provides an editing method for a female gene, comprising the steps of:
1) obtaining parthenogenetic fertilized eggs: preparing parthenogenetic fertilized eggs only containing single parthenogenetic female chromosomes;
2) and editing parthenogenetic fertilized egg genes: editing the target gene of the female solitary zygote by using a gene editing technology in a prokaryotic stage;
3) isolation of parthenogenetic blastomeres after editing: performing embryo culture on the parthenogenetic female fertilized eggs edited in the step 2) to a stage with blastomeres, and separating one blastomere;
4) reconstructing parthenogenetic embryos: reconstructing the blastomere separated in the step 3) and the MII-stage oocyte to obtain a reconstructed parthenogenetic female zygote, and continuously carrying out embryo culture on the reconstructed parthenogenetic female zygote to a stage with the blastomere;
5) and (4) identification of an editing result: taking out one blastomere obtained by culturing in the step 4) for identifying an editing result, wherein the genome of the remaining blastomere obtained in the step 4) is completely consistent with the genome of the blastomere used for identification;
all cells and chromosomes used in the method are derived from non-human mammals.
In the above method, the method for preparing the parthenogenetic female fertilized egg in step 1) includes the steps described in the following a or b:
a. injecting a single sperm into an oocyte in an MII stage to activate fertilization, removing a male pronucleus in a pronucleus stage and reserving a female pronucleus;
b. carrying out parthenogenetic activation on the MII-stage oocyte.
In the above method, the activation method in step b is chemical activation and electric activation, preferably the chemical activation mode of calcium ionophore A23187 combined with puromycin.
In the above method, the gene editing technology in step 2) is Zinc Finger Nuclease (ZFN), transcription activator-like effector nuclease (TALEN) or CRISPR/Cas9 technology;
the editing mode is one or more of base modification, base insertion, base knockout and base replacement of the target gene.
In any of the above methods, the blastomere-bearing stage in step 3) and step 4) is a 2-cell stage, a 4-cell stage, an 8-cell stage, a 16-cell stage, a 32-cell stage, a fusogenic embryonic stage, a morula stage, or a blastocyst stage.
In any of the above methods, the reconstitution in step 4) is by one or more of electrofusion, viral fusion, and cell injection.
In any of the above methods, the method for reconstructing parthenogenetic eggs in step 4) includes any one of the following steps i to iii:
i. injecting the blastomeres separated in the step 3) into the perivitelline space of the oocytes in the MII stage, activating fertilization after fusion, and removing pronuclei from the oocytes in the pronucleus stage;
ii, after removing the spindle of the MII stage oocyte, injecting the blastomere separated in the step 3) into the perivitelline space of the oocyte, and activating fertilization after fusion;
iii, injecting the separated blastomeres in the step 3) into the perivitelline space of the oocyte in the MII stage, activating fertilization after fusion, and removing the second polar body together with the nucleus from the oocyte after the second polar body is discharged.
In the method, the activation method is chemical activation and electric activation, and preferably the calcium ionophore A23187 is activated in combination with the chemical activation mode of puromycin.
In any of the methods described above, said identifying in step 5) identifies one or more of success or failure of gene editing, editing efficacy, cellular status, methylation and transcriptome.
The method described in any of the above, further comprising, after step 5), the steps of:
6) application of post-editing cells: and (4) carrying out subsequent application on the residual blastomeres cultured in the step 4) which is successfully edited.
In any of the above methods, the subsequent application in step 6) is one or more of reconstituting a fertilized egg, reconstituting an embryo, establishing an embryonic stem cell line, studying gene function, or cell therapy.
In any of the methods described above, the cells and chromosomes used in the method are derived from one of rabbit, rat or mouse.
In the method of any of the above, the spindle comprises a nucleus.
The method provided by the invention is used for gene editing, and has the following obvious advantages:
firstly, targeted editing can be carried out on female genes;
secondly, the editing result of the gene can be accurately judged;
thirdly, the successfully edited cells can be applied subsequently.
In a word, the method can indirectly realize the function of editing the mammalian ovum, can ensure the final gene editing effect, and is beneficial to the transformation of the gene editing technology to clinical medicine.
Drawings
FIG. 1 is a schematic flow chart of a method for female gene editing by gene editing in conjunction with nuclear transfer;
wherein 1 is the obtaining of parthenogenetic female fertilized eggs; 2, editing parthenogenetic fertilized egg genes; 3, culturing parthenogenetic embryos; 4, separating parthenogenetic embryo blastomeres; 5, obtaining a reconstructed parthenogenetic female fertilized egg; 6, culturing the reconstructed parthenogenetic embryo; 7, identifying an editing result; 8 is the application of the edited cells.
Fig. 2 is a schematic flow diagram of a method for obtaining parthenogenetic fertilized eggs;
9, performing parthenogenetic activation on the MII-stage oocyte to obtain a parthenogenetic fertilized egg; and 10, injecting the MII stage oocyte by using a single sperm to activate fertilization, removing a male pronucleus at a pronucleus stage and reserving a female pronucleus to obtain the parthenogenetic zygote.
Fig. 3 is a schematic flow diagram of an acquisition method of a reconstructed parthenogenetic fertilized egg;
wherein 11, the reconstructed parthenogenetic fertilized egg is obtained by a method of removing a spindle of an MII-stage oocyte (including a nucleus of the MII-stage oocyte), injecting and fusing blastomeres, and activating fertilization; 12, injecting and fusing a blastomere into the oocyte in the MII stage, activating and fertilizing, and removing the second polar body and the nucleus of the oocyte after the second polar body is discharged to obtain a reconstructed parthenogenetic ovum; and 13, injecting and fusing blastomeres into the MII-stage oocytes, activating and fertilizing, and removing pronucleus from the oocytes in the pronucleus stage to obtain the parthenogenetic zygotes.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
The mice in the following examples are Kunming mice, SPF grade, body mass 30 +/-2 g, age of the mice 6-8 weeks, and purchased from Experimental animals center of Chinese military medical academy of sciences.
The rabbits in the following examples are New Zealand rabbits, SPF grade, body mass 2.26-3.41 kg, and purchased from Qingdaokang Biotech, Inc.
Calcium ionophore A23187 was from Sigma under catalog number C7522.
The "female gene" in the present invention refers to genetic material derived from a female.
The term "haploid female chromosome" as used herein means a female chromosome that contains only a single copy.
The term "parthenogenetic female fertilized egg" as used herein refers to a fertilized egg containing only a single copy of a female chromosome.
"parthenogenetic haploid cells" in the context of the present invention refer to cells that contain only a single copy of a female chromosome, such as the blastomeres in the examples described below.
Example 1
1. Obtaining parthenogenetic fertilized eggs: parthenogenetic activation of mice MII stage oocytes by means of calcium ionophore A23187 in combination with puromycin results in parthenogenetic zygotes containing only female chromosomes (i.e., haploid female chromosomes) (see 1 in FIG. 1 and 9 in FIG. 2).
2. And editing parthenogenetic fertilized egg genes: and (3) editing the target gene of the parthenogenetic female fertilized egg obtained in the step (1) by using a CRISPR/Cas9 gene editing technology in a prokaryotic stage (see 2 in the figure 1).
3. Isolation of parthenogenetic blastomeres after editing: and (3) continuing embryo culture on the edited parthenogenetic ovum obtained in the step (2) until the parthenogenetic ovum reaches 8 cell stages (see figure 1). One blastomere in the embryo was aspirated with an embryo biopsy needle with an inner diameter of 30 microns (see 4 in FIG. 1).
4. Reconstructing parthenogenetic embryos: removing a spindle body (including a nucleus of the MII stage oocyte) of the mouse MII stage oocyte to obtain an enucleated oocyte; injecting the blastomeres sucked in the step 3 into the perivitelline space of the enucleated oocyte, activating and fertilizing in a mode of combining calcium ionophore A23187 with puromycin after fusion, and obtaining the reconstructed parthenogenetic zygote (see 5 in the figure 1 and 11 in the figure 3). The reconstructed parthenogenetic zygotes were further subjected to embryo culture to 8-cell stage (see 6 in fig. 1).
5. And (4) identification of an editing result: and (3) sucking one blastomere in the embryo cultured in the step (4) by using an embryo biopsy needle with the inner diameter of 30 microns for identifying the editing effect (see 7 in the figure 1), wherein the genome of the residual blastomere obtained in the step (4) is completely consistent with the genome of the blastomere for identifying.
6. Application of post-editing cells: and (4) using the residual blastomeres obtained by the culture in the step (4) for reconstructing fertilized eggs, embryos or other scientific researches according to the editing effect (see 8 in the figure 1).
Example 2
1. Obtaining parthenogenetic fertilized eggs: and (2) injecting the mouse MII stage oocyte with mouse single sperm to activate fertilization, removing male pronucleus and reserving female pronucleus after the two pronucleus appear (pronucleus stage), and finally forming the parthenogenetic female zygote only containing female chromosomes (see 1 in figure 1 and 10 in figure 2).
2. And editing parthenogenetic fertilized egg genes: and (3) editing the target gene of the parthenogenetic female fertilized egg obtained in the step (1) by using a CRISPR/Cas9 gene editing technology in a prokaryotic stage (see 2 in the figure 1).
3. Isolation of parthenogenetic blastomeres after editing: and (3) continuing embryo culture on the edited parthenogenetic ovum obtained in the step (2) until the parthenogenetic ovum reaches 8 cell stages (see figure 1). One blastomere in the embryo was aspirated with an embryo biopsy needle with an inner diameter of 30 microns (see 4 in FIG. 1).
4. Reconstructing parthenogenetic embryos: removing a spindle body (including a nucleus of the MII stage oocyte) of the mouse MII stage oocyte to obtain an enucleated oocyte; injecting the blastomeres sucked in the step 3 into the perivitelline space of the enucleated oocyte, activating and fertilizing in a mode of combining calcium ionophore A23187 with puromycin after fusion, and obtaining the reconstructed parthenogenetic zygote (see 5 in the figure 1 and 11 in the figure 3). The reconstructed parthenogenetic zygotes were further subjected to embryo culture to 8-cell stage (see 6 in fig. 1).
5. And (4) identification of an editing result: one blastomere in the embryo cultured in step 4 was aspirated with an embryo biopsy needle with an inner diameter of 30 μm for identification of editing effect (see 7 in fig. 1).
6. Application of post-editing cells: and (4) using the residual blastomeres obtained by the culture in the step (4) for reconstructing fertilized eggs, embryos or other scientific researches according to the editing effect (see 8 in the figure 1).
Example 3
1. Obtaining parthenogenetic fertilized eggs: the MII-stage oocyte of the mouse is subjected to parthenogenetic activation by means of combining calcium ionophore A23187 and puromycin to form a parthenogenetic fertilized egg only containing female chromosomes (see 1 in figure 1 and 9 in figure 2).
2. And editing parthenogenetic fertilized egg genes: and (3) editing the target gene of the parthenogenetic female fertilized egg obtained in the step (1) by using a CRISPR/Cas9 gene editing technology in a prokaryotic stage (see 2 in the figure 1).
3. Isolation of parthenogenetic blastomeres after editing: and (3) continuing embryo culture on the edited parthenogenetic ovum obtained in the step (2) until the parthenogenetic ovum reaches 8 cell stages (see figure 1). One blastomere in the embryo was aspirated with an embryo biopsy needle with an inner diameter of 30 microns (see 4 in FIG. 1).
4. Reconstructing parthenogenetic embryos: injecting the blastomeres sucked in the step 3 into the perivitelline space of the oocyte in the MII stage of the mouse, activating fertilization by using a calcium ionophore A23187 in combination with puromycin after fusion, and removing the second polar body together with the nucleus from the oocyte after the second polar body is discharged to obtain the reconstructed parthenogenetic zygote (see 5 in the figure 1 and 12 in the figure 3). The reconstructed parthenogenetic zygotes were further subjected to embryo culture to 8-cell stage (see 6 in fig. 1).
5. And (4) identification of an editing result: one blastomere in the embryo cultured in step 4 was aspirated with an embryo biopsy needle with an inner diameter of 30 μm for identification of editing effect (see 7 in fig. 1).
6. Application of post-editing cells: and (4) using the residual blastomeres obtained by the culture in the step (4) for reconstructing fertilized eggs, embryos or other scientific researches according to the editing effect (see 8 in the figure 1).
Example 4
1. Obtaining parthenogenetic fertilized eggs: the MII-stage oocyte of the mouse is subjected to parthenogenetic activation by means of combining calcium ionophore A23187 and puromycin to form a parthenogenetic fertilized egg only containing female chromosomes (see 1 in figure 1 and 9 in figure 2).
2. And editing parthenogenetic fertilized egg genes: and (3) editing the target gene of the parthenogenetic zygote obtained in the step (1) by using a CRISPR/Cas9 gene editing technology in a prokaryotic stage (see 2 in the figure 1).
3. Isolation of parthenogenetic blastomeres after editing: and (3) continuing embryo culture on the edited parthenogenetic ovum obtained in the step (2) until the parthenogenetic ovum reaches 8 cell stages (see figure 1). One blastomere in the embryo was aspirated with an embryo biopsy needle with an inner diameter of 30 microns (see 4 in FIG. 1).
4. Reconstructing parthenogenetic embryos: injecting the blastomeres sucked in the step 3 into the perivitelline space of the oocyte in the MII stage of the mouse, activating and fertilizing in a mode of combining calcium ionophore A23187 with puromycin after fusion to form double pronucleus (pronucleus stage), and removing pronucleus from the oocyte to obtain the reconstructed parthenogenetic zygote (see 5 in the figure 1 and 13 in the figure 3). The activated parthenogenetic zygotes are further subjected to embryo culture to 8-cell stage (see 6 in fig. 1).
5. And (4) identification of an editing result: one blastomere in the embryo cultured in step 4 was aspirated with an embryo biopsy needle with an inner diameter of 30 μm for identification of editing effect (see 7 in fig. 1).
6. Application of post-editing cells: and (4) using the residual blastomeres obtained by the culture in the step (4) for reconstructing fertilized eggs, embryos or other scientific researches according to the editing effect (see 8 in the figure 1).
Example 5
1. Obtaining parthenogenetic fertilized eggs: parthenogenetic activation of rabbit MII stage oocytes by means of calcium ionophore a23187 in combination with puromycin results in parthenogenetic zygotes containing only female chromosomes (see 1 in fig. 1 and 9 in fig. 2).
2. And editing parthenogenetic fertilized egg genes: and (3) editing the target gene of the parthenogenetic zygote obtained in the step (1) by using a CRISPR/Cas9 gene editing technology in a prokaryotic stage (see 2 in the figure 1).
3. Isolation of parthenogenetic blastomeres after editing: and (3) continuing embryo culture on the edited parthenogenetic ovum obtained in the step (2) until the parthenogenetic ovum reaches 8 cell stages (see figure 1). One blastomere in the embryo was aspirated with an embryo biopsy needle with an inner diameter of 30 microns (see 4 in FIG. 1).
4. Reconstructing parthenogenetic embryos: removing spindle bodies of the rabbit MII stage oocyte (including the nucleus of the MII stage oocyte) to obtain an enucleated oocyte; injecting the blastomeres sucked in the step 3 into the perivitelline space of the enucleated oocyte, activating and fertilizing in a mode of combining calcium ionophore A23187 with puromycin after fusion, and obtaining the reconstructed parthenogenetic zygote (see 5 in the figure 1 and 11 in the figure 3). The reconstructed parthenogenetic zygotes were further subjected to embryo culture to 8-cell stage (see 6 in fig. 1).
5. And (4) identification of an editing result: one blastomere in the embryo cultured in step 4 was aspirated with an embryo biopsy needle with an inner diameter of 30 μm for identification of editing effect (see 7 in fig. 1).
6. Application of post-editing cells: and (4) using the residual blastomeres obtained by the culture in the step (4) for reconstructing fertilized eggs, embryos or other scientific researches according to the editing effect (see 8 in the figure 1).
Claims (10)
1. A method of editing for a female gene comprising the steps of:
1) obtaining parthenogenetic fertilized eggs: preparing parthenogenetic fertilized eggs only containing single parthenogenetic female chromosomes;
2) and editing parthenogenetic fertilized egg genes: editing the target gene of the female solitary zygote by using a gene editing technology in a prokaryotic stage;
3) isolation of parthenogenetic blastomeres after editing: performing embryo culture on the parthenogenetic female fertilized eggs edited in the step 2) to a stage with blastomeres, and separating one blastomere;
4) reconstructing parthenogenetic embryos: reconstructing the blastomere separated in the step 3) and the MII-stage oocyte to obtain a reconstructed parthenogenetic female zygote, and continuously carrying out embryo culture on the reconstructed parthenogenetic female zygote to a stage with the blastomere;
5) and (4) identification of an editing result: taking out one blastomere obtained by culturing in the step 4) for identifying an editing result, wherein the genome of the remaining blastomere obtained in the step 4) is completely consistent with the genome of the blastomere used for identification;
all cells and chromosomes used in the method are derived from non-human mammals.
2. The method of claim 1, wherein: the method for preparing the parthenogenetic fertilized egg in the step 1) comprises the following steps a or b:
a. injecting a single sperm into an oocyte in an MII stage to activate fertilization, removing a male pronucleus in a pronucleus stage and reserving a female pronucleus;
b. carrying out parthenogenetic activation on the MII-stage oocyte.
3. The method of claim 1, wherein: the gene editing technology in the step 2) is zinc finger nuclease, transcription activator-like effector nuclease or CRISPR/Cas9 technology;
the editing mode is one or more of base modification, base insertion, base knockout and base replacement of the target gene.
4. A method according to any one of claims 1-3, characterized in that: the stage with blastomeres in the steps 3) and 4) is 2 cell stage, 4 cell stage, 8 cell stage, 16 cell stage, 32 cell stage, fusion embryo stage, mulberry embryo stage or blastocyst stage.
5. A method according to any one of claims 1-3, characterized in that: the reconstruction mode in the step 4) is one or more of electrofusion, virus fusion and cell injection.
6. A method according to any one of claims 1-3, characterized in that: the method for reconstructing parthenogenetic eggs in the step 4) comprises the steps of any one of the following i-iii:
i. injecting the blastomeres separated in the step 3) into the perivitelline space of the oocytes in the MII stage, activating fertilization after fusion, and removing pronuclei from the oocytes in the pronucleus stage;
ii, after removing the spindle of the MII stage oocyte, injecting the blastomere separated in the step 3) into the perivitelline space of the oocyte, and activating fertilization after fusion;
iii, injecting the separated blastomeres in the step 3) into the perivitelline space of the oocyte in the MII stage, activating fertilization after fusion, and removing the second polar body together with the nucleus from the oocyte after the second polar body is discharged.
7. A method according to any one of claims 1-3, characterized in that: said identifying in step 5) identifies one or more of success or failure of gene editing, editing effect, cellular status, methylation and transcriptome.
8. A method according to any one of claims 1-3, characterized in that: the following steps are also provided after step 5):
6) application of post-editing cells: and (4) carrying out subsequent application on the residual blastomeres cultured in the step 4) which is successfully edited.
9. The method of claim 8, wherein: the subsequent application in step 6) is one or more of reconstruction of fertilized eggs, reconstruction of embryos, establishment of embryonic stem cell lines, research of gene functions or cell therapy.
10. A method according to any one of claims 1-3, characterized in that: the cells and chromosomes used in the method are derived from one of rabbit, rat or mouse.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001030978A1 (en) * | 1999-10-28 | 2001-05-03 | University Of Massachusetts | Gynogenetic or androgenetic production of pluripotent cells and cell lines, and use thereof to produce differentiated cells and tissues |
| CN103361304A (en) * | 2012-03-27 | 2013-10-23 | 中国科学院上海生命科学研究院 | Androgenesis haploid stem cell line as well as preparation method and application thereof |
| CN105755047A (en) * | 2016-03-30 | 2016-07-13 | 魏红江 | Method for obtaining cloned pigs with modified genes through continuous cloning |
| CN106676075A (en) * | 2015-11-11 | 2017-05-17 | 中国科学院上海生命科学研究院 | PG-haESCs (parthenogenetic-haploid embryonic stem cells) as well as preparation and application thereof |
| CN107699571A (en) * | 2017-09-06 | 2018-02-16 | 湖北省农业科学院畜牧兽医研究所 | A kind of porcine somatostatin gene editing site and its application |
-
2018
- 2018-03-21 CN CN201810237058.6A patent/CN108504691B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001030978A1 (en) * | 1999-10-28 | 2001-05-03 | University Of Massachusetts | Gynogenetic or androgenetic production of pluripotent cells and cell lines, and use thereof to produce differentiated cells and tissues |
| CN103361304A (en) * | 2012-03-27 | 2013-10-23 | 中国科学院上海生命科学研究院 | Androgenesis haploid stem cell line as well as preparation method and application thereof |
| CN106676075A (en) * | 2015-11-11 | 2017-05-17 | 中国科学院上海生命科学研究院 | PG-haESCs (parthenogenetic-haploid embryonic stem cells) as well as preparation and application thereof |
| WO2017080452A1 (en) * | 2015-11-11 | 2017-05-18 | 中国科学院上海生命科学研究院 | Parthenogenetic haploid embryonic stem cells, preparation method therefor, and application thereof |
| CN105755047A (en) * | 2016-03-30 | 2016-07-13 | 魏红江 | Method for obtaining cloned pigs with modified genes through continuous cloning |
| CN107699571A (en) * | 2017-09-06 | 2018-02-16 | 湖北省农业科学院畜牧兽医研究所 | A kind of porcine somatostatin gene editing site and its application |
Non-Patent Citations (4)
| Title |
|---|
| CRISPR /Cas9 系统介导的一步法胚胎注射获得猪 GGTA1 敲除胚胎;尹智等;《畜牧与兽医》;20161231;第48卷(第4期);第16页第1.4-1.5节、第17页2.2节、第18页右栏第1段 * |
| Genetic Modification and Screening in Rat Using Haploid Embryonic Stem Cells;Wei Li et al.;《Cell Stem Cell》;20140306;第14卷;第404-414页 * |
| 单倍体干细胞研究进展;李伟等;《生命科学》;20160831;第28卷(第8期);第862-866页 * |
| 卵子介导细胞重编程的基础与应用研究;李庆等;《生命科学》;20171031;第29卷(第10期);第986页右栏第2-4段及第987页左栏第1段、第987页图2 * |
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| CN108504691A (en) | 2018-09-07 |
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