CN116640803A - A method for inducing polyploid pigs by colchicine - Google Patents
A method for inducing polyploid pigs by colchicine Download PDFInfo
- Publication number
- CN116640803A CN116640803A CN202310428544.7A CN202310428544A CN116640803A CN 116640803 A CN116640803 A CN 116640803A CN 202310428544 A CN202310428544 A CN 202310428544A CN 116640803 A CN116640803 A CN 116640803A
- Authority
- CN
- China
- Prior art keywords
- colchicine
- polyploid
- cell
- pigs
- pig
- Prior art date
- Legal status (The legal status 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 status listed.)
- Granted
Links
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 title claims abstract description 158
- 229960001338 colchicine Drugs 0.000 title claims abstract description 79
- 208000020584 Polyploidy Diseases 0.000 title claims abstract description 59
- 241000282887 Suidae Species 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 26
- 230000001939 inductive effect Effects 0.000 title claims abstract description 15
- 210000004027 cell Anatomy 0.000 claims abstract description 67
- 230000006698 induction Effects 0.000 claims abstract description 15
- 210000002950 fibroblast Anatomy 0.000 claims abstract description 14
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 14
- 208000035199 Tetraploidy Diseases 0.000 claims abstract description 13
- 238000012546 transfer Methods 0.000 claims abstract description 12
- 238000011282 treatment Methods 0.000 claims description 24
- 210000002257 embryonic structure Anatomy 0.000 claims description 18
- 238000004458 analytical method Methods 0.000 claims description 14
- 238000012216 screening Methods 0.000 claims description 13
- 210000002459 blastocyst Anatomy 0.000 claims description 11
- 210000001840 diploid cell Anatomy 0.000 claims description 11
- 230000001605 fetal effect Effects 0.000 claims description 11
- 210000001850 polyploid cell Anatomy 0.000 claims description 9
- 238000003776 cleavage reaction Methods 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 8
- 230000007017 scission Effects 0.000 claims description 8
- 210000001082 somatic cell Anatomy 0.000 claims description 8
- 230000003698 anagen phase Effects 0.000 claims description 7
- 210000000349 chromosome Anatomy 0.000 claims description 7
- 238000011161 development Methods 0.000 claims description 6
- 230000018109 developmental process Effects 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 5
- 239000012895 dilution Substances 0.000 claims description 5
- 238000010367 cloning Methods 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- 238000000684 flow cytometry Methods 0.000 claims description 4
- 238000005457 optimization Methods 0.000 claims description 4
- 230000004927 fusion Effects 0.000 claims description 3
- 238000011419 induction treatment Methods 0.000 claims 1
- 239000013049 sediment Substances 0.000 claims 1
- 238000002054 transplantation Methods 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 13
- 238000009395 breeding Methods 0.000 abstract description 7
- 230000001488 breeding effect Effects 0.000 abstract description 7
- 238000010374 somatic cell nuclear transfer Methods 0.000 abstract description 6
- 230000001737 promoting effect Effects 0.000 abstract 1
- 241001465754 Metazoa Species 0.000 description 5
- 238000010370 cell cloning Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 208000027223 tetraploidy syndrome Diseases 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 210000000287 oocyte Anatomy 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 241000938605 Crocodylia Species 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000013373 clone screening Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- XGVJWXAYKUHDOO-DANNLKNASA-N lycorine Chemical compound C1CN2CC3=CC=4OCOC=4C=C3[C@H]3[C@H]2C1=C[C@H](O)[C@H]3O XGVJWXAYKUHDOO-DANNLKNASA-N 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000021121 meiosis Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000010449 nuclear transplantation Methods 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 230000008672 reprogramming Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- 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
- C12N15/8778—Swine embryos
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0273—Cloned vertebrates
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0656—Adult fibroblasts
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/108—Swine
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Veterinary Medicine (AREA)
- Biodiversity & Conservation Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Rheumatology (AREA)
- Cell Biology (AREA)
- Animal Husbandry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
Description
技术领域technical field
本发明申请涉及生物工程技术领域，具体涉及一种通过秋水仙素诱导多倍体猪的方法。The application of the present invention relates to the technical field of bioengineering, in particular to a method for inducing polyploid pigs by colchicine.
背景技术Background technique
生猪种业是国家基础性、战略性的核心产业，育种周期长、投入大、效率低是制约其产业发展的重要瓶颈。多倍体在育种高效性上表现出了巨大的应用潜力，并已在甲壳类、鱼类育种领域取得了重要进展。然而，除两栖类、爬行类及部分鱼类，多倍化现象在脊椎动物中极为罕见，多倍体猪个体及应用至今尚未见报道。The pig breeding industry is a national basic and strategic core industry. Long breeding cycle, large investment and low efficiency are the important bottlenecks restricting the development of its industry. Polyploidy has shown great application potential in breeding efficiency, and has made important progress in the fields of crustacean and fish breeding. However, except for amphibians, reptiles and some fishes, polyploidy is extremely rare in vertebrates, and the individual and application of polyploid pigs have not been reported so far.
目前动物多倍体产生的途径主要有核移植与细胞融合、体细胞染色体加倍及卵母细胞的减数分裂异常三种。秋水仙素（Colchicine）已被用在诱导哺乳动物细胞多倍化的科学研究中，最早相关领域科学家已在兔子、小鼠的多倍化研究应用中取得了进展，随后在小鼠的多倍化诱导研究中也有应用研究的报道。近年来秋水仙素也成功应用到了小鼠成纤维细胞、人成纤维细胞等体细胞多倍化诱导及基础理论研究的应用中，但至今尚未报道通过秋水仙素成功诱导多倍体猪细胞、胚胎或新生个体。At present, animal polyploidy is mainly produced in three ways: nuclear transfer and cell fusion, chromosome doubling in somatic cells, and abnormal meiosis of oocytes. Colchicine has been used in the scientific research of inducing mammalian cell polyploidy. The earliest scientists in related fields have made progress in the application of polyploidy research in rabbits and mice, and subsequently in polyploidy of mice. There are also reports of applied research in chemical induction research. In recent years, colchicine has also been successfully applied to mouse fibroblasts, human fibroblasts and other somatic cell polyploidy induction and basic theoretical research, but it has not been reported that colchicine has successfully induced polyploid pig cells, Embryo or newborn individual.
体细胞核移植重编程是细胞工程常用的技术手段，即利用显微注射的方法把终末分化的体细胞核移入到去核的卵母细胞中，使其发生再程序化并重编程为新的全能性发育胚胎的过程，并最终发育为完整的动物个体，该技术成功颠覆了 传统发育生物学认为的动物胚胎发育只有受精卵才具备全能性的理论，是现今唯一能利用终末期分化体细胞产生全能性胚胎的技术，目前通过体细胞核移植技术先后已获得了包括猪等在内的20多种哺乳动物个体，在现代畜牧业的发展中具有重要的前景及应用潜力。若通过秋水仙素处理诱导获得猪多倍体细胞系，再结合体细胞核移植技术将其进行重编程获得新生的多倍体猪，将是现代畜牧业育种研究领域内的一项重大突破，将为开展多倍体猪的基础理论研究及在多倍体育种领域的应用研究奠定重要的基础。Somatic cell nuclear transfer reprogramming is a commonly used technical method in cell engineering, that is, the terminally differentiated somatic cell nucleus is transferred into the enucleated oocyte by microinjection, so that it can be reprogrammed and reprogrammed into a new totipotent The process of developing embryos and eventually developing into complete animal individuals. This technology has successfully subverted the theory of traditional developmental biology that only fertilized eggs can be totipotent in animal embryo development. It is the only one that can use terminally differentiated somatic cells to produce totipotency. At present, more than 20 kinds of mammalian individuals including pigs have been obtained through somatic cell nuclear transfer technology, which has important prospects and application potential in the development of modern animal husbandry. If the porcine polyploid cell line is induced by colchicine treatment, and then combined with somatic cell nuclear transfer technology to reprogram it to obtain a newborn polyploid pig, it will be a major breakthrough in the field of modern animal husbandry breeding research. It lays an important foundation for the basic theoretical research of polyploid pigs and the application research in the field of polyploid breeding.
发明内容Contents of the invention
针对以上技术问题，本发明提供了一种通过秋水仙素诱导多倍体猪的方法，本方法通过不同浓度的秋水仙素处理诱导猪细胞系后，通过不同倍性细胞的比例等指标筛选秋水仙素处理诱导的最佳浓度，并利用最佳浓度的秋水仙素处理猪细胞系后，检测其细胞的加倍效率，经单细胞克隆筛选纯化获得多倍体猪细胞系，并以此为体细胞核移植的核供体构建克隆胚，经胚胎移植生产多倍体猪，为加快猪多倍体发育生物学的基础理论研究提供研究模型及材料，对推动猪多倍体的实践应用的探索研究具有重要的现实意义。Aiming at the above technical problems, the present invention provides a method for inducing polyploid pigs by colchicine. After the method induces pig cell lines by different concentrations of colchicine, the polyploid pigs are screened by indicators such as the ratio of different ploidy cells. The optimal concentration induced by narcissine treatment, and after the porcine cell line was treated with the optimal concentration of colchicine, the doubling efficiency of the cells was detected, and the polyploid porcine cell line was obtained by single-cell clone screening and purification, and used as a body Create cloned embryos from nuclear donors for nuclear transplantation, and produce polyploid pigs through embryo transfer, providing research models and materials for accelerating the basic theoretical research on the developmental biology of porcine polyploidy, and exploring and researching the practical application of porcine polyploidy has important practical significance.
为达到上述目的，本发明提供如下技术方案：To achieve the above object, the present invention provides the following technical solutions:
一种通过秋水仙素诱导多倍体猪的方法，具体步骤为：A method for inducing polyploid pigs by colchicine, the specific steps are:
1）秋水仙素诱导最佳浓度的筛选1) Screening for the optimal concentration of colchicine induction
将猪胎儿成纤维细胞传代至对数生长期，持续一段时间分别通过不同浓度的秋水仙素处理后，检测不同浓度的细胞倍性情况、染色体数目，再根据四倍性（4n）所占比例的大小筛选秋水仙素处理诱导的最佳浓度；The pig fetal fibroblasts were passaged to the logarithmic growth phase, and after being treated with different concentrations of colchicine for a period of time, the cell ploidy and chromosome number of different concentrations were detected, and then according to the proportion of tetraploidy (4n) The size screened the optimal concentration induced by colchicine treatment;
2）秋水仙素诱导体系的优化2) Optimization of colchicine induction system
将猪胎儿成纤维细胞传代至对数生长期，经上步所述最佳浓度的秋水仙素处理后，将被处理的靶细胞采用抖落法收集细胞沉淀后，再继续利用秋水仙素诱导处理，进一步降低处理后二倍性细胞残留的比例；The pig fetal fibroblasts were passaged to the logarithmic growth phase, and after being treated with the optimal concentration of colchicine described in the previous step, the treated target cells were collected by shaking off the cell pellet, and then continued to be induced by colchicine. treatment to further reduce the proportion of residual diploid cells after treatment;
3）多倍体细胞的筛选与纯化3) Screening and purification of polyploid cells
将经秋水仙素处理后的细胞群进行单细胞克隆极度稀释培养，单细胞克隆经细胞倍性检测后，再进行细胞核型分析，获得多倍体猪细胞系；The colchicine-treated cell population was subjected to extremely diluted single-cell clone culture, and the single-cell clone was tested for cell ploidy, followed by karyotype analysis to obtain a polyploid pig cell line;
4）多倍体细胞系的克隆及胚胎移植4) Cloning of polyploid cell lines and embryo transfer
以筛选纯化获得的四倍体细胞系为核供体进行体细胞克隆，检测克隆胚发育能力后进行胚胎移植，以进一步生产多倍体猪。Somatic cell cloning was carried out using the tetraploid cell line obtained by screening and purification as nuclear donors, and embryo transfer was carried out after testing the developmental ability of cloned embryos to further produce polyploid pigs.
进一步的，步骤1）和2）中所述猪胎儿成纤维细胞为二倍体。Further, the pig fetal fibroblasts in steps 1) and 2) are diploid.
进一步的，步骤1）中所述一段时间为4天，所述不同浓度的秋水仙素为0.05~0.2µg/mL的秋水仙素。Further, the period of time in step 1) is 4 days, and the different concentrations of colchicine are 0.05-0.2 μg/mL colchicine.
进一步的，步骤1）和2）中所述最佳浓度为0.2µg/mL。Further, the optimal concentration described in steps 1) and 2) is 0.2 µg/mL.
进一步的，步骤1）中，第一次秋水仙素处理的时间为17小时，第二次秋水仙素处理的时间为3天。Further, in step 1), the time for the first colchicine treatment is 17 hours, and the time for the second colchicine treatment is 3 days.
进一步的，步骤3）中所述单细胞克隆培养的方法包括极度稀释和流式细胞仪分选。Further, the method for culturing single cell clones in step 3) includes extreme dilution and sorting by flow cytometry.
进一步的，步骤4）中所述检测克隆胚发育能力的指标包括融合率、卵裂率、囊胚率及囊胚细胞数。Further, the indicators for detecting the developmental ability of cloned embryos described in step 4) include fusion rate, cleavage rate, blastocyst rate and blastocyst cell number.
应当理解的是，以上的一般描述和后文的细节描述仅是示例性和解释性的，并不能限制本发明申请。It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application.
本发明的有益技术效果：Beneficial technical effect of the present invention:
本发明通过不同浓度的秋水仙素诱导处理猪细胞系，通过细胞倍性分析、核型分析筛选了最佳的秋水仙素处理浓度，并进一步以最佳浓度的秋水仙素处理诱导猪细胞后，优化了秋水仙素处理的方法，检测了诱导后猪细胞倍性变化情况，经单细胞克隆培养成功筛选获得了四倍体猪细胞系，并以此为体细胞核移植的核供体，开展了多倍体猪胚胎的构建，并进行了胚胎移植生产多倍体猪，对开展多倍体猪发育基础理论及应用研究具有重要的应用价值。The present invention induces and treats pig cell lines with different concentrations of colchicine, screens the optimal colchicine treatment concentration through cell ploidy analysis and karyotype analysis, and further treats the induced pig cells with the optimal concentration of colchicine , optimized the method of colchicine treatment, detected the ploidy changes of pig cells after induction, successfully screened and obtained tetraploid pig cell lines through single-cell clone culture, and used them as nuclear donors for somatic cell nuclear transfer. The construction of polyploid pig embryos was carried out, and embryo transfer was carried out to produce polyploid pigs, which has important application value for the basic theory and application research of polyploid pig development.
附图说明Description of drawings
图1是本发明的一个实施方式流程图。Fig. 1 is a flowchart of an embodiment of the present invention.
图2是0.05µg/mL、0.1µg/mL、0.2µg/mL秋水仙素处理二倍体细胞后倍性检测结果。Figure 2 shows the ploidy detection results of diploid cells treated with 0.05µg/mL, 0.1µg/mL, and 0.2µg/mL colchicine.
图3是0.05µg/mL、0.1µg/mL、0.2µg/mL秋水仙素处理二倍体细胞后不同倍性细胞所占比例。Figure 3 shows the proportion of different ploidy cells after diploid cells were treated with 0.05µg/mL, 0.1µg/mL, and 0.2µg/mL colchicine.
图4是0.05µg/mL秋水仙素处理二倍体细胞后核型分析结果；图中，A）细胞核型分析统计图；B）不同染色体数目出现频率统计结果。Figure 4 is the karyotype analysis results of diploid cells treated with 0.05µg/mL colchicine; in the figure, A) Statistical chart of cell karyotype analysis; B) Statistical results of frequency of different chromosome numbers.
图5是0.1µg/mL秋水仙素处理二倍体细胞后核型分析结果；图中，A）细胞核型分析统计图；B）不同染色体数目出现频率统计结果。Figure 5 shows the results of karyotype analysis of diploid cells treated with 0.1 µg/mL colchicine; in the figure, A) Statistical chart of cell karyotype analysis; B) Statistical results of frequency of different chromosome numbers.
图6是0.2µg/mL秋水仙素处理二倍体细胞后核型分析结果；图中，A）细胞核型分析统计图；B）不同染色体数目出现频率统计结果。Figure 6 is the result of karyotype analysis of diploid cells treated with 0.2 µg/mL colchicine; in the figure, A) Statistical chart of cell karyotype analysis; B) Statistical results of frequency of different chromosome numbers.
图7是猪四倍体核供体克隆胚发育能力检测结果；图中，A）克隆胚卵裂图；B）克隆胚囊胚图；C）克隆胚卵裂率；D）克隆胚囊胚率；E）克隆胚囊胚细胞数。Figure 7 shows the test results of the developmental ability of cloned embryos from pig tetraploid nuclear donors; in the figure, A) cleavage map of cloned embryos; B) cleavage map of cloned embryos; C) cleavage rate of cloned embryos; D) blastocysts of cloned embryos E) The number of blastocyst cells in cloned embryos.
具体实施方式Detailed ways
下面将参照附图更详细地描述本发明申请的可选实施方式。虽然附图中显示了本发明申请的可选实施方式，然而应该理解，可以以各种形式实现本发明申请而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了使本发明申请更加透彻和完整，并且能够将本发明申请的范围完整地传达给本领域的技术人员。Alternative embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although alternative embodiments of the present application are shown in the drawings, it should be understood that the present application can be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.
在本发明申请使用的术语是仅仅出于描述特定实施例的目的，而非旨在限制本发明申请。在本发明申请和所附权利要求书中所使用的单数形式的“一种”、“所述”和“该”也旨在包括多数形式，除非上下文清楚地表示其他含义。还应当理解，本文中使用的术语“和/或”是指并包含一个或多个相关联的列出项目的任何或所有可能组合。The terms used in the present application are for the purpose of describing particular embodiments only, and are not intended to limit the present application. As used in this application and the appended claims, the singular forms "a", "the", and "the" are also intended to include the plural forms unless the context clearly dictates otherwise. It should also be understood that the term "and/or" as used herein refers to and includes any and all possible combinations of one or more of the associated listed items.
以下结合附图对本发明申请通过秋水仙素诱导多倍体猪的方法进行详细说明，具体如下：Below in conjunction with accompanying drawing, the method for the application of the present invention to induce polyploid pig by colchicine is described in detail, specifically as follows:
如图1所示，本发明申请中通过秋水仙素诱导多倍体猪的方法，具体步骤为：As shown in Figure 1, the method for inducing polyploid pigs by colchicine in the application of the present invention, the specific steps are:
1）秋水仙素诱导最佳浓度的筛选1) Screening for the optimal concentration of colchicine induction
将猪胎儿成纤维细胞传代至对数生长期，持续一段时间分别通过不同浓度的秋水仙素处理后，检测不同浓度的细胞倍性情况、染色体数目，再根据四倍性（4n）所占比例的大小筛选秋水仙素处理诱导的最佳浓度；The pig fetal fibroblasts were passaged to the logarithmic growth phase, and after being treated with different concentrations of colchicine for a period of time, the cell ploidy and chromosome number of different concentrations were detected, and then according to the proportion of tetraploidy (4n) The size screened the optimal concentration induced by colchicine treatment;
2）秋水仙素诱导体系的优化2) Optimization of colchicine induction system
将猪胎儿成纤维细胞传代至对数生长期，经上步所述最佳浓度的秋水仙素处理后，将被处理的靶细胞采用抖落法收集细胞沉淀后，再继续利用秋水仙素诱导处理，进一步降低处理后二倍性细胞残留的比例；The pig fetal fibroblasts were passaged to the logarithmic growth phase, and after being treated with the optimal concentration of colchicine described in the previous step, the treated target cells were collected by shaking off the cell pellet, and then continued to be induced by colchicine. treatment to further reduce the proportion of residual diploid cells after treatment;
3）多倍体细胞的筛选与纯化3) Screening and purification of polyploid cells
将经秋水仙素处理后的细胞群进行单细胞克隆极度稀释培养，单细胞克隆经细胞倍性检测后，再进行细胞核型分析，获得多倍体猪细胞系；The colchicine-treated cell population was subjected to extremely diluted single-cell clone culture, and the single-cell clone was tested for cell ploidy, followed by karyotype analysis to obtain a polyploid pig cell line;
4）多倍体细胞系的克隆及胚胎移植4) Cloning of polyploid cell lines and embryo transfer
以筛选纯化获得的四倍体细胞系为核供体进行体细胞克隆，检测克隆胚发育能力后进行胚胎移植，以进一步生产多倍体猪。Somatic cell cloning was carried out using the tetraploid cell line obtained by screening and purification as nuclear donors, and embryo transfer was carried out after testing the developmental ability of cloned embryos to further produce polyploid pigs.
在本发明申请的一种实施方式中，步骤1）和2）中所述猪胎儿成纤维细胞为二倍体。In one embodiment of the present application, the pig fetal fibroblasts in steps 1) and 2) are diploid.
在本发明申请的一种实施方式中，步骤1）中所述一段时间为4天，所述不同浓度的秋水仙素为0.05~0.2µg/mL的秋水仙素。In one embodiment of the present application, the period of time in step 1) is 4 days, and the different concentrations of colchicine are 0.05-0.2 μg/mL colchicine.
在本发明申请的一种实施方式中，步骤1）和2）中所述最佳浓度为0.2µg/mL。In one embodiment of the present application, the optimal concentration described in steps 1) and 2) is 0.2 µg/mL.
在本发明申请的一种实施方式中，步骤1）中，第一次秋水仙素处理的时间为17小时，第二次秋水仙素处理的时间为3天。In one embodiment of the application of the present invention, in step 1), the time of the first colchicine treatment is 17 hours, and the time of the second colchicine treatment is 3 days.
在本发明申请的一种实施方式中，步骤3）中所述单细胞克隆培养的方法包括极度稀释和流式细胞仪分选。In one embodiment of the present application, the single cell clone culture method in step 3) includes extreme dilution and flow cytometry sorting.
在本发明申请的一种实施方式中，步骤4）中所述检测克隆胚发育能力的指标包括融合率、卵裂率、囊胚率及囊胚细胞数。In one embodiment of the application of the present invention, the indicators for detecting the developmental ability of cloned embryos in step 4) include fusion rate, cleavage rate, blastocyst rate and blastocyst cell number.
为更清楚起见，下面通过以下实施例进行详细说明。For more clarity, detailed description is given below through the following examples.
实施例1Example 1
一种通过秋水仙素诱导多倍体猪的方法，具体步骤为：A method for inducing polyploid pigs by colchicine, the specific steps are:
1）秋水仙素诱导最佳浓度的筛选1) Screening for the optimal concentration of colchicine induction
将二倍体猪胎儿成纤维细胞传代至对数生长期，分别通过0.05µg/mL、0.1µg/mL、0.2µg/mL的秋水仙素处理后，连续处理4天后，收集细胞通过流式细胞仪分别检测不同浓度的细胞倍性情况（图2），统计不同细胞倍性所占比例（图3），根据四倍性（4n）所占比例的大小筛选秋水仙素处理诱导的最佳浓度，即为0.2µg/mL；The diploid pig fetal fibroblasts were passaged to the logarithmic growth phase, and were treated with 0.05µg/mL, 0.1µg/mL, and 0.2µg/mL colchicine respectively. After continuous treatment for 4 days, the cells were collected and passed through flow cytometry. The instrument detects the cell ploidy of different concentrations (Figure 2), counts the proportion of different cell ploidy (Figure 3), and screens the optimal concentration induced by colchicine treatment according to the proportion of tetraploidy (4n) , which is 0.2µg/mL;
2）秋水仙素诱导体系的优化2) Optimization of colchicine induction system
将二倍体猪胎儿成纤维细胞传代至对数生长期，经0.2µg/mL秋水仙素处理后，经倍性检测后，处理的细胞群中仍残留二倍性细胞（图2），为进一步降低处理后二倍性细胞残留的比例，通过秋水仙素处理17小时后，将被处理的靶细胞采用抖落法收集细胞沉淀继续利用秋水仙素诱导处理3天，残留二倍体的细胞降低至了1.67%。The diploid pig fetal fibroblasts were passaged to the logarithmic growth phase, and after being treated with 0.2 μg/mL colchicine, diploid cells remained in the treated cell population after the ploidy detection (Figure 2), which was Further reduce the proportion of residual diploid cells after treatment. After 17 hours of colchicine treatment, the treated target cells were collected by shaking off the cell pellet and continued to be treated with colchicine induction for 3 days, and diploid cells remained reduced to 1.67%.
3）多倍体细胞的筛选与纯化3) Screening and purification of polyploid cells
将经秋水仙素处理后的细胞群进行单细胞克隆极度稀释培养，共获得了82个单细胞克隆，经细胞倍性检测后，71个单细胞克隆为四倍性（86.6%），再经细胞核型分析确认后，获得多倍体猪细胞系；The colchicine-treated cell population was subjected to extreme dilution and culture of single-cell clones, and a total of 82 single-cell clones were obtained. After cell ploidy detection, 71 single-cell clones were tetraploid (86.6%). After the confirmation of cell karyotype analysis, polyploid pig cell lines were obtained;
4）多倍体细胞系的克隆及胚胎移植4) Cloning of polyploid cell lines and embryo transfer
以筛选纯化获得的四倍体细胞系为核供体进行体细胞克隆，其克隆胚能正常卵裂并发育至囊胚（图7A&B），其卵裂率、囊胚率、囊胚细胞数与二倍体克隆胚均无显著差异（P>0.05），可进一步进行胚胎移植生产多倍体猪。The tetraploid cell line obtained by screening and purification was used as the nuclear donor for somatic cell cloning, and the cloned embryos could normally cleavage and develop into blastocysts (Figure 7A&B). The cleavage rate, blastocyst rate, blastocyst cell number and There was no significant difference in diploid cloned embryos (P>0.05), and further embryo transfer could be performed to produce polyploid pigs.
利用实施例中通过秋水仙素诱导多倍体猪的方法，本发明通过不同浓度的秋水仙素诱导处理猪细胞系，通过细胞倍性分析、核型分析筛选了最佳的秋水仙素处理浓度，并进一步以最佳浓度的秋水仙素处理诱导猪细胞后，优化了秋水仙素处理的方法，检测了诱导后猪细胞倍性变化情况，经单细胞克隆培养成功筛选获得了四倍体猪细胞系，并以此为体细胞核移植的核供体，开展了多倍体猪胚胎的构建，并进行了胚胎移植生产多倍体猪，对开展多倍体猪发育基础理论及应用研究具有重要的应用价值。Using the method for inducing polyploid pigs by colchicine in the examples, the present invention induces and treats pig cell lines with different concentrations of colchicine, and screens the best colchicine treatment concentration through cell ploidy analysis and karyotype analysis , and further treated the induced pig cells with the optimal concentration of colchicine, optimized the method of colchicine treatment, detected the ploidy changes of pig cells after induction, and successfully screened and obtained tetraploid pigs by single cell clone culture The cell line was used as the nuclear donor for somatic cell nuclear transfer to carry out the construction of polyploid pig embryos, and the embryo transfer was carried out to produce polyploid pigs, which is of great importance for the development of polyploid pig development basic theory and application research application value.
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that it can be described in terms of form and Various changes may be made in the details without departing from the scope of the invention defined by the claims.
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310428544.7A CN116640803B (en) | 2023-04-20 | 2023-04-20 | Method for inducing polyploid pigs through colchicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310428544.7A CN116640803B (en) | 2023-04-20 | 2023-04-20 | Method for inducing polyploid pigs through colchicine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116640803A true CN116640803A (en) | 2023-08-25 |
| CN116640803B CN116640803B (en) | 2024-09-13 |
Family
ID=87614262
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310428544.7A Active CN116640803B (en) | 2023-04-20 | 2023-04-20 | Method for inducing polyploid pigs through colchicine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116640803B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080222745A1 (en) * | 2007-03-07 | 2008-09-11 | Utah State University | Colcemid-Treatment of Oocytes to enhance Nuclear Transfer Cloning |
| CN101798569A (en) * | 2009-02-09 | 2010-08-11 | 北京华盛兴邦生物技术有限公司 | Novel method for therapeutic cloning with substitution of oocyte by developing embryo after cleavage |
| CN104357483A (en) * | 2014-11-04 | 2015-02-18 | 东北农业大学 | Method for increasing pig cell reprogramming capability and application |
-
2023
- 2023-04-20 CN CN202310428544.7A patent/CN116640803B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080222745A1 (en) * | 2007-03-07 | 2008-09-11 | Utah State University | Colcemid-Treatment of Oocytes to enhance Nuclear Transfer Cloning |
| CN101798569A (en) * | 2009-02-09 | 2010-08-11 | 北京华盛兴邦生物技术有限公司 | Novel method for therapeutic cloning with substitution of oocyte by developing embryo after cleavage |
| CN104357483A (en) * | 2014-11-04 | 2015-02-18 | 东北农业大学 | Method for increasing pig cell reprogramming capability and application |
Non-Patent Citations (4)
| Title |
|---|
| LAI LIANGXUE等: ""Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells"", 《MOLECULAR REPRODUCTION AND DEVELOPMENT》, vol. 62, no. 3, 17 July 2002 (2002-07-17), pages 2 - 4 * |
| 吴孟超主编: "《吴孟超肝脏外科基础与临床》", 31 May 2007, 同济大学出版社, pages: 761 * |
| 李宁主编: "《动物克隆与基因组编辑》", 30 June 2012, 中国农业大学出版社, pages: 99 - 100 * |
| 汪旭等: ""三种有丝分裂抑制剂诱发小鼠骨髓细胞非整倍体的比较研究"", 《遗传学报》, vol. 18, no. 4, 29 August 1991 (1991-08-29), pages 1 - 3 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116640803B (en) | 2024-09-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bryja et al. | Derivation of mouse embryonic stem cells | |
| RU2015101740A (en) | GENETICALLY MODIFIED ANIMALS AND METHODS FOR PRODUCING THEM | |
| CN111778287B (en) | A kind of preparation method of chimeric embryo or animal | |
| Kadarmideen et al. | Genomic selection of in vitro produced and somatic cell nuclear transfer embryos for rapid genetic improvement in cattle production | |
| CN107299113A (en) | Application processes of the H3K27me3 and its demethylase KDM6A/B in mouse nuclear transfer reconstructed embryo | |
| Yang et al. | Machine learning in time-lapse imaging to differentiate embryos from young vs old mice | |
| CN113249456B (en) | Method for rapidly screening gene editing pig positive cell line | |
| CN116640803A (en) | A method for inducing polyploid pigs by colchicine | |
| CN104109652B (en) | Artificially induced tetraploid crucian carp cell line and its culture method and application | |
| CN113881623A (en) | A method for in vitro differentiation of parthenogenetically activated embryonic stem cells to form eggs | |
| Jochems et al. | Preimplantation genetic testing for aneuploidy (PGT-A) reveals high levels of chromosomal errors in in vivo-derived pig embryos, with an increased incidence when produced in vitro | |
| Seshagiri et al. | Enabling hamster embryo culture system: development of preimplantation embryos | |
| CN114736847B (en) | Method for rapidly obtaining primary gonad cells of giant salamander | |
| WO2013159313A1 (en) | Animal embryonic stem cell line, and preparation method and application thereof | |
| CN113684282B (en) | A method and its application for detecting hairless traits in pigs based on EDAR gene expression | |
| Li et al. | The genetic heterozygosity and fitness of tetraploid embryos and embryonic stem cells are crucial parameters influencing survival of mice derived from embryonic stem cells by tetraploid embryo aggregation | |
| CN101886059A (en) | Culture solution used for embryo vitro production and method for bovine embryo vitro production | |
| CN115336556A (en) | A kind of breeding method and application of gynogenetic mirigal carp | |
| CN117683817A (en) | Somatic cell nuclear transfer techniques to overcome pre-implantation and post-implantation epigenetic disorders | |
| CN111690684A (en) | Composition and application thereof | |
| KR20090112109A (en) | How to Obtain Germline Stem Cells from Mammalian Testis | |
| Zhang et al. | Powering mammalian genetic screens with mouse haploid embryonic stem cells | |
| Sahab et al. | An Efficient Fluorescence‐Activated Protoplast Sorting (FAPS) and Regeneration Protocol for Canola (Brassica napus) | |
| CN119162090A (en) | A gonadal cell line of hybrid sturgeon and its application | |
| Sasidhara | Animal biotechnology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |