CN116640804B - Method for constructing triploid pig based on somatic cell nuclear transfer technology - Google Patents
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Abstract
本发明涉及一种基于体细胞核移植技术构建三倍体猪的方法。本发明在体外建立二倍体猪细胞系，以此为核供体进行体细胞核移植，经电融合、电激活后构建克隆胚，检测其克隆胚的体外发育潜力，进一步移植至代孕母猪中继续发育，适时根据B超监测结果获取胎儿，建立胎儿成纤维细胞系进行倍性鉴定及核型分析。以三倍体猪胎儿成纤维细胞为核供体再次体细胞核移植，经电融合、电激活后构建克隆胚，检测其克隆胚的体外发育能力，并进一步移植至代孕母猪体内，生产三倍体猪。本发明克服了猪三倍体胚胎发育效率低、成功率低的问题，对三倍体猪的生产及应用具有重要的意义。
The present invention relates to a method for constructing a triploid pig based on somatic cell nuclear transplantation technology. The present invention establishes a diploid pig cell line in vitro, uses it as a nuclear donor for somatic cell nuclear transplantation, constructs a cloned embryo after electric fusion and electric activation, detects the in vitro development potential of the cloned embryo, further transplants it into a surrogate sow for continued development, obtains the fetus according to the B-ultrasound monitoring results in a timely manner, and establishes a fetal fibroblast cell line for ploidy identification and karyotype analysis. Somatic cell nuclear transplantation is performed again using triploid pig fetal fibroblasts as nuclear donors, constructs a cloned embryo after electric fusion and electric activation, detects the in vitro development ability of the cloned embryo, and further transplants it into a surrogate sow to produce a triploid pig. The present invention overcomes the problems of low efficiency and low success rate of pig triploid embryo development, and is of great significance to the production and application of triploid pigs.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for constructing triploid pigs based on somatic cell nuclear transfer technology.
Background
In nature, organisms containing three or more complete chromosomes are generally called polyploids, the polyploid phenomenon widely exists in plants, fungi, amphibians, reptiles and other lower animals, and about 60% -70% of angiosperms in the organism world, such as wheat, cotton, coffee and the like, are polyploids. In animals, polyploid has shown attractive prospects and application value in the field of shellfish and fish biological breeding research. However, in vertebrates, polyploid phenomenon occurs very rarely except for amphibians, reptiles, and some fish. In the study of polyploid pigs, triploid pig embryos or surviving triploid pig individuals that developed to a late stage have not yet been reported.
The lack of triploid pig research model and materials is still an important factor for restricting the analysis of triploid embryo development disorder. At present, many researches on the development of the polyploid embryo of the pig and the non-survival mechanism of individuals are only based on the excavation of trace polyploid evidence data, even the speculation according to a mathematical modeling simulation system and the like, a systematic basic research theory system of the polyploid embryo of the pig is not formed yet, and the mechanism analysis of the development disorder of the polyploid embryo of the pig, sex determination, gene expression regulation and control, cell division and growth and development processes are unclear, so that the polyploid embryo of the pig cannot survive and develop for a long time, and the deep analysis of the development disorder of the polyploid embryo of the pig and the innovative research and application in the breeding field are severely limited to a great extent.
Reprogramming somatic cell nuclear transfer provides a powerful tool for modulation of epigenetic modifications during mammalian embryo development. Genomic imprinting is an important epigenetic modification in mammalian development, and unbalanced expression of imprinted genes is typical of triploid mammals. The 3D chromatin framework is reprogrammed Cheng Xianxiang at the chromatin level in the development process of somatic cell nuclear transfer embryo, the formation and occurrence of polyploid organisms often undergo a great deal of biological processes such as genome structure change, epigenetic modification and related phenotypic change, and the chromatin structure remodeling, DNA replication and recombination and gene expression regulation are closely related to the growth and development process of organisms. Therefore, the reprogramming of somatic cell nuclear transfer provides a possibility for regulating epigenetic modifications such as abnormal expression, wrong expression or non-expression of imprinted genes in the development process of triploid pig embryos, and provides an important tool for the construction of triploid pigs.
Disclosure of Invention
Aiming at the technical problems, the invention provides a method for constructing triploid pigs based on somatic cell nuclear transfer technology, which comprises the steps of establishing a diploid pig cell line in vitro, taking the diploid pig cell line as a nuclear donor to carry out somatic cell nuclear transfer, constructing cloned embryos after electrofusion and electroactivation, detecting the in vitro development potential of the cloned embryos, further transferring the cloned embryos into a surrogate sow to continue to develop, acquiring a fetus according to a B-ultrasonic monitoring result at proper time, and establishing a fetal fibroblast line to carry out ploidy identification and karyotype analysis. And (3) taking the triploid pig fetal fibroblasts as nuclear donors for re-somatic cell nuclear transplantation, constructing cloned embryos after electrofusion and electroactivation, detecting the in-vitro development capacity of the cloned embryos, and further transplanting the cloned embryos into a pregnant sow to produce the triploid pig. The invention solves the problems of low development efficiency and low success rate of the pig triploid embryo, and has important significance for the production and application of the triploid pig.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for constructing triploid pigs based on somatic cell nuclear transfer technology comprises the following specific steps:
1) Establishment of diploid porcine cell lines
And taking diploid pig tissues to separate a pig cell line, and carrying out in-vitro subculture to establish the diploid pig cell line as a donor nucleus for producing triploid pig somatic cell nuclear transplantation.
2) Construction of pig triploid embryo
Injecting a diploid pig cell nuclear donor into a mature non-enucleated pig oocyte, obtaining a reconstructed embryo after electrofusion and electroactivation, detecting the embryo development capability of the reconstructed embryo, and further transplanting the reconstructed embryo into a pregnant sow to continue to develop and produce a triploid pig.
3) Identification of pig triploid
After the transplanted pregnant sow is pregnant for a period of time, the fetus is obtained and separated into a fibroblast line, ploidy identification and karyotype analysis are carried out, and the triploid pig fetus is obtained through identification.
4) Production of pig triploid
And continuously cloning again by taking the obtained triploid pig fetal fibroblasts as a nuclear donor, detecting the development capacity of cloned embryos, and further transplanting the triploid pig fetal fibroblasts into a surrogate sow body until a survival triploid pig individual is obtained.
Further, in step 1), the cell lines include different porcine tissue, fetal-derived fibroblast cell lines.
Further, in step 2), the detection indicators of embryo development ability include fusion rate, cleavage rate, blastocyst rate and blastocyst cell number.
Further, the specific method in the step 3) is that a fetal isolated fibroblast line is obtained according to a timely B-ultrasonic detection result, cell ploidy identification is carried out through a flow cytometer, the number of chromosomes is determined through karyotype analysis, and the chromosome source is verified based on microsatellites.
Further, in step 4), the continuous cloning may be performed a plurality of times until a viable triploid pig individual is obtained.
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 invention as claimed.
The beneficial technical effects of the invention are as follows:
The invention takes a diploid pig cell line as a nuclear donor to carry out somatic cell nuclear transplantation, obtains a reconstructed embryo after electrofusion and electroactivation, detects the in-vitro development potential of the reconstructed embryo, further transplants the reconstructed embryo into a surrogate sow, acquires a fetus according to a timely B-ultrasonic monitoring result, and establishes a fetal fibroblast line to carry out ploidy identification and karyotype analysis. Further, the triploid pig fetal fibroblasts are used as nuclear donors for continuous cloning, the in vitro development potential of cloned embryo is detected, and the cloned embryo is further transplanted into a surrogate sow for continuous development to produce triploid pig individuals. The technical problems of low development rate and low success rate of the triploid embryo of the pig are overcome to a certain extent, the limit of the development of the triploid embryo is broken through, and the method has important significance for the production and application research of the triploid pig.
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FIG. 1 is a flow chart of one embodiment of the present invention.
Detailed Description
Alternative embodiments of the present application will be described in more detail below with reference to the accompanying drawings. While the drawings illustrate alternative embodiments of the present application, it should be understood that the present application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
The terminology used in the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any or all possible combinations of one or more of the associated listed items.
The method for constructing triploid pigs based on somatic cell nuclear transfer technology is described in detail below with reference to the accompanying drawings, and is specifically as follows:
as shown in fig. 1, the method for constructing triploid pigs based on somatic cell nuclear transfer technology in the application comprises the following specific steps:
1) Establishment of diploid porcine cell lines
And taking diploid pig tissues to separate a pig cell line, and carrying out in-vitro subculture to establish the diploid pig cell line as a donor nucleus for producing triploid pig somatic cell nuclear transplantation.
2) Construction of pig triploid embryo
Injecting a diploid pig cell nuclear donor into a mature non-enucleated pig oocyte, obtaining a reconstructed embryo after electrofusion and electroactivation, detecting the embryo development capability of the reconstructed embryo, and further transplanting the reconstructed embryo into a pregnant sow to continue to develop and produce a triploid pig.
3) Identification of pig triploid
After the transplanted pregnant sow is pregnant for a period of time, the fetus is obtained and separated into a fibroblast line, ploidy identification and karyotype analysis are carried out, and the triploid pig fetus is obtained through identification.
4) Production of pig triploid
And continuously cloning again by taking the obtained triploid pig fetal fibroblasts as a nuclear donor, detecting the development capacity of cloned embryos, and further transplanting the triploid pig fetal fibroblasts into a surrogate sow body until a survival triploid pig individual is obtained.
In one embodiment of the present application, in step 1), the cell lines comprise different porcine tissue, fetal derived fibroblast cell lines.
In one embodiment of the present application, in step 2), the detection indicators of embryo development ability include fusion rate, cleavage rate, blastocyst rate and blastocyst cell number.
In one embodiment of the present application, the specific method of step 3) is to obtain a fetal isolated fibroblast line according to the timely B-ultrasonic detection result, perform cell ploidy identification by a flow cytometer, determine the chromosome number by using a karyotype analysis, and verify the chromosome source based on microsatellites.
In one embodiment of the present application, in step 4), the continuous cloning may be performed multiple times until a viable triploid individual pig is obtained.
For clarity, the following examples are provided in detail.
Example 1
A method for constructing triploid pigs based on somatic cell nuclear transfer technology comprises the following specific steps:
1) Establishment of diploid porcine cell lines
Taking diploid pig ear or fetal tissue, storing in DMEM containing 5% of green-streptomycin solution, placing on crushed ice to carry back to a laboratory, washing with PBS containing 5% of green-streptomycin solution in a super clean bench, sterilizing, shearing, digesting with 0.1% of IV type collagenase, establishing a pig tissue fibroblast cell line, and performing in vitro subculture to obtain triploid pig by performing somatic cell nuclear transplantation as nuclear donor.
2) Construction and identification of pig triploid embryo
The diploid pig fibroblast nuclear donor is injected into the non-enucleated pig oocyte which is mature, and after electrofusion and electroactivation, 368 reconstructed embryos are constructed, the cleavage rate (73.66+/-14.42%), the blastocyst rate (26.65+/-9.29%) of the diploid pig fibroblast nuclear donor are not significantly different from the cleavage rate (73.22 +/-7.45%) of the oocyte enucleated control group (19.46+/-5.31%), but the blastocyst cell number (27.22+/-8.47) of the diploid pig fibroblast donor is significantly lower than that of the oocyte enucleated control group (41.58+/-15.45, P < 0.05), so that the reconstructed embryos have normal development potential. Further transplanting the obtained product to 4 pregnant sows for further development, detecting 3 pregnant sows by B ultrasonic at 23 days, wherein the pregnancy rate is 75%, obtaining 6 fetal tissue samples by laparotomy at 26 days of gestation ages, wherein 3 tissue samples successfully establish a fetal fibroblast line, detecting 1 fetus as a triploid by a flow cytometer, and further carrying out microsatellite analysis on the 6 tissue samples, wherein the result shows that 2 fetuses are the triploid.
3) Production of triploid pigs
The triploid pig fetal fibroblasts are used as nuclear donors for somatic cell nuclear transplantation, 1208 cloned embryos are constructed after electrofusion and electroactivation, the fusion rate (95.26+/-2.41%), the cleavage rate (77.93+/-7.63%), the blastocyst rate (24.64+/-3.90%) and the fusion rate (97.22+/-1.51%), the cleavage rate (79.85+/-3.30%) and the blastocyst rate (21.60+/-3.08%) of the diploid pig fetal fibroblasts are all not significantly different (P > 0.05), but the blastocyst cell number (33.59 +/-12.74) is significantly lower than that of the diploid pig fetal fibroblasts serving as nuclear donors for constructing cloned embryos (45.01 +/-15.42, P < 0.05). It is shown that it has normal embryo development ability and can be further embryo transferred until a viable triploid pig is obtained.
The method for constructing triploid pigs based on the somatic cell nuclear transfer technology in the embodiment is characterized in that a diploid pig cell line is used as a nuclear donor for somatic cell nuclear transfer, a reconstructed embryo is obtained after electrofusion and electroactivation, the in-vitro development potential of the reconstructed embryo is detected, the reconstructed embryo is further transferred into a pregnant sow, a fetus is obtained according to a timely B-ultrasonic monitoring result, and a fetal fibroblast line is established for ploidy identification and karyotype analysis. Further, the triploid pig fetal fibroblasts are used as nuclear donors for continuous cloning, the in vitro development potential of cloned embryo is detected, and the cloned embryo is further transplanted into a surrogate sow for continuous development, so as to produce triploid pig individuals. The technical problems of low development rate and low success rate of the triploid embryo of the pig are overcome to a certain extent, the limit of the development of the triploid embryo is broken through, and the method has important significance for the production and application research of the triploid pig.
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
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Citations (2)
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| CN104894166A (en) * | 2014-03-04 | 2015-09-09 | 云南农业大学 | Method for production of parthenogenesis mammals by continuous cloning technology |
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| CA2381124A1 (en) * | 1999-09-13 | 2000-09-13 | Steven L. Stice | Cloning pigs using donor cells or nuclei from differentiated cells and production of pluripotent porcine |
| CN105755047A (en) * | 2016-03-30 | 2016-07-13 | 魏红江 | Method for obtaining cloned pigs with modified genes through continuous cloning |
| CN107937444A (en) * | 2017-07-25 | 2018-04-20 | 北京希诺谷生物科技有限公司 | The method of somatic cell clone dog |
| CN111979243B (en) * | 2020-08-24 | 2023-05-23 | 大连大学 | Method for constructing TAP gene-deleted pig T2 cells by using CRISPR/Cas9 system |
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| CN103343142A (en) * | 2013-07-15 | 2013-10-09 | 云南农业大学 | Method for producing parthenogenetic cloned pig by means of continuous cloning technology |
| CN104894166A (en) * | 2014-03-04 | 2015-09-09 | 云南农业大学 | Method for production of parthenogenesis mammals by continuous cloning technology |
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