CN113249456A - Method for rapidly screening gene editing pig positive cell line - Google Patents
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Abstract
The invention relates to a method for rapidly screening a gene editing pig positive cell line, belonging to the technical field of animal transgenosis. The method aims at the known pig positive fibroblast lines with different gene editing types, PCR, enzyme digestion and sequencing identification are carried out, and the lowest cell amount required by cell genotype identification is obtained by screening based on the gray value of a cell PCR strip; transfecting a target gene expression vector to pig fibroblasts, carrying out single cell culture screening, picking the cells when a monoclonal cell line is formed, carrying out amplification culture on the cells on a culture plate, carrying out genotype identification according to the minimum cell count, immediately carrying out somatic cell nuclear transplantation after identification, taking out a fetus, carrying out separation culture and identification, and obtaining a large number of transgenic pig positive fibroblast lines. The invention overcomes the problems of long screening period, low efficiency and low success rate of the gene editing pig positive cell line, and has important significance for promoting the production of transgenic pigs.
Description
Technical Field
The invention belongs to the technical field of animal transgenosis, and particularly relates to a method for rapidly screening a gene editing pig positive cell line.
Background
The somatic cell nuclear transfer technology is the only technology which can obtain the pig embryo with development totipotency through pig somatic cells in the current research since birth till now and is the only technology in the research of the pig embryo with development totipotency, and in addition to the emergence of the animal gene editing technology, the somatic cell nuclear transfer technology is also the main method for producing different gene editing pig individuals and is widely applied to the research of animal gene genetics biology, human disease models and xenogeneic organ transplantation, therefore, the pig somatic cell nuclear transfer technology transplantation is the hotspot and the focus of attention of the research of the life science community, and the screening of a large number of gene editing pig positive cells is the premise and the basis of the clone production of various gene editing pigs.
At present, fetal fibroblasts are mostly selected as nuclear transfer donor cells for pig somatic cell nuclear transfer, and the method breaks through the limitation that large animals such as pigs and the like cannot produce transgenic pigs due to lack of embryonic stem cells. However, the in vitro cultured porcine fibroblasts have short life span, are easy to age, are not easy to subculture for a long time, cannot be subjected to some complex gene modification and long-term screening identification, have very low efficiency for realizing gene editing, long required cell screening period and complex screening process, and at the present stage, a great number of reports have been made on the successful acquisition of cloned pigs by selecting porcine fetal fibroblasts as donor cells for nuclear transplantation.
In order to obtain the cloned pig individual with target gene modification, the gene modification is carried out on pig fetal fibroblasts in vitro, cells with target genes stably modified are screened out from a cell population with a large amount of random gene modifications and are used as donor cells for somatic cell nuclear transplantation, the method comprises the steps of diluting the cell population with a large amount of random gene modifications, carrying out single cell culture to obtain a plurality of monoclonal cell lines with different genotypes, carrying out genotype identification on different cloned cell lines, screening the monoclonal cell line with the target genes stably modified to be used as the donor cells for nuclear transplantation, and the traditional identification method needs to provide a large amount of cell lines for extracting DNA for genotype identification, so that the number of the residual monoclonal cell lines is reduced, the cell vitality is reduced, the cells are aged, and the cell proliferation is slowed down due to the attenuation of signal transmission among the cells, even after the subculture, the monoclonal cell line screened for a long time is dead, so that the success rate of cell screening is greatly reduced, and the production efficiency of the transgenic cloned pig is severely restricted.
Disclosure of Invention
Aiming at the technical problems, the invention provides a method for rapidly screening a gene editing positive cell line, which is characterized in that on the basis of the existing pig positive cell lines with different gene editing types, a micromanipulation system is utilized to accurately count the known pig positive fibroblast lines with different gene editing types according to different cell quantity requirements, then PCR, enzyme digestion and sequencing identification are carried out, and the lowest cell quantity required by cell genotype identification is obtained by screening based on the cell PCR strip gray value. In order to verify the feasibility of identifying the clone point cell line result by using the required minimum cell amount, a target gene expression vector is transfected to pig fibroblasts and is subjected to single cell culture screening, when a monoclonal cell line is formed, the monoclonal cell line is picked and cultured in a 96-well plate, genotype identification is carried out according to the minimum cell amount, somatic cell nuclear transplantation is immediately carried out after identification, and a large number of transgenic pig positive fibroblast lines are obtained after fetal separation culture and identification are taken out. The invention overcomes the problems of low screening efficiency and low success rate of the gene-edited pig positive cell line, can efficiently screen and obtain a large amount of gene-edited pig fibroblast lines in the shortest time, and has important significance for promoting the production and batch cloning of transgenic pigs.
In order to achieve the purpose, the invention provides the following technical scheme:
the method for rapidly screening the gene editing pig positive cell line comprises the following specific steps:
1) determination of the minimum cell mass required for genotyping porcine fibroblasts
Carrying out PCR, enzyme digestion and sequencing identification on the known pig positive fibroblast lines with different gene editing types, and screening to obtain the minimum cell amount required by cell genotype identification based on the gray value of a cell PCR strip;
2) screening of transgenic Positive pig fibroblast lines
Based on animal gene editing technology, constructing a target gene expression vector by adopting different gene editing modes aiming at different genes, transfecting the target gene expression vector to a pig fibroblast line, carrying out single cell culture screening after extreme dilution, and carrying out amplification culture on a culture plate after digging when a monoclonal cell line is formed, and carrying out genotype identification on cell lines of different cloning points by taking the minimum cell amount required by the genotype identification of the cells;
taking the monoclonal cell line identified as positive as donor cell to immediately perform somatic cell nuclear transfer and embryo transfer, taking out the editing condition of the fetus identification target gene after the pregnant sow is pregnant, and obtaining the transgenic pig fetus positive cell line after separation culture. Further preferably, in step 1), the minimum number of cells required for genotyping is 50 cells.
Further preferably, in step 2), the donor cells for pig somatic cell nuclear transfer are derived from a small number of cells cultured in a 96-well plate and identified as positive by counting 50 cells; the number of the surrogate pregnant sows is 3, the pregnancy condition needs to be monitored by B ultrasonic in due time, fetuses are taken out during pregnancy, the gene editing condition of the fetuses is identified, the identified positive fetuses can be isolated and cultured to obtain a large number of gene editing pig positive fetal fibroblast lines, and the requirement of the homozygous fetal fibroblast of the pig for subsequent somatic cell nuclear transfer gene editing can be completely met.
The invention has the beneficial effects that:
the invention obtains the minimum quantity required by the identification of the cell genotype by screening on the basis of the known pig fibroblast lines with different gene editing types, identifies and screens the cell genotype of unknown genotype by utilizing the minimum quantity of the cells required by the identification of the cell genotype, overcomes the problems of low screening efficiency and low success rate of the positive cell line of the pig subjected to gene editing to a certain extent, can efficiently screen and obtain a large number of pig fetal fibroblast lines subjected to gene editing in the shortest time, and has important significance for promoting the production and batch cloning of transgenic pigs.
Drawings
FIG. 1 is a flow chart of the present invention.
FIG. 2 shows the results of genotyping positive cells for gene editing with known cell mass; in the figure, a) results of GHRKO cell counting for different gradient cell volumes; b) PCR results of GHRKO cells with different gradient cell amounts; c) carrying out enzyme digestion on GHRKO cells T7EN1 with different gradient cell amounts; d) quantification results of PCR and T7EN1 enzyme digestion bands of different numbers of GHRKO cells; f) results of sequencing of GHR genotype Sanger in 50 GHRKO cells.
FIG. 3 is a screen of porcine IPO13KO gene editing positive single cell clones; in the figure, a) schematic representation of pig IPO13 gene targeting; b) editing 50 cell PCR results of single cell clones with 20 IPO13 genes; c) editing single cell clone 50 cells by 20 IPO13 genes, and performing enzyme digestion on T7EN 1; d-e) sequencing results of Sanger sequencing of IPO13KO positive cloning points.
FIG. 4 is a cloned fetal and piglet IPO13KO genotype identification; in the figure, a) 8 IPO13KO cloned fetuses; b) PCR results for 8 cloned fetal IPO13 genes; c) carrying out enzyme digestion detection on 8 cloned fetus IPO13 genes T7EN 1; d) 6 surviving IPO13KO cloned pigs; e) sequencing results of 8 cloned fetuses and 9-head born IPO13KO cloned piglets.
FIG. 5 is a schematic of a gene editing pig positive fibroblast screening; in the figure, a) a schematic diagram of a general screening method; b) the screening method of the invention is shown schematically; c) comparing screening periods; d) the power comparison is screened.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding of the skilled person.
Examples
A method for rapidly screening a gene-editing pig positive cell line is characterized by comprising the following steps: the method comprises the following specific steps:
1) determining the minimum cell mass required for genotyping porcine fibroblasts based on cell lines of known different gene editing types
Aiming at the known GHRKO pig positive fibroblast cell line, the pig positive fibroblast cell lines with different gene editing types are accurately counted by a micromanipulation system according to 20, 50, 100, 150 and 200 cells, each gradient is repeated for 3 times, then PCR, T7E1 enzyme digestion and DNA sequencing identification are respectively carried out, and the gray values of PCR bands of the cells with different numbers are statistically analyzed. As shown in FIG. 2, a clear PCR band and a T7ENI cleavage band can be seen in 50 cells, and the sequencing result of 50 cells is accurate and reliable; thus, the minimum cell mass required for genotyping of the cells can be determined for 50 cells.
2) Screening by using the minimum cell quantity required for identifying the known cell genotype to obtain the transgenic positive pig fibroblast cell line
Based on CRISPR/Cas9 gene editing technology, an sgRNA knockout expression vector is constructed for an IPO13 gene, the knockout expression vector is transfected to a pig fibroblast line by an electroporation method, single cell culture screening is carried out after extreme dilution, when a single clone cell line is formed, after digging, amplification culture is carried out on a 96-well plate, and genotype identification is carried out on different formed clone point cell lines after counting according to 50 cells. The results show that clone nos. 4 and 17 have the point of IPO13 bi-allelic knockout monoclonal cell line, and the results are shown in fig. 3.
Taking the number 17 monoclonal cell line identified as positive as a donor cell to immediately perform somatic cell nuclear transfer and embryo transfer, taking 8 fetuses after 35 days of gestation of a surrogate sow, identifying all fetuses as IPO13 double allele knockout fetuses and enabling gene sequences to be identical with the number 17 monoclonal cell line, obtaining a large number of IPO13 double allele knockout fetal fibroblast cell lines after separation culture, further performing somatic cell nuclear transfer by taking the cells as the donor cell, and obtaining cloned piglet individuals of IPO13KO in batches, wherein the result is shown in figure 4.
The invention discloses a method for rapidly screening a gene-editing pig positive cell line by utilizing an embodiment, which is characterized in that on the basis of known pig fibroblast lines with different gene editing types, the minimum quantity required by cell genotype identification is obtained by screening, and then the cell line genotype of unknown gene editing is identified and screened by utilizing the minimum quantity of cells required by the cell genotype identification, so that the problems of low screening efficiency and low screening success rate of the gene-editing pig positive cell line are overcome to a certain extent, a large number of gene-editing pig fetal fibroblast lines can be efficiently screened and obtained in the shortest time, the screening period of the gene-editing pig positive cell line is greatly shortened, the screening period is shortened by about 15 days, the screening success rate is improved by about 4 times (figure 5), and the method has important significance for accelerating the production of transgenic pigs.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (3)
1. A method for rapidly screening a gene-editing pig positive cell line is characterized by comprising the following steps: the method comprises the following specific steps:
1) determination of the minimum cell mass required for genotyping porcine fibroblasts
Carrying out PCR, enzyme digestion and sequencing identification on the known pig positive fibroblast lines with different gene editing types, and screening to obtain the minimum cell amount required by cell genotype identification based on the gray value of a cell PCR strip;
2) screening of transgenic Positive pig fibroblast lines
Transfecting a target gene expression vector to pig fibroblasts, performing single cell culture screening, digging and then performing expanded culture on a culture plate when a monoclonal cell line is formed, and performing genotype identification on cell lines of different cloning points by taking the minimum cell amount required by the genotype identification of the cells;
taking the monoclonal cell line identified as positive as donor cell to immediately perform somatic cell nuclear transfer and embryo transfer, taking out the editing condition of the fetus identification target gene after the pregnant sow is pregnant, and obtaining the transgenic pig fetus positive cell line after separation culture.
2. The method of claim 1, wherein the screening is performed by using a panel of positive cell lines of pig: in step 1), the minimum cell number required for genotyping is 50 cells.
3. The method of claim 1, wherein the screening is performed by using a panel of positive cell lines of pig: in the step 2), donor cells for pig somatic cell nuclear transfer come from a small number of cells which are cultured in a 96-well plate and are positive after being counted by 50 cells; the number of the surrogate pregnant sows is 3, the pregnancy condition needs to be monitored by B ultrasonic in due time, fetuses are taken out during pregnancy, the gene editing condition of the fetuses is identified, the identified positive fetuses can be isolated and cultured to obtain a large number of gene editing pig positive fetal fibroblast lines, and the requirement of the homozygous fetal fibroblast of the pig for subsequent somatic cell nuclear transfer gene editing can be completely met.
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| CN114134216A (en) * | 2021-11-03 | 2022-03-04 | 深圳大学 | Method for rapidly identifying MSC gene modification based on PCR and application |
| CN114807333A (en) * | 2022-05-31 | 2022-07-29 | 华南农业大学 | Method for identifying whole genome variation of gene editing animal |
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