CN112813031A - Construction method and application of stable-transfer expression SpCas9 protein white cell line - Google Patents
Construction method and application of stable-transfer expression SpCas9 protein white cell line Download PDFInfo
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Abstract
The invention relates to a construction method of a white cell line stably expressing SpCas9 protein and application thereof, wherein the construction method comprises the following steps: packaging and purifying lentivirus, including HEK 293T cell preparation, transfection packaging plasmid, collecting cell culture supernatant after transfection culture, and concentrating virus liquid; and (3) screening the obtained concentrated virus infected cells by drug concentration, screening lentivirus infected cells by drugs, and selecting a monoclonal for identification. The mouse myoblast C2C12 stable-transformation expression SpCas9 protein leukocyte line and the mouse embryonic stem cell E14 stable-transformation expression SpCas9 protein leukocyte line are obtained by adopting a construction method of stably-transformation expression SpCas9 protein leukocyte line, can stably express SpCas9 protein, can be used as a rapid verification tool for performing rapid functional verification on designed SgRNA to detect whether the SgRNA design is successful, and can also be used as a functional gene screening system for subsequent large-scale functional gene screening.
Description
Technical Field
The invention relates to a method for constructing a cell line stably expressing SpCas9 protein and application thereof, belonging to the technical field of cell biology.
Background
In the existing SgRNA verification and functional gene screening technical systems, a single plasmid system is mostly adopted to express SpCas9 protein, and a single plasmid system is a eukaryotic expression system, so that effective SgRNA functional verification and functional gene screening cannot be carried out in certain primary cells or certain cell lines with low transfection efficiency. How to effectively express SpCas9 protein is a technical problem which needs to be solved urgently.
Disclosure of Invention
Technical problem to be solved
In order to solve the problems in the prior art, the invention provides a method for constructing a white cell line stably expressing SpCas9, and provides a white cell line stably expressing SpCas9 by a mouse myoblast C2C12 and a white cell line stably expressing SpCas9 by a mouse embryonic stem cell E14.
(II) technical scheme
In order to achieve the purpose, the invention adopts the main technical scheme that:
a method for constructing a white cell line stably expressing SpCas9 protein comprises the following steps:
s1, HEK 293T cell preparation: selecting HEK 293T cells with clear sources and good growth states, and treating the revived cells for two generations by using a mycoplasma inhibitor to ensure the cell states;
s2, transfection packaging plasmid: transfection was performed according to the cation-mediated cell transfection method, and the ratio of virus-packaging plasmids was LentiCas 9: psPAX 2: pmd2.g ═ 4: 3: 1, transfecting HEK 293T cells in a mass ratio, replacing a culture medium after 6-8 h of transfection, and selecting a proper culture medium for liquid replacement according to a cell line constructed as required;
s3, virus collection: after transfection is carried out for 36-48 h, cell culture supernatant can be collected, a 0.45-micron filter is used for filtering to remove cell debris, and a Millipore column is used for concentrating virus liquid to obtain purified concentrated slow virus liquid;
s4, inoculating the cells of the cell line to be constructed in a six-well plate at low density, setting a concentration gradient of a screening marker medicament Blasticidin after the cells grow adherent to the six-well plate, adding the concentration gradient into the six-well plate for 24h, observing the survival condition of the cells, and selecting the concentration of the cells when the cells die by nearly 90%;
s5, lentivirus-infected cells: resuscitating cells needing to construct a cell line, inoculating the cells in a culture dish when the growth state is good, replacing the cells with a culture medium containing polybrene (polybrene) with the final concentration of 8 mug/mL after the cells are attached to the wall, adding concentrated virus solution, and replacing the cells with a fresh normal culture medium after infection;
s6, drug screening: after infection culture for a period of time, adding a culture medium containing the concentration of the screening drug selected in the step S4 for screening until no dead cells appear;
s7, selecting a single clone: and (3) counting cells after the digestion of the cells subjected to drug screening, culturing for one week to obtain macroscopic cell clones, picking the monoclone to a 96-well plate, carrying out amplification culture, and collecting a cell sample for identification.
The construction method as described above, preferably, in step S1, the mycoplasma inhibitor is treated with Biomyc-3.
The construction method as described above, preferably, in step S2, the cation-mediated cell transfection method employs the following steps:
inoculating cells with proper density before transfection;
secondly, performing transfection when the cell density reaches 70-90%, and replacing the culture medium 1h before transfection;
③ sucking 100 mu L of 0.9 percent sterile normal saline into a 1.5mL EP tube, and adding 5 mu g of plasmid to be transfected;
fourthly, sucking 100 mu L of 0.9 percent sterilized normal saline into a 1.5mL EP tube again, adding 2 mu L of VigoFect transfection reagent, mixing evenly and standing for 5min at room temperature;
fifthly, dropwise adding the VigoFect diluted in the fourth step into the plasmid in the third step, and standing for 15min at room temperature;
sixthly, uniformly dripping the mixed solution after standing into a cell culture medium and uniformly mixing;
seventhly, the culture solution is replaced after transfection for 6-8 hours.
In the above construction method, preferably, in step S4, the low-density inoculation is performed when the density of the adherent cells after inoculation reaches 30% to 40%; the concentration gradient of the Blasticidin is set to be 0, 2, 4, 6, 8 and 10 mu g/mL;
in step S5, the infection time is changed to be 6-8 h, and then the medium is changed to be fresh and normal;
in step S6, the infection culture period refers to 32-48 h of culture, and passage treatment is performed when the cell density is too high in the screening process, so that the cell density is not more than 80% in the screening process;
in step S7, 3000 to 5000 cells are seeded in a 10cm dish.
The construction method as described above, preferably, in step S8, the SpCas9 protein expressed by western blotting detection is identified or the genome of the cell is detected by PCR using primers for detecting the SpCas9 sequence.
The construction method as described above, preferably, the primers for detecting the SpCas9 sequence include a forward primer ATGGACAAGAAGTACAGCATCGG and a reverse primer GTCGCCTCCCAGCTGAGAC.
The construction method as described above, preferably, the PCR amplification condition is pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 15s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1kb/min, and 25-30 cycles; extension at 72 ℃ for 10 min.
In the above-described construction method, preferably, the cell line to be constructed is a mouse myoblast cell C2C12 cell or a mouse embryonic stem cell E14 cell.
A mouse myoblast C2C12 stably expresses SpCas9 protein white cell line, and is preferably obtained by constructing the cell line to be constructed into the mouse myoblast C2C12 cell by adopting the construction method.
A mouse embryonic stem cell E14 stably expresses SpCas9 protein white cell line, and is preferably obtained by constructing a cell line to be constructed into a mouse embryonic stem cell E14 cell by adopting the construction method.
(III) advantageous effects
The invention has the beneficial effects that:
the invention provides a construction method for stably expressing a SpCas9 protein leukocyte line, and the obtained cell line can stably express a SpCas9 protein; a method for verifying the function of SgRNA and effectively screening functional genes is provided for primary cells with low transfection efficiency and partial cell lines; after detection, the constructed SpCas9 protein cell line is proved not to influence the original cell dryness of the E14 cells.
The method for constructing the white cell line stably expressing the SpCas9 protein constructs a cell line stably expressing the SpCas9 protein in cell lines C2C12 and E14 with low transfection efficiency. Compared with the transient transfected cells, the expression level of SpCas9 protein of the prepared stable transfected cell line has no difference among the cells, and false positive results cannot be generated due to the protein expression difference among the cells in the subsequent functional gene screening and single cell sequencing.
The mouse myoblast C2C12 stably expresses SpCas9 egg white cell line and the mouse embryonic stem cell E14 stably expresses SpCas9 egg white cell line, can stably express SpCas9 protein, can be used as a rapid verification tool for performing rapid functional verification on designed SgRNA to detect whether the SgRNA design is successful, and can be used as a functional gene screening system for performing subsequent large-scale functional gene screening.
Drawings
FIG. 1 shows the results of a monoclonal western blotting assay of the C2C12-Cas9 cell line;
FIG. 2 shows the detection result of the constructed C2C12-Cas9 cell line with the sequence primer of SpCas 9;
FIG. 3 shows the expression of SpCas9 protein of the E14-SpCas9 cell line;
FIG. 4 shows the expression of SpCas9 protein of the E14-SpCas9 cell line;
FIG. 5 shows the expression of the constructed E14-SpCas9 cell lines SpCas9, Nanog, Oct4 and Sox2 proteins;
FIG. 6 shows the result of cell dryness test of the constructed E14-SpCas9 cell line.
Detailed Description
For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.
Example 1
In the embodiment, a mouse myoblast C2C12 stable-transfer expression SpCas9 protein leukocyte line is constructed, a cell line is constructed mainly by adopting a lentivirus infection mode, and the used lentivirus expression plasmid is LentiCas9-Blast and Addgene official website number is # 52962.
The packaging and purification of lentiviruses was first performed:
(1) HEK 293T cell preparation: because the lentivirus packaging process is very cell dependent, it is necessary to select HEK 293T cells with well-defined sources and good growth status, and use the recovered cells with the mycoplasma inhibitor Biomyc-3 (purchased from Biological Industries); the use method is that the ratio of the total amount of the cells in the cell culture process is 1: 100 was added to the medium. The cell state is guaranteed after two generations of treatment.
(2) Transfection packaging plasmid: transfecting according to a cation-mediated cell transfection method, specifically, inoculating HEK 293T cells with appropriate density before transfecting;
secondly, performing transfection when the cell density reaches 70-90%, and replacing the culture medium 1h before transfection;
③ sucking 100 mu L of 0.9 percent sterile normal saline into a 1.5mL EP tube, and adding 5 mu g of plasmid to be transfected; wherein, the plasmid to be transfected is a plasmid which is prepared from LentiCas9 according to the mass ratio: psPAX 2: pmd2.g ═ 4: 3: 1 mixed plasmids;
fourthly, sucking 100 mu L of 0.9 percent sterilized normal saline into a 1.5mL EP tube again, adding 2 mu L of VigoFect transfection reagent, mixing evenly and standing for 5min at room temperature;
fifthly, dropwise adding the VigoFect diluted in the fourth step into the plasmid in the third step, and standing for 15min at room temperature;
sixthly, uniformly dripping the mixed solution after standing into a cell culture medium and uniformly mixing;
and (c) transfecting the cells for 6-8 hours, and then, adding 10% serum into the DMEM medium.
Wherein the proportion of the virus packaging plasmid is LentiCas 9: psPAX 2: pmd2.g ═ 4: 3: 1, transfecting HEK 293T cells for 6-8 h, then replacing the culture medium, selecting a proper culture medium for replacing liquid according to the cells to be infected, wherein the culture medium for C2C12 cells is a DMEM culture medium containing 10% fetal calf serum. (psPAX 2: Addgene official gazette number 12260; pMD2. G: Addgene official gazette number 12259; LentiCas9-Blast, Addgene official gazette number # 52962; LentiCas9 plasmid is lentivirus backbone, can express SpCas9 protein.)
(3) And (3) virus harvesting: after transfection for 36-48 h, cell culture supernatant can be collected, cell debris is removed by filtering with a 0.45 μm filter, virus solution is concentrated by a Millipore column, and the virus solution is stored for a long time at-80 ℃ after subpackaging. (0.45 μm filter 4612 from Pall Corporation; concentration column from Millipore ACS510024)
The resulting concentrated virus was used to infect cells:
(1) screening the concentration of the drug: C2C12 cells are inoculated in a six-well plate at low density, after adherent growth, a screening marker drug Blasticidin is arranged to be added into the six-well plate according to concentration gradients of 0, 2, 4, 6, 8 and 10 mu g/mL, the survival condition of the cells is observed after 24 hours of addition, and the concentration of the Blasticidin when the cells are close to 90% death is 4 mu g/mL. (Blasticidin is a product of Thermo corporation, cat # 461120)
(2) Lentivirus infected cells: recovering the cells needing to construct a cell line C2C12, when the growth state is good, inoculating the cells, enabling the density of the cells to reach 30% -40% after the cells are attached to the wall, placing the cells in a 6cm culture dish, replacing the cells with a culture medium containing polybrene with the final concentration of 8 mu g/mL after the cells are attached to the wall, adding about 20 mu L of concentrated virus solution, and replacing the cells with a fresh normal culture medium after infecting for 6-8 hours.
(3) Drug screening: after 36h of infection, the medium containing the selected screening drug at 4 μ g/mL was added for screening until no dead cells appeared. And (3) carrying out passage treatment when the cell density is too high in the screening process, and ensuring that the cell density does not exceed 80% in the screening process. The experimental waste needs high pressure treatment in the whole cell line construction process.
(4) Picking a single clone: and (3) counting cells after the digestion of the cells subjected to drug screening, inoculating 3000-5000 cells into a 10cm culture dish according to the cell growth speed and the cell size, culturing for a week, then, allowing the cells to be visible to the naked eye, picking the monoclone to a 96-well plate, performing amplification culture, and then, collecting a cell sample to perform western blotting identification. (the SpCas9 protein antibody is Abcam product, cat # ab191468, and the cell culture dish is Corning product.)
C2C12 cells are infected after the SpCas9 expression plasmid is subjected to lentivirus packaging, a single clone is picked after the Blasticidin screening to verify that 13 clones can stably express SpCas9 protein, the result is shown in figure 1, GAPDH in the figure is glyceraldehyde-3-phosphate dehydrogenase and serves as an experimental reference in a western blotting experiment, and then a cell line 3 and a cell line 5 are selected and respectively marked as C2C12-Cas9-3 and C2C12-Cas 9-5. Extracting genomes of C2C12-Cas9-3 and C2C12-Cas9-5, and designing a sequence primer of SpCas 9: the sequence of the forward primer is ATGGACAAGAAGTACAGCATCGG, the sequence of the reverse primer is GTCGCCTCCCAGCTGAGAC, and the genome PCR reaction system is as follows: 2 × HiFi mix 25 μ L; 50ng of genomic DNA; 2 mu L of upstream and downstream primers; ddH2The content of O is filled to 50 mu L. Reaction procedure for amplification: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 15s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1kb/min, and 25-30 cycles; extension at 72 ℃ for 10 min. The results are shown in FIG. 2, and the two cell lines show a band at the same position as the positive control LentiCas9-Blast, indicating that the genome of the cell line stably integrates the DNA sequence of the SpCas9 protein.
Example 2
In the embodiment, the embryonic stem cell E14 stably expresses SpCas9 protein leukocyte line, the cell line is constructed mainly by adopting a lentivirus infection mode, and the used lentivirus expression plasmid is LentiCas9-Blast and Addgene official website number is # 52962.
The packaging and purification of lentiviruses was first performed:
(1) HEK 293T cell preparation: because the slow virus packaging process has strong dependence on cells, HEK 293T cells with clear sources and good growth states need to be selected, and the recovered cells are applied to mycoplasma inhibitor Biomyc-3, a product of Biological Industries company; the use method is that the ratio of the total amount of the cells in the cell culture process is 1: 100 was added to the medium. The cell state is guaranteed after two generations of treatment.
(2) Transfection packaging plasmid: transfection was performed according to the cation-mediated cell transfection method, and the ratio of virus-packaging plasmids was LentiCas 9: psPAX 2: pmd2.g ═ 4: 3: 1, transfecting HEK 293T cells for 6-8 h, and then replacing the cells with a DMEM medium containing 15% fetal calf serum. (psPAX 2: Addgene official gazette No. 12260; pMD2. G: Addgene official gazette No. 12259, LentiCas9-Blast, Addgene official gazette no # 52962).
(3) And (3) virus harvesting: after transfection for 36-48 h, cell culture supernatant can be collected, cell debris is removed by filtering with a 0.45 μm filter, virus solution is concentrated by a Millipore column, and the virus solution is stored for a long time at-80 ℃ after subpackaging. (0.45 μm filter 4612 from Pall Corporation; concentration column from Millipore ACS510024)
The resulting concentrated virus was used to infect cells:
(1) screening the concentration of the drug: e14 cells are inoculated in a six-well plate at low density, after adherent growth, a screening marker drug Blasticidin is added into the six-well plate according to concentration gradients of 0, 2, 4, 6, 8 and 10 mu g/mL, the survival condition of the cells is observed after 24 hours of addition, and the concentration of 2 mu g/mLBlastidin when the cells are close to 90% death is selected. (Blasticidin is a product of Thermo corporation, cat # 461120)
(2) Lentivirus infected cells: recovering cells needing to construct a cell line E14, inoculating cells with proper density in a 6cm culture dish when the growth state is good, replacing the cells with a culture medium containing polybrene with the final concentration of 8 mug/mL after the cells adhere to the wall, adding about 20 mug L of concentrated virus solution, and replacing the cells with a fresh normal culture medium after infecting for 6-8 h.
(3) Drug screening: after 36h of infection, medium containing the selected drug concentration (i.e., 2. mu.g/mLBlastidin) was added for selection until no dead cells appeared. And (3) carrying out passage treatment when the cell density is too high in the screening process, and ensuring that the cell density does not exceed 80% in the screening process. The experimental waste needs high pressure treatment in the whole cell line construction process.
(4) Picking a single clone: and (3) counting cells after the digestion of the cells subjected to drug screening, inoculating 3000-5000 cells into a 10cm culture dish according to the cell growth speed and the cell size, culturing for a week, then, allowing the cells to be visible to the naked eye, picking the monoclone to a 96-well plate, performing amplification culture, and then, collecting a cell sample to perform western blotting identification. (all cell culture dishes used are products of Corning Co.)
LentiCas9-Blast plasmid is packaged by lentivirus, then the virus is purified and concentrated to detect the virus titer and infect wild mouse embryonic stem cells E14, single clones are picked after blasticidin screening, the picked single clones are subjected to western blotting experiment to detect the SpCas9 protein expression level, the results of picking labels E14-Cas9Clone1, 3, 4, 5, 6, 9, 16 and 18 are shown in figure 3, and the SpCas9 protein can be well expressed. Meanwhile, PCR detection is carried out by using the primers for detecting the sequence of SpCas9 designed in example 1, and the result shows that the cell lines of E14-Cas9Clone1, 3, 4, 5, 6, 9, 16 and 18 have bands at the same positions as the positive control LentiCas9-Blast, which indicates that the genome of the cell lines stably integrates the DNA sequence of the SpCas9 protein.
The literature reports that overexpression of SpCas9 protein can generate toxicity on embryonic stem cells and can affect the sternness of the embryonic stem cells, so that clones No. 5, 6, 16 and 18 (marked as E14-SpCas9-5, E14-SpCas9-6, E14-SpCas9-16 and E14-SpCas9-18) with good cell states are selected to detect sternness factors Sox2, Oct4 and Nanog expression conditions, antibodies are used for detecting the Sox2, Oct4 and Nanog expression conditions by using a western blotting experiment, and alkaline phosphatase staining and an embryoid body forming experiment are carried out to detect whether the sternness of the embryonic stem cells is affected. The specific procedure for alkaline phosphatase staining was as follows: (1) cell inoculation: the stem cells with the appropriate cell number are inoculated in a six-well plate, and a relatively low cell density is ensured for staining five days after inoculation and growth, and 200-500 cells are generally used for E14.
(2) Cell fixation: after five days of growth, the medium is discarded, 1mL of PBS is added to each well for washing once, 1mL of 4% paraformaldehyde is added, room temperature fixation is carried out for 1-2 min, and the fixative is discarded.
(3) Cleaning: 1mL of TBST was added, and the mixture was left to stand for 5min and discarded.
(4) Cell staining: FRV: naphthol: water 2: 1: 1, and storing in dark, adding 1mL of staining solution into each well, and dyeing for 15min in dark. (the kit is a product of Millipore company, the product number is SCR004)
(5) Cleaning after dyeing: the staining solution was discarded, washed once with 1mL TBST, repeated twice, and PBS was added.
(6) The result of the staining was that cells with dry color were stained purple red, and differentiated cells were not stained or stained very light in color.
Embryoid body formation experiments the procedure was to collect the cells for cell counting by digestion and dilute the cells to 2X 10 using LIF-free E14 cell culture medium7mL, and the cells were added to a low-adsorption 96-well plate at 100 μ L per well, and then placed in a cell incubator for culture, and the culture medium was supplemented every other day until the EB sphere was observed and collected for examination on the seventh day, to examine whether the dryness of the embryonic stem cells was affected. The sternness factors in stem cells such as Sox2, Oct4 and Nanog play an important role in maintaining the sternness of stem cells, and whether stem cells are differentiated or not and whether the sternness of stem cells is changed or not can be objectively judged by detecting the expression condition of the sternness factors. Alkaline phosphatase staining is a well-known experimental method for effectively identifying the dryness of stem cells, and undifferentiated stem cells can highly express alkaline phosphatase. Alkaline phosphatase catalyzes phosphodiester bonds in an alkaline environment, which allows the substrate in the detection reagent to hydrolyze and develop color reaction, while the differentiated cells do not develop color or have weak color. The embryoid body experiment simulates the development process of in vivo embryos, and embryoid bodies are difficult to form by culturing embryonic stem cells without dryness. Through the three experiments, whether the stable expression of the SpCas9 protein in the E14 cell influences the cell dryness of the E14 cell can be effectively verified.
Detecting whether the cell dryness of the constructed E14-SpCas9 cell line is changed or not, and detecting the expression conditions of cell dryness factors Nanog, Oct4 and Sox2 by a western blotting experiment, wherein the result is shown in figure 4, wherein the result of E14-WT shows that the expression levels of three dryness factor proteins are not changed compared with the wild type for the monoclones marked as E14-Cas9Clone 5, E14-Cas9Clone 6, E14-Cas9Clone 16 and E14-Cas9Clone 18; the result of the alkaline phosphatase staining experiment is shown in fig. 5, and all four cell lines can be stained to a deeper degree; the results of the embryoid body formation experiment are shown in FIG. 6, which shows that all four cell lines can form embryoid bodies and have complete stem cell differentiation functions. (wherein Nanog is a product of BETHYL, Cat. No. A300-397A; Oct4 is a product of Santa Cruz Biotech, Cat. No. sc-5279; Sox2 is a product of Millipore, Cat. No. AB 5603; alkaline phosphatase is a product of Millipore, Cat. No. SCR004)
The expression of stem cell sternness factors of the four cell lines was tested (fig. 4), compared with the wild-type E14 cell, the protein expression levels of the four cell lines Nanog, Oct4 and Sox2 were not changed, and the expression levels of SpCas9 protein of the four cell lines were not consistent under the condition of short development exposure time. Alkaline phosphatase staining of four cell lines (fig. 5) it was observed that all four cell lines were stained to the same extent as wild type E14 cells. Embryoid body formation experiments (FIG. 6) were also observed with formation of embryoid bodies in all four cell lines. Therefore, it is considered that the cell sternness of the E14-SpCas9 stable expression cell line is not different from that of the wild-type E14 cell, or strictly speaking, the SpCas9 protein does not influence the cell sternness of the embryonic stem cell in a short time.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in other forms, and any person skilled in the art can change or modify the technical content disclosed above into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (10)
1. A method for constructing a white cell line stably expressing SpCas9 protein, which is characterized by comprising the following steps:
s1, HEK 293T cell preparation: selecting cells with clear sources and good growth states, and treating the recovered cells for two generations by using a mycoplasma inhibitor to ensure the cell states;
s2, transfection packaging plasmid: transfection was performed according to the cation-mediated cell transfection method, and the ratio of virus-packaging plasmids was LentiCas 9: psPAX 2: pmd2.g ═ 4: 3: 1, transfecting cells according to a mass ratio, replacing a culture medium after 6-8 h of transfection, and selecting a proper culture medium for liquid replacement according to a cell line to be constructed;
s3, virus collection: after transfection is carried out for 36-48 h, cell culture supernatant can be collected, a 0.45-micron filter is used for filtering to remove cell debris, and a Millipore column is used for concentrating virus liquid to obtain purified concentrated slow virus liquid;
s4, inoculating the cells of the cell line to be constructed in a six-well plate at low density, setting a concentration gradient of a screening marker medicament Blasticidin after the cells grow adherent to the six-well plate, adding the concentration gradient into the six-well plate for 24h, observing the survival condition of the cells, and selecting the concentration of the cells when the cells die by nearly 90%;
s5, lentivirus-infected cells: resuscitating cells needing to construct a cell line, inoculating the cells in a culture dish when the growth state is good, replacing the cells with a culture medium containing polybrene with the final concentration of 8 mug/mL after the cells are attached to the wall, adding concentrated virus solution, and replacing the cells with a fresh normal culture medium after infection;
s6, drug screening: after infection culture for a period of time, adding a culture medium containing the concentration of the screening drug selected in the step S4 for screening until no dead cells appear;
s7, selecting a single clone: and (3) counting cells after the digestion of the cells subjected to drug screening, culturing for one week to obtain macroscopic cell clones, picking the monoclone to a 96-well plate, carrying out amplification culture, and collecting a cell sample for identification.
2. The method of claim 1, wherein in step S1, the mycoplasma inhibitor is treated with Biomyc-3;
in step S2, the cation-mediated cell transfection method employs the following steps:
inoculating cells with proper density before transfection;
secondly, performing transfection when the cell density reaches 70-90%, and replacing the culture medium 1h before transfection;
③ sucking 100 mu L of 0.9 percent sterile normal saline into a 1.5mL EP tube, and adding 5 mu g of plasmid to be transfected;
fourthly, sucking 100 mu L of 0.9 percent sterilized normal saline into a 1.5mL EP tube again, adding 2 mu L of VigoFect transfection reagent, mixing evenly and standing for 5min at room temperature;
fifthly, dropwise adding the VigoFect diluted in the fourth step into the plasmid in the third step, and standing for 15min at room temperature;
sixthly, uniformly dripping the mixed solution after standing into a cell culture medium and uniformly mixing;
seventhly, the culture solution is replaced after transfection for 6-8 hours.
3. The method of claim 1, wherein in step S4, the low density seeding is performed such that the density of the cells after seeding adherence reaches 30-40%; the concentration gradient of the Blasticidin is set to be 0, 2, 4, 6, 8 and 10 mu g/mL;
in step S5, the infection time is 6-8 h before changing to a fresh normal culture medium.
4. The construction method according to claim 1, wherein in step S6, the infection culture period is 32-48 h, and when the cell density is too high in the screening process, the cell density is ensured not to exceed 80%;
in step S7, 3000 to 5000 cells are seeded in a 10cm dish.
5. The method of claim 1, wherein in step S8, the identification is performed by western blotting detection of expressed SpCas9 protein or PCR detection of the genome of the cell using primers that detect the SpCas9 sequence.
6. The method of construction of claim 5, wherein the primers for detecting the sequence of SpCas9 comprise a forward primer ATGGACAAGAAGTACAGCATCGG and a reverse primer GTCGCCTCCCAGCTGAGAC.
7. The method of claim 5, wherein the PCR amplification conditions are 94 ℃ pre-denaturation for 10 min; denaturation at 94 ℃ for 15s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1kb/min, and 25-30 cycles; extension at 72 ℃ for 10 min.
8. The method of claim 1, wherein the cell line to be constructed is a mouse myoblast cell C2C12 cell or a mouse embryonic stem cell E14 cell.
9. The mouse myoblast C2C12 stably expresses SpCas9 protein white cell line, and is obtained by constructing the cell line needing to be constructed into the mouse myoblast C2C12 cell by the construction method as claimed in claim 1.
10. A mouse embryonic stem cell E14 stably expresses SpCas9 protein leukocyte line, which is obtained by constructing a cell line to be constructed as a mouse embryonic stem cell E14 cell by the construction method as claimed in claim 1.
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