CN113234684A - LLC-PK1 cell line for stably expressing Cas9 protein and construction method thereof - Google Patents
LLC-PK1 cell line for stably expressing Cas9 protein and construction method thereof Download PDFInfo
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Abstract
The invention discloses an LLC-PK1 cell line for stably expressing Cas9 protein and a construction method thereof, wherein LLC-PK1 cells are obtained by transfecting a lentiviral expression vector containing Cas9 by taking a pig proximal tubular epithelial cell LLC-PK1 as a host cell, and screening by puromycin of 5 mu g/mL to obtain a transformant capable of stably expressing Cas9 protein, and the construction method comprises the following steps: preparing pLentiCRISPRV2-Blast endotoxin-free plasmid; step two, packaging the lentivirus; step three, cell transfection; step four, multi-cell cloning and screening; step five, single cell cloning and screening; the LLC-PK1-Cas9 over-expression positive polyclonal cell strain screened by the invention comprises a complete Cas9 gene sequence, and the LLC-PK1-Cas9EDG120 over-expression monoclonal cell strain successfully expresses Cas9 protein.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to an LLC-PK1 cell line for stably expressing Cas9 protein and a construction method thereof.
Background
Compared with the traditional gene knockout method, the CRISPR-Cas9 is a new-generation gene editing means, and has the advantages that the CRISPR-Cas9 is not introduced with an exogenous DNA fragment except for higher efficiency and specificity, and the obtained cell line or animal individual does not contain the exogenous DNA fragment; LLC-PK1 cells, namely porcine proximal tubular epithelial cells, can grow adherently, can be passaged stably under normal conditions, LLC-PK1 cells have wide research in the fields of medicine, agriculture and the like, are good cell models, and in addition, LLC-PK1 cells are good in vitro virus susceptible cells and can be well adapted to infection of various viruses, and the existing genetic engineering lacks a method for constructing an LLC-PK1 cell line for stably expressing Cas9 protein.
Disclosure of Invention
The invention aims to provide an LLC-PK1 cell line capable of stably expressing Cas9 protein and a construction method thereof, so as to construct an LLC-PK1 cell line capable of stably expressing Cas9 protein.
In order to achieve the purpose, the invention provides the following technical scheme: LLC-PK1 cell line for stably expressing Cas9 protein, LLC-PK1 cell is a swine proximal tubular epithelial cell LLC-PK1 as a host cell, a lentiviral expression vector containing Cas9 is transfected, and a transformant capable of stably expressing Cas9 protein is obtained by screening 5 mu g/mL puromycin.
The construction method of the LLC-PK1 cell line for stably expressing the Cas9 protein comprises the following steps: preparing pLentiCRISPRV2-Blast endotoxin-free plasmid; step two, packaging the lentivirus; step three, cell transfection; step four, multi-cell cloning and screening; step five, single cell cloning and screening;
wherein the first step comprises the following steps:
1) centrifuging 10mL of bacterial solution at room temperature and 5000rpm to collect thalli, and sucking supernatant as much as possible;
2) adding 3mL of solution P1, and suspending the bacterial pellets;
3) adding 3mL of solution P2, immediately and gently turning up and down for 6-8 times, and standing at room temperature for 5 min;
4) adding 3mL of solution P4, immediately and gently turning up and down for 6-8 times, fully and uniformly mixing until white flocculent precipitate appears, then standing at room temperature for about 10min, centrifuging at 12000rpm for 10min, and forming precipitate at the bottom of a centrifuge tube;
5) adding the supernatant collected in the last step into a filter column CS in batches, centrifuging for 2min at 12000rpm, and collecting the filtrate in a clean 2ml centrifuge tube;
6) adding a certain amount of isopropanol into the filtrate, turning upside down, mixing uniformly, and transferring into an adsorption column CP 4;
7) centrifuging at 12000rpm for 1min at room temperature, pouring off waste liquid in the collecting tube, and putting the adsorption column back into the collecting tube;
8) adding 500 μ L deproteinized solution PD into adsorption column CP4, centrifuging at 12000rpm for 1min, pouring off waste liquid in the collection tube, and placing adsorption column CP4 into the collection tube;
9) adding 600 μ L of rinsing liquid PW 4 into adsorption column CP4, centrifuging at 12000rpm for 1min, pouring off waste liquid in the collection tube, and placing adsorption column CP4 into the collection tube;
10) adding 600 μ L of rinsing liquid PW 4 into adsorption column CP4, centrifuging at 12000rpm for 1min, and removing waste liquid in the collection tube;
11) the adsorption column CP4 is replaced into the collection tube again, and is centrifuged at 12000rpm for 2min, so that residual rinsing liquid in the adsorption column is removed;
12) placing adsorption column CP4 in a clean centrifuge tube, suspending and dripping 100 μ L elution buffer TB into the middle part of the adsorption membrane, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, collecting plasmid solution in the centrifuge tube, and storing at-20 deg.C;
13) measuring the plasmid concentration by a Qubit nucleic acid protein quantifier, and adjusting the plasmid concentration to 1.0 mug/muL;
wherein in the second step, the method comprises the following steps:
1) culturing 293T cells to ensure that the cells are in a vigorous growth state;
2) before virus packaging, discarding the old culture solution, adding a new culture medium, and culturing for 5h in an incubator;
3) preparing a plasmid mixed solution;
4) preparing PEI-Opti-MEM;
5) respectively adding PEI-Opti-MEM into the plasmid mixed solution, uniformly mixing the walls of the flicked tubes, and standing and incubating for 15min at room temperature;
6) dropwise adding the mixed liquid in the previous step into a culture medium, gently shaking and shaking uniformly, and continuously culturing in an incubator for 18 h;
7) after 18h, the medium was aspirated off and 5mL of a freshly prepared complete medium was added;
8) culturing for 48h, and collecting culture supernatant;
9) centrifuging the supernatant, filtering with a filter membrane, and quickly freezing and storing with liquid nitrogen;
wherein in the third step, the method comprises the following steps:
1) preparing a complete culture medium containing Polybrene;
2) LLC-PK1 cells were digested to prepare a 1.5X 105 cells/well cell suspension;
3) adding 500 mu L of virus solution into a six-hole plate, immediately adding LLC-PK1 cells, and shaking up;
4) standing and culturing the cells in an incubator for 48 hours;
wherein in the fourth step, the method comprises the following steps:
1) after cell transfection, the complete medium containing the blicidin S was changed, followed by one change 1-2 d;
2) after screening for 5d, the cells of the control group are all dead, and the positive cells of the experimental group are subjected to expanded culture; wherein in the fifth step, the method comprises the following steps:
1) counting cells, and diluting the cell suspension to 5 cells/mL;
2) after uniformly mixing, adding 100 mu L of cell suspension into each hole of a 96-hole plate, and culturing in an incubator;
3) after the static culture, the complete medium containing puromycin was replaced, and the formation of monoclonals was observed and recorded daily.
Preferably, in the step one 2), the solution P1 contains RNase A.
Preferably, in the step one 6), the amount of the isopropanol is 0.3 times of the volume of the filtrate.
Preferably, in the first step 11), the adsorption column CP4 is left at room temperature for several minutes, and the residual rinsing liquid in the adsorption material is dried.
Preferably, in the second step 9), the pore diameter of the filter membrane is 0.45 μm, and the storage temperature is-80 ℃.
Preferably, in the step three 1), the concentration of Polybrene in the complete medium is 10. mu.g/mL.
Preferably, in the step five 3), the concentration of puromycin in the complete culture medium is 5 μ g/mL.
Compared with the prior art, the invention has the beneficial effects that: the LLC-PK1-Cas9 over-expression positive polyclonal cell strain screened by the invention contains a complete Cas9 gene sequence, and the LLC-PK1-Cas9EDG120 over-expression monoclonal cell strain successfully expresses Cas9 protein.
Drawings
FIG. 1A is a diagram showing the PCR results of pLentiCRISPRV2-Blast endotoxin free plasmid;
FIG. 1B is a plasmid map of psPAX2(Addgene # 12260);
FIG. 1C is a plasmid map of pMD2.G (Addgene # 12259);
FIG. 2A is a schematic diagram of a six-well plate in LLC-PK1 cell Blasticidin S resistance concentration exploration experiments;
FIG. 2B is a schematic diagram of a plate paving in an experiment for investigating the resistance concentration of LLC-PK1 cells to Puromycin;
FIG. 3A is a PCR reaction result diagram of Cas9 gene sequence of a positive polyclonal cell strain;
FIG. 3B is a diagram showing the sequencing result of Cas9 gene sequence of the positive polyclonal cell strain;
FIG. 4A is a PCR reaction result diagram of Cas9 gene of positive monoclonal cell strain;
FIG. 4B is a graph showing the result of WB positive monoclonal cell line;
FIG. 5 is a graph of LLC-PK1-Cas9 cell growth;
FIG. 6 is a flow chart of a method of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1A-6, an embodiment of the present invention is shown: LLC-PK1 cell line for stably expressing Cas9 protein, LLC-PK1 cell is porcine proximal tubular epithelial cell LLC-PK1 as host cell, a lentivirus expression vector containing Cas9 is transfected, and a transformant capable of stably expressing Cas9 protein is obtained by screening 5 mu g/mL puromycin.
The construction method of the LLC-PK1 cell line for stably expressing the Cas9 protein comprises the following steps: preparing pLentiCRISPRV2-Blast endotoxin-free plasmid; step two, packaging the lentivirus; step three, cell transfection; step four, multi-cell cloning and screening; step five, single cell cloning and screening;
in the first step, SpCas9 gene is amplified from pX330(Addge #42230), a fusion fragment of EFS (SpCas 9: Flag: P2A: Blast) for expressing Cas9 gene and Blasticidin (Blasticidin) resistance gene is constructed, and a lentiviral expression vector pLentiCRISPRV2-Blast containing Cas9 is inserted into a lentiviral vector to obtain a lentiviral expression vector pLentiCRISPRV2-Blast (FIG. 1A); and the packaging plasmid psPAX2(Addgene #12260, figure 1B) and the envelope plasmid pMD2.G (Addgene #12259, figure 1C) are purchased to form a lentivirus packaging system, and the specific operation is as follows:
1) centrifuging 10mL of bacterial solution at room temperature and 5000rpm to collect thalli, and sucking supernatant as much as possible;
2) adding 3mL of solution P1, and suspending the bacterial pellet, wherein the solution P1 contains RNaseA;
3) adding 3mL of solution P2, immediately and gently turning up and down for 6-8 times, and standing at room temperature for 5 min;
4) adding 3mL of solution P4, immediately and gently turning up and down for 6-8 times, fully and uniformly mixing until white flocculent precipitate appears, then standing at room temperature for about 10min, centrifuging at 12000rpm for 10min, and forming precipitate at the bottom of a centrifuge tube;
5) adding the supernatant collected in the last step into a filter column CS in batches, centrifuging for 2min at 12000rpm, and collecting the filtrate in a clean 2ml centrifuge tube;
6) adding isopropanol with the volume of 0.3 time of that of the filtrate into the filtrate, turning the filtrate upside down and uniformly mixing the filtrate, and transferring the mixture into an adsorption column CP 4;
7) centrifuging at 12000rpm for 1min at room temperature, pouring off waste liquid in the collecting tube, and putting the adsorption column back into the collecting tube;
8) adding 500 μ L deproteinized solution PD into adsorption column CP4, centrifuging at 12000rpm for 1min, pouring off waste liquid in the collection tube, and placing adsorption column CP4 into the collection tube;
9) adding 600 μ L of rinsing liquid PW 4 into adsorption column CP4, centrifuging at 12000rpm for 1min, pouring off waste liquid in the collection tube, and placing adsorption column CP4 into the collection tube;
10) adding 600 μ L of rinsing liquid PW 4 into adsorption column CP4, centrifuging at 12000rpm for 1min, and removing waste liquid in the collection tube;
11) putting the adsorption column CP4 back into the collection tube again, centrifuging at 12000rpm for 2min to remove the residual rinsing liquid in the adsorption column, placing the adsorption column CP4 at room temperature for several minutes, and drying the residual rinsing liquid in the adsorption material;
12) placing adsorption column CP4 in a clean centrifuge tube, suspending and dripping 100 μ L elution buffer TB into the middle part of the adsorption membrane, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, collecting plasmid solution in the centrifuge tube, and storing at-20 deg.C;
13) measuring the plasmid concentration by a Qubit nucleic acid protein quantifier, and adjusting the plasmid concentration to 1.0 mug/muL;
wherein in the second step, the method comprises the following steps:
1) culturing 293T cells to ensure that the cells are in a vigorous growth state;
2) before virus packaging, discarding the old culture solution, adding a new culture medium, and culturing for 5h in an incubator;
3) preparing a plasmid mixed solution, wherein pLentiCRISPRV2-Blast is 6 mu g, psPAX is 4 mu g, pMD2.G is 1.5 mu g, and Add Opti-MEMto is 250 mu L;
4) preparing PEI-Opti-MEM, wherein 34.5 mu L of PEI and 250 mu L of Add Opti-MEM to are prepared;
5) respectively adding PEI-Opti-MEM into the plasmid mixed solution, uniformly mixing the walls of the flicked tubes, and standing and incubating for 15min at room temperature;
6) dropwise adding the mixed liquid in the previous step into a culture medium, gently shaking and shaking uniformly, and continuously culturing in an incubator for 18 h;
7) after 18h, the medium was aspirated off and 5mL of a freshly prepared complete medium was added;
8) culturing for 48h, and collecting culture supernatant;
9) centrifuging the supernatant, filtering with filter membrane, quick freezing with liquid nitrogen for storage, wherein the aperture of the filter membrane is 0.45 μm, and the storage temperature is-80 deg.C;
wherein in the third step, the method comprises the following steps:
1) preparing a Polybrene complete culture medium containing 10 mu g/mL;
2) LLC-PK1 cells were digested to prepare a 1.5X 105 cells/well cell suspension;
3) adding 500 mu L of virus solution into a six-hole plate, immediately adding LLC-PK1 cells, and shaking up;
4) standing and culturing the cells in an incubator for 48 hours;
polyclonal cell screening concentration exploration: inoculate 1.5X 10 per well in 6-well plates52mL of complete medium per well; after standing and culturing for 48h, adding complete culture medium containing different concentrations of Blasticidin S as shown in FIG. 2A, and continuously culturing in a cell culture box; replacing the culture medium containing the Blasticidin S every 24-48h, and observing the growth and survival conditions of the cells; after screening for 5 days, trypan blue (0.04%) staining is carried out, the cell growth condition is observed under a microscope, and the cell survival condition is counted; the experimental result shows that the polyclonal cell of LLC-PK1 dies completely in 5 days under the concentration of 12 mu g/mL;
screening concentration of monoclonal cells was investigated: LLC-PK1 cell counts, plated as in FIG. 2B; culturing for 24h after plating, observing the adherent growth condition of cells, and adding 100 mu L of Puromycin with different concentrations into each hole; the culture medium is replaced every two days (48 h); observing and recording the change of the cells under different concentrations and different densities every day; after 5 days of administration, the culture solution is discarded, trypan blue is used for staining for 1-2min, and the survival condition of the cells is observed under a microscope; the experimental result shows that the monoclonal cells of LLC-PK1 die completely in 5 days at the concentration of 5 mu g/mL;
wherein in the fourth step, the method comprises the following steps:
1) 48h after transfection of the cells, the complete medium containing 12. mu.g/mLBlastidin S was replaced, followed by one replacement 1-2 d;
2) after screening for 5d, completely dying cells in a control group, carrying out amplification culture on positive cells in an experimental group, extracting nucleic acid of the positive cells in the experimental group, carrying out PCR reaction, amplifying a Cas9 fragment, wherein the result is shown in figure 3A, and sending a PCR product for sequencing, and the result is shown in figure 3B;
wherein in the fifth step, the method comprises the following steps:
1) counting cells, and diluting the cell suspension to 5 cells/mL;
2) after uniformly mixing, adding 100 mu L of cell suspension into each hole of a 96-hole plate, and culturing in an incubator;
3) after static culture, replacing a puromycin complete culture medium containing 5 mu g/mL, and observing and recording the formation condition of monoclone every day;
after 5 days, amplifying the positive monoclonal obtained by screening, performing PCR amplification on a Cas9 gene fragment of the positive monoclonal cell strain, and performing sample-taking sequencing on a PCR product, wherein the result is as follows: all the EDG115-EDG1217 LLC-PK1-Cas9 over-expression positive monoclonal cell strains have complete Cas9 gene fragments, wherein the positive is pLentiCRISPRV2-Blast vector amplification products;
the over-expression positive monoclonal cell strain verification is verified by Western blot, and the specific details are as follows: extracting total protein of positive monoclonal cell with RIPA lysate, determining protein concentration of each sample by BCA method, wherein the protein loading amount is 40 μ g, and primary antibody
The secondary antibody (goat-anti-mouse) in 3% skimmed milk powder solution is 1:10000, and the result is shown in fig. 4B, the size of the target protein Cas9 is about: 163.2 kDa; WB results show that LLC-PK1-Cas9EDG120 overexpression monoclonal cell strain successfully expresses Cas9 protein;
LLC-PK1-Cas9 cell growth curve assay for stably expressing Cas9: in order to further compare the growth characteristics of the Cas9 gene before and after cloning to LLC-PK cells, cells with good growth state are taken and digested to prepare single cell suspension, the cells are inoculated into a 24-well plate, added into wells according to the amount of 3000/well and 0.5 mL/well (reference: 500 LLC-PK1 cells in a 96-well plate are full of cells in about 8 days), the growth condition of the cells is recorded by taking pictures at different time points, 3-well cells are collected and counted, the average value is calculated for 3 times, and the result is shown in FIG. 5, and the data result shows that the related growth speed and the amplification period of the Cas9 gene before and after cloning to the LLC-PK cells have no obvious difference.
Based on the above, the method has the advantages that LLC-PK1 cells are cultured to construct a Cas9 gene overexpression polyclonal cell strain, the obtained overexpression positive polyclonal cell strain is subjected to sequencing verification, the obtained sequencing result shows that the polyclonal cell strain contains a complete and correct Cas9 gene, the gene knockout positive polyclonal cell strain is verified, and the determination result shows that LLC-PK1-Cas9 polyclonal cell strain successfully expresses the Cas9 protein.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (8)
1. LLC-PK1 cell line stably expressing Cas9 protein, characterized in that: the LLC-PK1 cell is a transformant which is obtained by transfecting a lentiviral expression vector containing Cas9 by taking a porcine proximal tubular epithelial cell LLC-PK1 as a host cell and screening by puromycin at 5 mu g/mL and can stably express the Cas9 protein.
2. The construction method of the LLC-PK1 cell line for stably expressing the Cas9 protein comprises the following steps: preparing pLentiCRISPRV2-Blast endotoxin-free plasmid; step two, packaging the lentivirus; step three, cell transfection; step four, multi-cell cloning and screening; step five, single cell cloning and screening; the method is characterized in that:
wherein the first step comprises the following steps:
1) centrifuging 10mL of bacterial solution at room temperature and 5000rpm to collect thalli, and sucking supernatant as much as possible;
2) adding 3mL of solution P1, and suspending the bacterial pellets;
3) adding 3mL of solution P2, immediately and gently turning up and down for 6-8 times, and standing at room temperature for 5 min;
4) adding 3mL of solution P4, immediately and gently turning up and down for 6-8 times, fully and uniformly mixing until white flocculent precipitate appears, then standing at room temperature for about 10min, centrifuging at 12000rpm for 10min, and forming precipitate at the bottom of a centrifuge tube;
5) adding the supernatant collected in the last step into a filter column CS in batches, centrifuging for 2min at 12000rpm, and collecting the filtrate in a clean 2ml centrifuge tube;
6) adding a certain amount of isopropanol into the filtrate, turning upside down, mixing uniformly, and transferring into an adsorption column CP 4;
7) centrifuging at 12000rpm for 1min at room temperature, pouring off waste liquid in the collecting tube, and putting the adsorption column back into the collecting tube;
8) adding 500 μ L deproteinized solution PD into adsorption column CP4, centrifuging at 12000rpm for 1min, pouring off waste liquid in the collection tube, and placing adsorption column CP4 into the collection tube;
9) adding 600 μ L of rinsing liquid PW 4 into adsorption column CP4, centrifuging at 12000rpm for 1min, pouring off waste liquid in the collection tube, and placing adsorption column CP4 into the collection tube;
10) adding 600 μ L of rinsing liquid PW 4 into adsorption column CP4, centrifuging at 12000rpm for 1min, and removing waste liquid in the collection tube;
11) the adsorption column CP4 is replaced into the collection tube again, and is centrifuged at 12000rpm for 2min, so that residual rinsing liquid in the adsorption column is removed;
12) placing adsorption column CP4 in a clean centrifuge tube, suspending and dripping 100 μ L elution buffer TB into the middle part of the adsorption membrane, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, collecting plasmid solution in the centrifuge tube, and storing at-20 deg.C;
13) measuring the plasmid concentration by a Qubit nucleic acid protein quantifier, and adjusting the plasmid concentration to 1.0 mug/muL;
wherein in the second step, the method comprises the following steps:
1) culturing 293T cells to ensure that the cells are in a vigorous growth state;
2) before virus packaging, discarding the old culture solution, adding a new culture medium, and culturing for 5h in an incubator;
3) preparing a plasmid mixed solution;
4) preparing PEI-Opti-MEM;
5) respectively adding PEI-Opti-MEM into the plasmid mixed solution, uniformly mixing the walls of the flicked tubes, and standing and incubating for 15min at room temperature;
6) dropwise adding the mixed liquid in the previous step into a culture medium, gently shaking and shaking uniformly, and continuously culturing in an incubator for 18 h;
7) after 18h, the medium was aspirated off and 5mL of a freshly prepared complete medium was added;
8) culturing for 48h, and collecting culture supernatant;
9) centrifuging the supernatant, filtering with a filter membrane, and quickly freezing and storing with liquid nitrogen;
wherein in the third step, the method comprises the following steps:
1) preparing a complete culture medium containing Polybrene;
2) LLC-PK1 cells were digested to prepare 1.5X 10 cells5cells/well cell suspension;
3) adding 500 mu L of virus solution into a six-hole plate, immediately adding LLC-PK1 cells, and shaking up;
4) standing and culturing the cells in an incubator for 48 hours;
wherein in the fourth step, the method comprises the following steps:
1) after cell transfection, the complete medium containing the blicidin S was changed, followed by one change 1-2 d;
2) after screening for 5d, the cells of the control group are all dead, and the positive cells of the experimental group are subjected to expanded culture;
wherein in the fifth step, the method comprises the following steps:
1) counting cells, and diluting the cell suspension to 5 cells/mL;
2) after uniformly mixing, adding 100 mu L of cell suspension into each hole of a 96-hole plate, and culturing in an incubator;
3) after the static culture, the complete medium containing puromycin was replaced, and the formation of monoclonals was observed and recorded daily.
3. The method of claim 2 for constructing LLC-PK1 cell line stably expressing Cas9 protein, wherein: in the step one 2), the solution P1 contains RNase A.
4. The method of claim 2 for constructing LLC-PK1 cell line stably expressing Cas9 protein, wherein: in the first step 6), the amount of isopropanol is 0.3 times the volume of the filtrate.
5. The method of claim 2 for constructing LLC-PK1 cell line stably expressing Cas9 protein, wherein: in the first step 11), the adsorption column CP4 is placed at room temperature for several minutes, and the residual rinsing liquid in the adsorption material is dried.
6. The method of claim 2 for constructing LLC-PK1 cell line stably expressing Cas9 protein, wherein: in the second step 9), the aperture of the filter membrane is 0.45 μm, and the storage temperature is-80 ℃.
7. The method of claim 2 for constructing LLC-PK1 cell line stably expressing Cas9 protein, wherein: in the step three 1), the concentration of Polybrene in the complete medium is 10. mu.g/mL.
8. The method of claim 2 for constructing LLC-PK1 cell line stably expressing Cas9 protein, wherein: in the step five 3), the concentration of puromycin in the complete culture medium is 5 mug/mL.
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| CN105907721A (en) * | 2016-05-13 | 2016-08-31 | 广东省农业科学院农业生物基因研究中心 | Pig intestinal endothelial cell line for stably expressing CaS9 protein |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105907721A (en) * | 2016-05-13 | 2016-08-31 | 广东省农业科学院农业生物基因研究中心 | Pig intestinal endothelial cell line for stably expressing CaS9 protein |
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