CN112522206A - Construction method and application of ROR1 gene knockout tumor cell strain - Google Patents
Construction method and application of ROR1 gene knockout tumor cell strain Download PDFInfo
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Abstract
The application relates to the field of cell culture, and particularly discloses a construction method and application of a ROR1 gene knockout tumor cell strain. The construction method of the ROR1 gene knockout tumor cell strain comprises the following steps: inoculating the tumor cells into a pore plate for culturing; transfecting a cell; cell surface staining, screening ROR1 negative cell population by a flow cytometry sorter, and inoculating the screened ROR1 negative cell population into a pore plate for continuous culture; and (4) performing surface staining again, screening again, and continuously culturing to obtain the ROR1 gene knockout tumor cell strain. The ROR1 gene knockout tumor cell strain constructed by the application can be used for verifying the specific killing effect of ROR1-CAR-T cells on target cells. The construction method can effectively shorten the time for constructing the ROR1 gene knockout tumor cell strain, and can obtain the ROR1 gene knockout tumor cell strain with stable inheritance for killing analysis of targeted tumor cells.
Description
Technical Field
The application relates to the field of cell culture, in particular to a construction method and application of a ROR1 gene knockout tumor cell strain.
Background
Receptor tyrosine kinase-like orphan receptor 1 (ROR 1) is an important member of type I receptor tyrosine kinase family, and is involved in the processes of intercellular signal exchange, intracellular signal transduction and the like, and the regulation of cell proliferation, differentiation and transfer. ROR1 is expressed in embryonic tissues and not expressed in adult normal tissues, however, when tumors appear, ROR1 is highly expressed in tumor tissues, plays an important role in tumor cell proliferation, metastasis, drug resistance and the like, and has great application potential as a target of cancer treatment drugs, particularly CAR-T cell treatment targeting solid tumors.
ROR1 genes on tumor cells are knocked out by using a CRISPR/Cas9 technology, and the killing capability of ROR1-CAR-T cells is judged by comparing the killing capability of ROR1-CAR-T cells on the tumor cells and the killing capability of ROR1 gene knocked out tumor cells, so that a new research platform can be provided for targeted tumor killing research.
When constructing ROR1 gene knockout tumor cell strain, the cloning culture technology of the cell usually adopts a limiting dilution method, the principle is that the cell strain to be cloned is sucked out from a culture hole and used for cell counting, the cell strain is diluted by HT culture solution and then inoculated into the culture hole, the rest cell suspension is diluted by HT culture solution in a multiple ratio and then inoculated into the rest culture hole; after culturing for a certain time, selecting the positive hole for single clone growth to clone again, and repeating the operation until the positive hole rate reaches 100 percent.
In view of the above-mentioned related art, the inventors considered that the screening of ROR 1-negative cell lines by limiting dilution requires a long time for culturing the cell lines for 2 to 3 weeks, which takes a long time.
Disclosure of Invention
In order to improve the efficiency of screening cell strains, the application provides a construction method and application of a ROR1 gene knockout tumor cell strain.
In a first aspect, the application provides a method for constructing a ROR1 gene knockout tumor cell strain, which adopts the following technical scheme:
a construction method of ROR1 gene knockout tumor cell strains comprises the following steps:
s1, cell culture: inoculating the tumor cells into a pore plate for culture and proliferation;
s2, transfection: transfecting tumor cells by using a Cas9 protein and a gRNA through a transfection reagent;
s3, first screening: surface staining is carried out on transfected cells, ROR1 negative cell populations are screened out through a flow cytometry sorter, and the screened ROR1 negative cell populations are inoculated into a pore plate for continuous culture;
s4, secondary screening: and (3) surface staining the cells in S3, screening ROR1 negative cell groups by a flow cytometry, inoculating a single ROR1 negative cell/hole into a pore plate, and continuing culturing for 45-50h to obtain the ROR1 gene knockout tumor cell strain.
By adopting the technical scheme, the method of limiting dilution and flow cytometry separation is combined, so that the time for constructing the ROR1 gene knockout tumor cell strain can be effectively shortened, and the ROR1 gene knockout tumor cell strain with stable inheritance can be obtained for killing analysis of the targeted tumor cells.
Preferably, the mass ratio of the Cas9 protein to the gRNA in the step S2 is (4.8-5.3): 1.
By adopting the technical scheme, cells are transfected by using the Cas9 protein and the gRNA in a specific ratio, so that the ROR1 gene can be directionally cut by guiding the Cas9 protein to the tumor cells through the gRNA, and the possibility of off-target effect is reduced.
Preferably, in step S2, the transfection is performed when the tumor cells in the well plate proliferate to 50-70% confluence.
By adopting the technical scheme, the transfection reagent generally has certain toxicity to cells, transfection is carried out when the confluence degree is low, the tolerance of the cells to the transfection reagent is low, and the subsequent culture and amplification of the cells can be influenced; transfection is performed when the confluency is too high, and although the tolerance of cells to the transfection reagent is high, the confluency is too high, which affects subsequent cell screening and reduces the transfection efficiency. By performing transfection when the degree of confluence of cells reaches a value within a specific range, the tolerance of the cells to the transfection reagent can be improved to some extent, and the possibility of the damage of cell activity due to the overgrowth of the cells can be reduced.
Preferably, in step S2, the transfection comprises the following steps:
(1) preparation of solution i: 1152-1272ng Cas9 protein, 240ng gRNA, 2.4-2.6 mu L Plus chamber Reagent and 24-26 mu L transfection medium are mixed evenly to obtain solution I;
(2) preparation of solution II: uniformly mixing 24-26 mu L of transfection medium and 1.4-1.6 mu L of transfection reagent to obtain solution II;
(3) dropwise adding the solution I into the solution II, uniformly mixing, and incubating at 23-27 ℃ for 8-12min to obtain a solution III;
(4) adding the solution III into a pore plate, and placing the pore plate in a carbon dioxide incubator to incubate, wherein the carbon dioxide incubator contains 5% of carbon dioxide and the temperature is 36-38 ℃.
By adopting the technical scheme, the combination of the Plus-responsive Reagent and the transfection Reagent can improve the transfection efficiency and the transfection effect; the solution I and the solution II are respectively prepared, and the solution I and the solution II are mixed before incubation, so that the influence of a transfection reagent on Cas9 protein can be reduced when Cas9 protein and the transfection reagent are directly mixed, and the Cas9 protein can be ensured to have better activity to a certain extent; incubation at a specific temperature to prepare solution iii, facilitating the formation of a complex of Cas9 protein, gRNA, and transfection reagent; incubation of cells at specific temperatures facilitates uptake of the complex by the cells, thereby enabling Cas9 protein to cleave the ROR1 gene in a targeted manner on tumor cells under the guidance of grnas.
Preferably, in step S3, the cell surface staining method includes the following steps:
(1) digesting the tumor cells by pancreatin, and counting the cells;
(2) take 0.8 x 105-0.9*105Loading the cells into a flow cell tube, adding buffer solution for washing once, and addingMouse anti-human ROR1 monoclonal antibody;
adding isotype control antibody mouse IgG1 into another control tube, incubating on ice for 18-22min, and washing with buffer solution for 2-3 times;
(3) adding a secondary antibody goat anti-mouse IgG (H + L) -FITC into the flow cell tube and the control tube in the step (2), placing on ice, incubating for 18-22min, and washing for 2-3 times by using a buffer solution;
(4) screening the cells incubated in (3) by using a flow sorting cytometer, collecting a ROR1 negative cell population, and inoculating the cell population into a pore plate for culture and amplification.
By adopting the technical scheme, the mouse anti-human ROR1 monoclonal antibody can specifically recognize and bind ROR1 gene on tumor cells, meanwhile, the secondary goat anti-mouse IgG (H + L) -FITC can be specifically bound with the mouse anti-human ROR1 monoclonal antibody, FITC (fluorescein isothiocyanate) can be bound with Cas9 protein in cells to present bright yellow green fluorescence, and therefore ROR1 negative cell populations can be screened by using a flow cytometer.
In a second aspect, the application provides a ROR1 gene knockout tumor cell strain, which adopts the following technical scheme:
a ROR1 gene knockout tumor cell strain is constructed by adopting a construction method of ROR1 gene knockout tumor cell strain.
In a third aspect, the application provides an application of a ROR1 gene knockout tumor cell strain, and the following technical scheme is adopted:
an application of ROR1 gene knockout tumor cell strain is used for verifying the specific killing effect of ROR1-CAR-T cells on target cells.
In summary, the present application has the following beneficial effects:
1. the method screens the ROR1 gene knockout tumor cell strains in a mode combining flow sorting and limiting dilution, effectively saves the operation period of screening the ROR1 gene knockout tumor cell strains, and can obtain stably inherited ROR1 gene knockout tumor cell strains.
2. The ROR1 gene knockout tumor cell strain obtained by the application can escape killing of CAR-T cells, and a new research platform is provided for tumor killing research.
Drawings
Fig. 1 is a flow cytometric assay for ROR1 protein expression of example 1 of the present application;
FIG. 2 is an immunofluorescence map of ROR1 protein expression of example 1 of the present application;
fig. 3 is a proliferation map of wild-type a549 cells and ROR1 gene knock-out a549 cells of example 1 of the present application;
fig. 4 is a graph of in vitro killing of wild-type a549 cells and ROR1 gene knockout a549 cells by ROR1-CAR-T cells of example 2 of the present application.
Detailed Description
The present application will be described in further detail with reference to the following drawings and examples.
The A549 cell is a human lung adenocarcinoma cell, belongs to a continuous cell line, and can be stably subcultured. The ROR1 protein is highly expressed on the surface of the A549 cell, and is a target cell or a tool cell which is good in killing capability in vivo and in vitro of a CAR-T cell (namely ROR1-CAR-T cell) targeting ROR1 protein, and how to quickly and effectively construct a ROR1 gene knockout A549 cell strain is researched on the basis of the application.
The sources of the raw materials in the present application are shown in table 1:
TABLE 1 sources of the respective raw materials
Preparation example of gRNA
A gRNA capable of targeting ROR1 gene in this application was synthesized by a method described in "establishment of a method for rapidly synthesizing and detecting a gRNA" (proceedings of Experimental animals in China, 6.2015, vol.23, No. 3). The gRNA is synthesized by sgRNA targeting ROR1 gene and tracrRNA, wherein the sequence of sgRNA targeting ROR1 gene is AACCTCGACACCACAGACAC.
Examples
Example 1
A construction method of ROR1 gene knockout tumor cell strains specifically comprises the following steps:
s1, cell culture: taking healthy A549 cells, and culturing at 2 × 105Laying ROR1 negative cells/hole in a 24-hole plate for culture and proliferation;
s2, transfection: transfection was initiated when cells in the 24-well plates had proliferated to 60% confluence by the following procedure:
(1) preparation of solution i: 1250ng Cas9 protein, 240ng gRNA, 2.5 mu L Plus chamber Reagent and 25 mu L Opti-MEM transfection medium are taken and mixed uniformly to obtain a solution I;
(2) preparation of solution II: taking 25 mu L of Opti-MEM transfection medium and 1.5 mu L of CRISPRMAX ™ reaction transfection Reagent, and mixing uniformly to obtain a solution II;
(3) dropwise adding the solution I into the solution II, uniformly mixing, and incubating at 25 ℃ for 10min to obtain a solution III;
(4) adding the solution III into a 24-well plate according to the concentration of 50 mu L/well, and incubating for 48h in a carbon dioxide incubator at the temperature of 37 ℃ and the carbon dioxide content of 5%.
S3, first screening: collecting transfected cells for cell surface staining, and specifically operating as follows:
(1) cell counts were performed 1min after digestion of a549 cells with pancreatin:
(2) according to 1 x 105Each cell/tube is filled into a BD flow cell tube, the BD flow cell tube is washed once by D-PBS buffer solution, and then a mouse anti-human ROR1 monoclonal antibody with the concentration of 50ug/ml is added, and 10 mu L of each BD flow cell tube is added; adding an equivalent amount of isotype control antibody mouse IgG1 into another control tube, wherein the concentration of the isotype control antibody mouse IgG1 is 50 ug/ml; placing the BD flow cell tube and the control tube on ice for incubation for 20min, and then washing with a D-PBS buffer solution twice;
(3) the BD flow cell tube and the control tube were incubated on ice for 20min and then washed twice with D-PBS buffer to obtain stained cells, wherein the secondary antibody goat anti-mouse IgG (H + L) -FITC was added to the BD flow cell tube and the control tube at a concentration of 50ug/ml, 10. mu.L was added to each tube.
After the cell surface staining was completed, the ROR1 negative cell population was selected by flow cytometry, and ROR1 negative cells were selected as 2 × 105Laying ROR1 negative cells/hole on a 24-hole plate for continuous culture and amplification for 48 h;
s4, secondary screening: and (3) repeating the steps (1), (2) and (3) in the first screening, collecting cells, screening out a ROR1 cell population by using a flow cytometry sorter, and inoculating a single ROR1 negative cell/well into a 96-well plate for continuous culture to obtain a ROR1 gene knockout A549 cell strain.
Further, the ROR1 gene knockout a549 cell strain was confirmed, and after the cells in step S3 were cultured and amplified for 48 hours, cells of 6 96-well plates were randomly selected for flow cytometry identification and confirmation, and the detection results are shown in fig. 1.
Further, re-confirmation of ROR1 negative cells, transferring ROR1 negative a549 cells into a 6-well plate, performing immunofluorescence assay by using a cell slide, and re-confirmation of ROR1 gene knockout, wherein the confirmation results are shown in fig. 2.
Further, ROR1 negative a549 cells and wild type a549 cells of one well plate were randomly selected and compared by a cell proliferation assay to observe the proliferation ability of ROR1 negative a549 cells, which was specifically performed as follows:
1) knocking out A549 cells of A459 cells and ROR1 gene according to 1 x 104Inoculating each cell/hole into a 24-hole plate respectively, wherein each group is provided with three repeated holes;
2) after inoculation, cells were digested with pancreatin 24h, 48h, 72h and 95h after inoculation, 50 μ L of cell suspension was mixed with 50 μ L of 0.4% trypan blue, and added dropwise to a hemocytometer to count viable cells, and the proliferation of cells was counted and analyzed, and the analysis results are shown in fig. 3.
Example 2
An application of a ROR1 gene knockout A549 cell strain is characterized in that the ROR1 gene knockout A549 cell strain is used for verifying the specific killing effect of ROR1-CAR-T cells on target cells, and a lactate dehydrogenase release method is used for verification, wherein the specific verification mode is as follows:
1) collecting wild type A549 cells, ROR1 gene knockout A549 cells and ROR1-CAR-T cells in logarithmic growth phase, and respectively counting for later use;
2) inoculating ROR1-CAR-T cells and wild type A549 cells, ROR1-CAR-T cells and ROR1 gene knockout A549 cells into a 96-well cell culture plate according to the effective target ratio of 1:1, 5:1 and 10:1 for co-culture, and repeating three times in each group; corresponding T cells and wild type A549 cells, T cells and ROR1 gene knockout A549 cells are inoculated in the control holes according to the same proportion for co-culture;
3) 1h before the cell co-culture reaches the preset detection time, taking out the cell culture plate, adding an LDH release reagent provided by a lactate dehydrogenase cytotoxicity detection kit into a sample maximum enzyme activity control hole, wherein the addition amount of the LDH release reagent is 10% of the volume of the original culture solution, repeatedly beating the LDH release reagent for several times, uniformly mixing the LDH release reagent and the LDH release reagent, and then continuously incubating the LDH release reagent in a cell culture box;
4) after the predetermined time is reached, the cell culture plate is centrifuged for 5min at 400g in a multi-well plate centrifuge, 120. mu.L of the supernatant from each well is added to a new 96-well cell culture plate, and then the sample measurement is performed, the measurement results are shown in FIG. 4.
Referring to fig. 1, compared with an isotype control, the peak generated by the ROR1 gene knockout a549 cell obtained in the present application is substantially coincident with the peak generated by the isotype control, and the peak generated by the a549 cell is separated from the peak generated by the isotype control, which indicates that the cell obtained by screening in the present application is the ROR1 gene knockout a549 cell.
Referring to fig. 2, in fig. 2, the transverse first row is an immunofluorescence map obtained after wild-type a549 cells are stained, the transverse second row is an immunofluorescence map obtained after ROR1 gene knockout a549 cells are stained, and after DAPI working solution staining, both the cell nuclei of wild-type a549 cells and the cell nuclei of ROR1 gene knockout a549 cells are stained; after the ROR1 protein is stained, fluorescence is expressed by wild type A549 cells, and fluorescence is not expressed by ROR1 gene knockout A549 cells, which indicates that the ROR1 protein of the ROR1 gene knockout A549 cells obtained by the application is knocked out.
Referring to fig. 3, the proliferation rate of the wild-type a549 cell and the proliferation rate of the ROR1 gene knockout a549 cell obtained in the present application are substantially consistent within a certain culture time, which indicates that the construction method disclosed in the present application can obtain a stably inherited ROR1 gene knockout a549 cell strain, and has no great influence on the proliferation capacity of the ROR1 gene knockout a549 cell strain.
Referring to FIG. 4, compared with the killing effect of T cells on wild-type A549 cells and the killing effect of T cells on ROR1 gene knockout A549 cells, the killing effect of ROR1-CAR-T cells on wild-type A549 cells is higher than that of T cells on wild-type A549 cells under different effect-to-target ratios, the killing effect of ROR1-CAR-T cells on ROR1 gene knockout A549 cells is basically the same as that of T cells on ROR1 gene knockout A549 cells, that is, ROR1-CAR-T cells have no substantial killing effect on ROR1 gene knockout A549 cells because ROR1 gene knockout A549 cells do not have a specific antigen (ROR 1 gene) recognized by ROR1-CAR-T cells, thereby causing ROR1 gene knockout A549 cells not to be activated to release effector molecules to be cracked into target cells, therefore, the ROR1 gene knockout A549 cell strain can be used for verifying the specific killing effect of ROR1-CAR-T cells on target cells.
Compared with the 2-3 weeks consumed for constructing the ROR1 gene knockout tumor cell strain by adopting a limiting dilution method in the related technology, the ROR1 gene knockout tumor cell strain with stable inheritance can be obtained only in 1 week, and the construction time of the cell strain is greatly shortened.
Example 3
This example differs from example 1 only in that: the A459 cells are replaced by NCI-H1975 cells, and the obtained ROR1 gene knockout NCI-H1975 cells are subjected to flow cytometric identification confirmation and immunofluorescence detection. In the flow cell identification confirmation, the result shows that the obtained ROR1 gene knockout NCI-H1975 cell generates a peak which is basically coincident with that generated by an isotype control, and the NCI-H1975 cell generates a peak which is separated from that generated by the isotype control, thereby indicating that the cell screened by the application is the ROR1 gene knockout NCI-H1975 cell. In an immunofluorescence test, fluorescence is expressed by wild-type NCI-H1975 cells, and ROR1 gene knockout NCI-H1975 cells do not show fluorescence, which indicates that the ROR1 protein of the obtained ROR1 gene knockout NCI-H1975 cells is knocked out.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (7)
1. A construction method of ROR1 gene knockout tumor cell strains is characterized by comprising the following steps:
s1, cell culture: inoculating the tumor cells into a pore plate for culture and proliferation;
s2, transfection: transfecting tumor cells by using a Cas9 protein and a gRNA through a transfection reagent;
s3, first screening: surface staining is carried out on transfected cells, ROR1 negative cell populations are screened out through a flow cytometry sorter, and the screened ROR1 negative cell populations are inoculated into a pore plate for continuous culture;
s4, secondary screening: and (3) surface staining the cells in S3, screening ROR1 negative cell groups by a flow cytometry, inoculating a single ROR1 negative cell/hole into a pore plate, and continuing culturing for 45-50h to obtain the ROR1 gene knockout tumor cell strain.
2. The method for constructing the ROR1 gene knockout tumor cell strain according to claim 1, which is characterized in that: in the step S2, the mass ratio of the Cas9 protein to the gRNA is (4.8-5.3): 1.
3. The method for constructing the ROR1 gene knockout tumor cell strain according to claim 1, which is characterized in that: in step S2, the transfection is performed when the tumor cells in the well plate proliferate to 50-70% confluence.
4. The method for constructing the ROR1 gene knockout tumor cell strain according to claim 1, which is characterized in that: in step S2, the transfection includes the following steps:
(1) preparation of solution i: 1152-1272ng Cas9 protein, 240ng gRNA, 2.4-2.6 mu L Plus chamber Reagent and 24-26 mu L transfection medium are mixed evenly to obtain solution I;
(2) preparation of solution II: uniformly mixing 24-26 mu L of transfection medium and 1.4-1.6 mu L of transfection reagent to obtain solution II;
(3) dropwise adding the solution I into the solution II, uniformly mixing, and incubating at 23-27 ℃ for 8-12min to obtain a solution III;
(4) adding the solution III into a pore plate, and placing the pore plate in a carbon dioxide incubator to incubate, wherein the carbon dioxide incubator contains 5% of carbon dioxide and the temperature is 36-38 ℃.
5. The method for constructing the ROR1 gene knockout tumor cell strain according to claim 1, which is characterized in that: in step S3, the cell surface staining method includes the following steps:
(1) digesting the tumor cells by pancreatin, and counting the cells;
(2) take 0.8 x 105-0.9*105Loading the cells into a flow cell tube, adding a buffer solution for washing once, and adding a mouse anti-human ROR1 monoclonal antibody;
adding isotype control antibody mouse IgG1 into another control tube, incubating on ice for 18-22min, and washing with buffer solution for 2-3 times;
(3) adding a secondary antibody goat anti-mouse IgG (H + L) -FITC into the flow cell tube and the control tube in the step (2), placing on ice, incubating for 18-22min, and washing for 2-3 times by using a buffer solution;
(4) screening the cells incubated in (3) by using a flow sorting cytometer, collecting a ROR1 negative cell population, and inoculating the cell population into a pore plate for culture and amplification.
6. A ROR1 gene knockout tumor cell strain is characterized in that: constructed using the method of any one of claims 1 to 5.
7. The use of the ROR1 gene knockout tumor cell line of claim 6, wherein: used for verifying the specific killing effect of ROR1-CAR-T cells on target cells.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480292A (en) * | 2022-01-24 | 2022-05-13 | 苏州恒康生命科学有限公司 | Method for constructing CAR-T cell by using shRNA to silence human Tim-3 gene and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104662044A (en) * | 2012-08-24 | 2015-05-27 | 加利福尼亚大学董事会 | Antibodies and vaccines for use in treating ror1 cancers and inhibiting metastasis |
| CN107236763A (en) * | 2017-06-26 | 2017-10-10 | 中南民族大学 | A kind of method for building Knockout cells system based on flow cytometry |
| CN107418974A (en) * | 2017-07-28 | 2017-12-01 | 新乡医学院 | It is a kind of to sort the quick method for obtaining CRISPR/Cas9 gene knockout stable cell lines using monoclonal cell |
| CN108441505A (en) * | 2018-05-28 | 2018-08-24 | 上海恒润达生生物科技有限公司 | A kind of Chimeric antigen receptor and application thereof of targeting ROR1 |
| EP3080279B1 (en) * | 2013-12-11 | 2018-09-26 | Regeneron Pharmaceuticals, Inc. | Methods and compositions for the targeted modification of a genome |
| CN109836498A (en) * | 2017-11-25 | 2019-06-04 | 深圳宾德生物技术有限公司 | It is a kind of to target the single-chain antibody of ROR1, Chimeric antigen receptor T cell and its preparation method and application |
| CN109837246A (en) * | 2017-11-25 | 2019-06-04 | 深圳宾德生物技术有限公司 | A kind of Chimeric antigen receptor T cell and its preparation method and application for the targeting ROR1 knocking out PD1 |
| WO2020057666A1 (en) * | 2018-09-21 | 2020-03-26 | 科济生物医药（上海）有限公司 | T-cell expressing chimeric receptor |
-
2020
- 2020-12-15 CN CN202011480649.XA patent/CN112522206A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104662044A (en) * | 2012-08-24 | 2015-05-27 | 加利福尼亚大学董事会 | Antibodies and vaccines for use in treating ror1 cancers and inhibiting metastasis |
| EP3080279B1 (en) * | 2013-12-11 | 2018-09-26 | Regeneron Pharmaceuticals, Inc. | Methods and compositions for the targeted modification of a genome |
| CN107236763A (en) * | 2017-06-26 | 2017-10-10 | 中南民族大学 | A kind of method for building Knockout cells system based on flow cytometry |
| CN107418974A (en) * | 2017-07-28 | 2017-12-01 | 新乡医学院 | It is a kind of to sort the quick method for obtaining CRISPR/Cas9 gene knockout stable cell lines using monoclonal cell |
| CN109836498A (en) * | 2017-11-25 | 2019-06-04 | 深圳宾德生物技术有限公司 | It is a kind of to target the single-chain antibody of ROR1, Chimeric antigen receptor T cell and its preparation method and application |
| CN109837246A (en) * | 2017-11-25 | 2019-06-04 | 深圳宾德生物技术有限公司 | A kind of Chimeric antigen receptor T cell and its preparation method and application for the targeting ROR1 knocking out PD1 |
| CN108441505A (en) * | 2018-05-28 | 2018-08-24 | 上海恒润达生生物科技有限公司 | A kind of Chimeric antigen receptor and application thereof of targeting ROR1 |
| WO2020057666A1 (en) * | 2018-09-21 | 2020-03-26 | 科济生物医药（上海）有限公司 | T-cell expressing chimeric receptor |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480292A (en) * | 2022-01-24 | 2022-05-13 | 苏州恒康生命科学有限公司 | Method for constructing CAR-T cell by using shRNA to silence human Tim-3 gene and application thereof |
| CN114480292B (en) * | 2022-01-24 | 2024-04-05 | 苏州恒康生命科学有限公司 | Method for constructing CAR-T cells by utilizing shRNA to silence human Tim-3 gene and application thereof |
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