U87-Cas9-IDH1 mut cell line as well as construction method and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a U87-Cas9-IDH1mut cell line as well as a construction method and application thereof. The cell line is a U87-Cas9-IDH1-R132H human brain astroblastoma cell strain, and is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 65385. According to the invention, a U87 cell is taken as a host cell, a lentiviral expression vector containing Cas9 is transfected, and a transformant for stably expressing Cas9 protein is obtained through plastin screening and positive cell monoclonal culture; a lentiviral expression vector containing IDH1mut is used for transfecting a U87-Cas9 cell, and a U87 cell line stably expressing Cas9 and IDH1mut protein is obtained through hygromycin screening. The cell line has a wide application prospect in the field of preparation of target cell strains / model animals by gene editing.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a U87-Cas9-IDH1 mut cell line and a construction method and application thereof. Background Art

[0002] Glioma is the most common primary intracranial malignant tumor in the world, with the characteristics of strong invasiveness, rapid growth, high recurrence and poor prognosis. Glioblastoma is the most malignant glioma among astrocytic tumors, and its traditional treatment methods include surgery, radiotherapy, temozolomide chemotherapy, etc. However, due to the rapid development, strong heterogeneity, treatment resistance and high recurrence rate of glioblastoma, the treatment effect is often poor and the prognosis is poor. Therefore, there is an urgent need to reveal the molecular mechanism of the onset and development of glioblastoma and to find more effective targets and biomarkers for precision treatment.

[0003] U87 cells are a type of brain neuroblastoma cell that has been widely studied in the fields of medicine and medicine. U87 cells originate from malignant tumors of the central nervous system and exhibit many hallmark features of glioblastoma, including rapid proliferation, high invasiveness, and significant genetic and phenotypic heterogeneity. This cell is one of the most widely used cell models in neurobiology, immuno-oncology, and cancer research, and is also an important tool for studying the molecular and cellular mechanisms and treatment strategies of brain tumors.

[0004] Isocitrate dehydrogenase (IDH) is a key rate-limiting enzyme in the tricarboxylic acid cycle, catalyzing the oxidative decarboxylation of isocitrate to generate a-ketoglutarate and CO2, providing energy for cellular metabolism and precursors for biosynthesis. IDH has three isomeric enzyme forms (IDH1 / 2 / 3), IDH1 and IDH2 form homodimers, and IDH3 forms heterotetramers. These enzymes function in specific locations. The R132H mutation of IDH1 (IDH1 mut) is one of the most common mutation types in gliomas. Compared with wild-type IDH1, the prognosis of patients with mutant gliomas is significantly improved, so IDH1-R132H is a highly potential therapeutic target.

[0005] CRISPR / Cas9 is a new generation of gene editing technology. Compared with traditional gene knockout methods, CRISPR has the advantages of high specificity, high efficiency and scalability. It can achieve complete knockout of targeted genes. When sgRNA targets the whole genome sequence, it can also be used as an efficient whole genome screening tool, playing a vital role in gene function research and target screening.

[0006] Therefore, constructing a U87 cell line that stably expresses Cas9 and IDH1 mut protein will help study new targets for glioma using the CRISPR / Cas9 gene editing system, and is expected to provide new therapeutic targets and combined treatment strategies for existing treatments. Summary of the invention

[0007] In view of this, one of the objects of the present invention is to provide a glioblastoma cell line that stably expresses Cas9 and IDH1 mut protein.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] Glioblastoma cell lines stably expressing Cas9 and IDH1 mut protein,

[0010] The glioblastoma cell line is named U87-Cas9-IDH1-R132H human brain astrocytic glioblastoma cell line, which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC No: 65385.

[0011] Furthermore, the brain glioma cell line is U87 cell line.

[0012] A second object of the present invention is to provide a method for constructing a glioblastoma cell line that stably expresses Cas9 and IDH1 mut protein.

[0013] To achieve the above object, the present invention adopts the following technical solutions:

[0014] The method for constructing a glioblastoma cell line stably expressing Cas9 and IDH1 mut proteins comprises the following steps: using U87 cells as host cells, transfecting a lentiviral expression vector containing Cas9, and obtaining transformants stably expressing Cas9 protein through blasticidin screening and monoclonal culture of positive cells; transfecting U87-Cas9 cells with a lentiviral expression vector containing IDH1 mut, and obtaining a U87 cell line stably expressing Cas9 and IDH1 mut proteins through hygromycin screening.

[0015] Furthermore, the final concentration of the blasticidin screening was 10 μg / mL.

[0016] Furthermore, the final concentration of hygromycin for screening is 250 μg / mL.

[0017] Furthermore, the construction method comprises the following steps:

[0018] (1) Preparation of LentiCas9-Blast and IDH1(human)-R132H-3×FLAG-Hygromycin plasmids;

[0019] (2) Cas9, IDH1 mut lentiviral packaging;

[0020] (3) Cas9 lentiviral transfection;

[0021] (4) Multi-cell cloning screening of U87-Cas9 cells;

[0022] (5) Single-cell cloning to screen Cas9 stable expression cell lines;

[0023] (6) IDH1 mut lentiviral transfection;

[0024] (7) Polyclonal cell screening of U87-Cas9 IDH1 mut cell lines.

[0025] Further, steps (1) to (2) include: transforming the LentiCas9-Blast plasmid with blasticidin resistance and the IDH1(human)-R132H-3×FLAG-Hygromycin plasmid with hygromycin resistance respectively, and then forming a lentiviral packaging system together with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G.

[0026] Furthermore, in step (3) and step (6), Polybrene reagent is added during the lentiviral transfection process; the final concentration of the Polybrene is 0.8 μg / mL.

[0027] As a preferred solution, step (1) is specifically as follows:

[0028] 1) Centrifuge the bacterial solution at 5000 rpm for 3 min at room temperature to collect the bacterial precipitate and remove the supernatant as much as possible;

[0029] 2) Add solution P1 to resuspend the bacterial pellet;

[0030] 3) Add solution P2, and immediately and gently turn the tube upside down 6-8 times to fully lyse the bacteria, and leave it at room temperature for 5 minutes;

[0031] 4) Add solution P4, and immediately gently invert the tube 6-8 times to mix thoroughly until a white flocculent precipitate is formed. Then, place the tube at room temperature for about 10 minutes, centrifuge it at 8000 rpm for 10 minutes, and collect the supernatant.

[0032] 5) filtering the supernatant obtained in step 4), adding the endotoxin-removing solution ER to the obtained filtrate, and mixing by inversion;

[0033] 6) adding 0.3 times the volume of the filtrate to the solution obtained in step 5) and mixing by inverting the mixture, and then transferring the mixture to the adsorption column CP6;

[0034] 7) Centrifuge the adsorption column CP6 at 8000 rpm for 2 min at room temperature, and then wash it with rinse solution ED and rinse solution PW;

[0035] 8) Place the air-dried adsorption column CP6 in a clean centrifuge tube, add elution buffer TB to the middle part of the adsorption membrane, place at room temperature for 5 minutes, centrifuge at 8000 rpm for 5 minutes, collect the plasmid solution and measure the plasmid concentration.

[0036] Preferably, in step 2), RNase A is added to the solution P1 in advance.

[0037] Preferably, in step 7), anhydrous ethanol is added to the rinsing liquid PW in advance.

[0038] As a preferred solution, step (2) is specifically as follows:

[0039] 1) 293T cells were seeded into a 100 mm cell culture dish the day before and allowed to grow to 50%-60% on the second day;

[0040] 2) Add the PLUS reagent mixture to the lentiviral plasmid mixture, invert to mix, and incubate at room temperature for 5 minutes;

[0041] 3) Add the lentiviral plasmid mixture and PLUS reagent mixture to the Lipo2000 transfection reagent mixture, invert to mix, and incubate at room temperature for 20 minutes;

[0042] 4) Transfer the lentiviral transfection mixture into a culture dish, shake it gently using the cross method, and continue culturing in a cell culture incubator for 6 hours; discard the medium containing the lentivirus, add fresh complete medium, and continue culturing for 48 hours;

[0043] 5) After 48 hours, collect the supernatant and store it at 4°C;

[0044] 6) Add 10 mL of fresh complete medium to the culture dish, continue culturing for 24 hours, and collect the supernatant;

[0045] 7) The supernatants collected from the two times were mixed, centrifuged and filtered through a filter membrane, and then aliquoted and frozen.

[0046] Preferably, in the lentiviral plasmid mixture, Cas9 and IDH1 mut are 5 μg, psPAX2 is 4 μg, pMD2.G is 2 μg, and Opti-MEM is added to 500 μL.

[0047] Preferably, in the PLUS reagent mixture, Plus is 50 μL, and Opti-MEM is added to 500 μL.

[0048] Preferably, in the Lipo2000 transfection reagent mixture, Lipo2000 is 27.5 μL, and Opti-MEM is added to 500 μL.

[0049] As a preferred solution, step (3) includes: inoculating U87 cells into a 6-well plate and growing them to 70% confluence on the second day; discarding the old culture medium, adding Cas9 lentivirus solution and complete culture medium to 3 wells of the 6-well plate in a ratio of 1:1, adding Polybrene reagent, and culturing for 24 hours.

[0050] As a preferred solution, step (4) includes: after cell transfection is completed, all 6-well plates are replaced with complete culture medium containing Blasticidin S, and then replaced every 1-2 days; after screening for 3 days, the positive cells in the experimental group are expanded and cultured.

[0051] As a preferred solution, step (5) is specifically as follows:

[0052] 1) Count the positive cells, dilute the cell suspension to 1-2 cells / 100 μL, and mix well;

[0053] 2) 100 μL per well was plated into a 96-well plate and placed in a cell culture incubator for continued culture;

[0054] 3) After static culture, replace the complete medium containing Blasticidin S, and observe and record the formation of monoclonal cells daily;

[0055] 4) After a single clone is formed, the cells are transferred to 24-well plates, 6-well plates and 100 mm cell culture dishes in turn for further expansion and culture.

[0056] 5) Determine protein expression by WB and select cell lines with high expression levels for freezing or continued cultivation.

[0057] As a preferred solution, step (6) comprises:

[0058] U87 Cas9 cells were seeded into a 6-well plate and grown to 70% confluence the next day; the old culture medium was discarded, and the IDH1mut lentivirus solution and complete culture medium were added to 3 wells of the 6-well plate at a ratio of 1:1, and Polybrene reagent was added, and cultured in an incubator for 24 hours.

[0059] As a preferred solution, step (7) comprises:

[0060] After cell transfection, the 6-well plates were replaced with complete culture medium containing hygromycin, and then replaced every 2 days. After 3 days of screening, the positive cells in the experimental group were expanded and cultured. Cell protein expression was determined by WB.

[0061] The third object of the present invention is to provide a use of the aforementioned glioblastoma cell line in screening IDH1 mutant glioma-specific target genes.

[0062] A fourth object of the present invention is to provide a method for screening candidate drugs for treating glioblastoma.

[0063] To achieve the above object, the present invention adopts the following technical solutions:

[0064] A method for screening candidate drugs for treating glioblastoma comprises constructing a glioblastoma animal model using the aforementioned glioblastoma cell line; administering a test drug to the glioblastoma animal model, and the test drug that improves or cures the symptoms of glioblastoma after administration is a candidate drug for treating glioblastoma.

[0065] The beneficial effects of the present invention are:

[0066] The present invention provides a U87-Cas9-IDH1-R132H human brain astrocytic glioblastoma cell line and a construction method thereof. The U87-Cas9-IDH1 mut overexpression positive polyclonal cell line screened by the present invention contains complete Cas9 and IDH1mut gene sequences and successfully expresses the corresponding protein. The U87-Cas9-IDH1 mut cell line can be used to screen IDH1 mutant glioma-specific target genes or screen anti-glioblastoma drug candidates, and can also provide new means for studying specific gene functions and other aspects, and has broad application prospects in the field of gene editing to prepare target cell lines / model animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is the result of Cas9 and IDH1 mut endotoxin-free plasmid gel;

[0068] Figure 2 This is the plasmid map of Cas9 (Addgene #52962);

[0069] Figure 3 is the IDH1 mut plasmid map;

[0070] Figure 4 This is the plasmid map of psPAX2 (Addgene #12260);

[0071] Figure 5 This is the plasmid map of pMD2.G (Addgene #12259);

[0072] Figure 6 This is a microscopic image of a 24-well plate in the U87 cell Blasticidin S resistance concentration exploration experiment. Figure 6-A is the microscopic result of 24-well plate of U87 cells with Blasticidin S resistance concentration of 0 μg / mL. Figure 6 -B is the microscopic result of 24-well plate of U87 cells with Blasticidin S resistance concentration of 8μg / mL. Figure 6 -C is a microscopic image of a 24-well plate of U87 cells with Blasticidin S resistance at a concentration of 10 μg / mL;

[0073] Figure 7 This is a microscopic result diagram of cas9 positive cell screening, where: Figure 7 -A is the microscopic result of the experimental group (+Cas9+BlasticidinS), Figure 7 -B is the microscopic result of the control group (-Cas9+Blasticidin S);

[0074] Figure 8 This is the WB result of the positive polyclonal cell line U87 Cas9;

[0075] Fig. 9 This is the WB result of the positive monoclonal cell line U87 Cas9;

[0076] Fig.10 This is a microscopic image of a 24-well plate in the U87 cell Hgromycin antibiotic concentration exploration experiment. Fig.10 -A is the microscopic result of 24-well plate of U87 cells with Hgromycin antibiotic concentration of 0 μg / mL. Fig.10 -B is the microscopic result of 24-well plate of U87 cells with Hgromycin antibiotic concentration of 100 μg / mL. Fig.10 -C is the microscopic result of 24-well plate of U87 cells with Hgromycin antibiotic concentration of 250 μg / mL;

[0077] Fig.11 This is the microscopic result of IDH1 mut-positive cell screening, where: Fig.11 -A is the microscopic result of the experimental group (+IDH1mut+Hgromycin), Fig.11 -B is the microscopic result of the control group (-IDH1 mut+Hgromycin);

[0078] Fig.12 This is the WB result of the positive polyclonal cell line U87-Cas9 IDH1 mut;

[0079] Fig.13 The figure is a flow chart of the method for constructing the U87 cell line stably expressing Cas9 and IDH1-R132H of the present invention. DETAILED DESCRIPTION

[0080] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0081] In the embodiment of the present invention, the relevant plasmid Addgene sequence information is shown in Table 1.

[0082] Table 1

[0083] Plasmids Addgene sequence number Manufacturer / Article No. LentiCas9-Blast Plasmid Plasmid#52962 MIAOLINGBIOLOGY / P0805 psPAX2 Plasmid Plasmid#12260 MIAOLINGBIOLOGY / P0261 pMD2.G plasmid Plasmid#12259 MIAOLINGBIOLOGY / P0262

[0084] In the examples of the present invention, IDH1(human)-R132H-3×FLAG-Hygromycin plasmid was purchased from MIAOLINGBIOLOGY with the catalog number P47988.

[0085] Example 1. Construction of a U87 cell line stably expressing Cas9 and IDH1 mut protein

[0086] according to Fig.13 The operation flow shown in the figure constructs a U87 cell line that stably expresses Cas9 and IDH1 mut protein, which specifically includes the following steps:

[0087] (1) Preparation of LentiCas9-Blast and IDH1(human)-R132H-3×FLAG-Hygromycin plasmids

[0088] The purchased LentiCas9-Blast plasmid with blasticidin resistance and the IDH1(human)-R132H-3×FLAG-Hygromycin plasmid with hygromycin resistance were transformed, and a large number of plasmids were extracted and verified; they were combined with the purchased packaging plasmid psPAX2 and envelope plasmid pMD2.G to form a lentiviral packaging system. Among them, the plasmid maps of LentiCas9-Blast, IDH1(human)-R132H-3×FLAG-Hygromycin, psPAX2 and pMD2.G are as follows: Figure 2 to Figure 5 The specific operations are as follows:

[0089] 1) Take 100 ml of bacterial solution and centrifuge at 5000 rpm for 3 min at room temperature to collect bacterial precipitate, and remove the supernatant as much as possible;

[0090] 2) Add 10 ml of solution P1 to resuspend the bacterial pellet; RNase A was added to solution P1 in advance;

[0091] 3) Add 10 ml of solution P2, immediately and gently invert the tube 6-8 times to fully lyse the cells, and leave at room temperature for 5 min;

[0092] 4) Add 10 ml of solution P4, and immediately and gently invert the tube 6-8 times to mix thoroughly until a white flocculent precipitate is formed. Then, place the tube at room temperature for about 10 min, and centrifuge at 8000 rpm for 10 min. At this time, a precipitate is formed at the bottom of the centrifuge tube.

[0093] 5) Add the supernatant collected in the previous step to the filter CS in portions, push the push handle to filter, and collect the filtrate in a clean 50 mL centrifuge tube;

[0094] 6) Add 3 mL of endotoxin-free solution ER, invert and mix, and the solution will become a uniform transparent yellow color.

[0095] 7) Add 0.3 times the volume of isopropanol to the filtrate, mix by inversion, and transfer to the adsorption column CP6;

[0096] 8) Centrifuge at 8000 rpm for 2 min at room temperature, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0097] 9) Add 10 mL of rinse solution ED to the adsorption column CP6, centrifuge at 8000 rpm for 2 min, pour out the waste liquid in the collection tube, and place the adsorption column CP6 in the collection tube;

[0098] 10) Add 10 mL of rinse solution PW (anhydrous ethanol has been added to the rinse solution PW in advance) to the adsorption column CP6, centrifuge at 8000 rpm for 2 min, pour out the waste liquid in the collection tube, and place the adsorption column CP6 in the collection tube;

[0099] 11) Add 10 mL of rinse solution PW to the adsorption column CP6, centrifuge at 8000 rpm for 2 min, and discard the waste liquid in the collection tube;

[0100] 12) Place the adsorption column CP6 back into the collection tube and centrifuge at 8000 rpm for 1 min to remove the remaining rinse solution in the adsorption column. Place the adsorption column CP6 at room temperature for several minutes to dry the remaining rinse solution in the adsorption material.

[0101] 13) Place the adsorption column CP6 in a clean 50 mL centrifuge tube, add 1 mL of elution buffer TB to the middle part of the adsorption membrane, place at room temperature for 5 min, centrifuge at 8000 rpm for 5 min, collect the plasmid solution and transfer it to a clean 1.5 mL centrifuge tube, and store at -20°C;

[0102] 14) The plasmid concentration was measured by Qubit Nucleic Acid Protein Quantifier and verified by gel electrophoresis. Figure 1 As shown, the marker is Bio-Tech's DNA Ladder (0.2-12 kb, 12 bands), the size of LentiCas9-Blast is 12860 bp, and the size of IDH1(human)-R132H-3×FLAG-Hygromycin is 9107 bp.

[0103] (2) Cas9, IDH1 mut lentiviral packaging

[0104] 1) 293T cells were seeded into a 100 mm cell culture dish the day before and allowed to grow to 50%-60% on the second day;

[0105] 2) Prepare a plasmid mixture: 5 μg of Cas9 and IDH1 mut, 4 μg of psPAX2, 2 μg of pMD2.G, and add Opti-MEM to 500 μL;

[0106] 3) Prepare the PLUS reagent mixture and invert to mix: 50 μL of Plus, add Opti-MEM to 500 μL;

[0107] 4) Add the PLUS reagent mixture to the lentiviral plasmid mixture, invert to mix, and incubate at room temperature for 5 minutes;

[0108] 5) Prepare Lipo2000 transfection reagent mixture and invert to mix: Lipo2000 is 27.5 μL (plasmid μg: Lipo2000 μL = 1:2.5), add Opti-MEM to 500 μL;

[0109] 6) Add the lentiviral plasmid mixture and PLUS reagent mixture to the Lipo2000 transfection reagent mixture, invert to mix, and incubate at room temperature for 20 minutes;

[0110] 7) Use a pipette to transfer 8 mL of the lentiviral transfection mixture into each 100 mm culture dish, shake gently using the cross method, and place in a cell culture incubator for 6 hours;

[0111] 8) After 6 hours, discard the medium containing lentivirus, add 10 mL of fresh complete medium, and continue culturing in the incubator for 48 hours;

[0112] 9) After 48 hours, collect the supernatant and store it at 4°C;

[0113] 10) Add 10 mL of fresh complete culture medium to the culture dish, continue culturing for 24 hours, and collect the supernatant;

[0114] 11) The supernatants collected twice were mixed, centrifuged, filtered through a 0.45 μm filter membrane, and stored at -80°C for a long time after aliquoting to avoid repeated freezing and thawing.

[0115] (3) Cas9 lentiviral transfection

[0116] 1) U87 cells were seeded into 6-well plates and grown to 70% confluence the next day;

[0117] 2) Discard the old culture medium, add the Cas9 lentiviral solution and the prepared complete culture medium to 3 wells of the 6-well plate at a ratio of 1:1, and do not add the lentiviral solution to the remaining wells as a control;

[0118] 3) Add Polybrene at a final concentration of 0.8 μg / mL to increase the efficiency of lentiviral infection;

[0119] 4) Culture the cells in an incubator for 24 h.

[0120] (4) Multi-cell cloning screening of U87-Cas9 cells

[0121] 24 hours after cell transfection, the complete medium containing Blasticidin S (final concentration of 10 μg / mL) was replaced in all 6-well plates, and then replaced every 1-2 days. After 3 days of screening, Figure 7 As shown in the figure, all cells in the control group died, and the positive cells in the experimental group were collected for further expansion and culture and Western blot verification, specifically: trypsin digestion was used to collect positive polyclonal cells, 1×SDSlysis buffer was used to extract total protein, and the concentration of each sample was determined by Qubit nucleic acid protein quantification instrument, and then electrophoresis was performed according to 40μg protein loading, 5% skim milk powder was blocked, the primary antibody was CRISPR Cas91:3000, and the secondary antibody (goat anti-rabbit) was configured with 5% skim milk powder at 1:10000. The results are shown in the figure. Figure 8 As shown, Cas9 protein was successfully expressed.

[0122] (5) Single-cell cloning and screening of Cas9 stable expression cell lines

[0123] Count the positive cells, dilute the cell suspension to 1-2 cells / 100 μL, and mix well; plate 100 μL per well in a 96-well plate and continue to culture in a cell culture incubator; after static culture, replace the complete culture medium containing Blasticidin S (final concentration of 10 μg / mL), observe and record the formation of monoclones every day; after the formation of monoclones, transfer to 24-well plates, 6-well plates and 100 mm culture dishes in turn to continue to expand the culture. Overexpression positive monoclonal cell lines were verified by Western blot, specifically: positive monoclonal cells were collected by trypsin digestion, total protein was extracted with 1×SDS lysis buffer, the concentration of each sample was determined by Qubit nucleic acid protein quantification instrument, and then electrophoresis was performed at a protein loading of 30 μg, blocked with 5% skimmed milk powder, the primary antibody was CRISPRCas91:3000, and the secondary antibody (sheep anti-rabbit) was configured with 5% skimmed milk powder at 1:10000. Fig. 9 As shown, the target protein size is about 160kD. WB results show that the U87-Cas9 overexpression monoclonal cell line successfully expresses Cas9 protein. Finally, the cell line with the highest expression level is selected for freezing or continued culture.

[0124] (6) IDH1 mut lentiviral transfection

[0125] 1) U87 Cas9 cells were seeded into 6-well plates and grown to 70% confluence the next day;

[0126] 2) Discard the old culture medium, add the IDH1 mut lentiviral solution and the prepared complete culture medium into 3 wells of a 6-well plate at a ratio of 1:1, and do not add lentiviral solution to the remaining wells as a control;

[0127] 3) Add Polybrene at a final concentration of 0.8 μg / mL to increase the efficiency of lentiviral infection;

[0128] 4) Place the cells in an incubator and culture for 24 hours;

[0129] (7) Polyclonal cell screening of U87-Cas9 IDH1 mut cell lines

[0130] 1) 24 hours after cell transfection, replace the complete medium containing Hygromycin (final concentration 250 μg / mL) in all 6-well plates, and then replace it every 2 days;

[0131] 2) After 3 days of screening, all cells in the control group died, such as Fig.11 As shown, the positive cells in the experimental group were expanded and cultured;

[0132] 3) WB determination of IDH1 mut protein overexpression, specifically: trypsin digestion to collect positive monoclonal cells, 1× SDSlysis buffer to extract total protein, Qubit nucleic acid protein quantification instrument to determine the concentration of each sample, electrophoresis according to 40μg protein loading, 5% skim milk powder blocking, primary antibodies were IDH11:3000, DDDDK-Tag 1:3000, 5% skim milk powder to configure secondary antibody (goat anti-rabbit) 1:10000. Fig.12 As shown, the target protein IDH1-Tag fusion protein is about 49kD (exogenous, DDDDK-Tag size is 2.7kD), and the endogenous IDH1 is 46kD. WB results show that exogenous IDH1 R132H is successfully expressed.

[0133] In summary, the results show that the present invention successfully constructed a U87 cell line that stably expresses Cas9 and IDH1 mut proteins. U87 cells are brain glioma cells, which are sequentially transfected with lentiviral expression vectors containing Cas9 and IDH1 mut, and the transformants that can stably express Cas9 and IDH1 mut proteins are obtained by screening with 10 μg / mL of blasticidin and 250 μg / mL of hygromycin. The present invention is named U87-Cas9-IDH1-R132H human brain astrocytic glioblastoma cell line, and is preserved in the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with a preservation address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, and a preservation date of October 31, 2024. The deposit applicant is Sichuan University of Light Industry, and the deposit number is GDMCC No: 65385. The classification of the biomaterial is named human brain astrocytic glioblastoma cell line (Homo sapiens).

[0134] Example 2. Blasticin S antibiotic screening concentration exploration

[0135] Before the step "(4) screening U87-Cas9 cells with multiple cell clones" in Example 1, the concentration of Blasticin S antibiotic screening was explored. Specifically, the U87 cells were counted at 3×10 5 Each well of the 6-well plate was inoculated with 10 cells, and the plate was placed in a cell culture incubator for 24 hours. Complete medium containing different gradient concentrations of Blasticidin S (0, 2, 4, 6, 8, 10 μg / mL) was added, and the culture was continued in the cell culture incubator. The complete medium containing Blasticidin S was replaced every 1-2 days, and the cell growth and survival were observed. After 3 days of screening, the cells were observed under a microscope and the cell survival was counted.

[0136] The experimental results are as follows Figure 6As shown in the figure, all cells of the U87 cell line transfected with Cas9 lentivirus have died at a concentration of 10 μg / mL, so 10 μg / mL is the lowest screening concentration.

[0137] Example 3. Hygromycin antibiotic screening concentration exploration

[0138] Before the step “(7) Polyclonal cell screening of U87-Cas9 IDH1 mut cell line” in Example 1, the concentration of hygromycin antibiotic screening was explored. Specifically, the U87 cells were counted at 3×10 5 Each well of the 6-well plate was inoculated with 10 cells, and the plate was placed in a cell culture incubator for 24 hours. Complete medium containing different gradient concentrations of Hygromycin (0, 100, 250, 500, 750, 1000 μg / mL) was added, and the culture was continued in the cell culture incubator. The complete medium containing Hygromycin was replaced every 1-2 days, and the cell growth and survival were observed. After 3 days of screening, the cells were observed under a microscope and the cell survival was counted.

[0139] The experimental results are as follows Fig.10 As shown, all cells of the U87 cell line transfected with IDH1 mut lentivirus died at a concentration of 250 μg / mL, so 250 μg / mL is the lowest screening concentration.

Claims

1. A glioblastoma cell line stably expressing Cas9 and IDH1 mut protein, characterized in that: The glioblastoma cell line is named U87-Cas9-IDH1-R132H human brain astrocytic glioblastoma cell line, which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC No: 65385.

2. The glioblastoma cell line according to claim 1, characterized in that The brain glioma cell line is U87 cell line.

3. The method for constructing a glioblastoma cell line according to any one of claims 1 to 2, characterized in that: Using U87 cells as host cells, transformants stably expressing Cas9 protein were obtained by transfection with a lentiviral expression vector containing Cas9, followed by blasticidin screening and monoclonal culture of positive cells. U87-Cas9 cells were transfected with a lentiviral expression vector containing IDH1 mut, and a U87 cell line stably expressing Cas9 and IDH1 mut proteins was obtained by hygromycin screening.

4. The construction method according to claim 3, characterized in that: The final concentration of the blasticidin screening was 10 μg / mL.

5. The construction method according to claim 3, characterized in that: The final concentration of hygromycin for screening was 250 μg / mL.

6. The construction method according to claim 3, characterized in that: The construction method comprises the following steps: (1) Preparation of LentiCas9-Blast and IDH1(human)-R132H-3×FLAG-Hygromycin plasmids; (2) Cas9, IDH1 mut lentiviral packaging; (3) Cas9 lentiviral transfection; (4) Multi-cell cloning screening of U87-Cas9 cells; (5) Single-cell cloning to screen Cas9 stable expression cell lines; (6) IDH1 mut lentiviral transfection; (7) Polyclonal cell screening of U87-Cas9 IDH1 mut cell lines.

7. The construction method according to claim 6, characterized in that: Steps (1) to (2) include: transforming the LentiCas9-Blast plasmid with blasticidin resistance and the IDH1(human)-R132H-3×FLAG-Hygromycin plasmid with hygromycin resistance respectively, and then forming a lentiviral packaging system together with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G.

8. The method according to claim 6, characterized in that In step (3) and step (6), Polybrene reagent is added during the lentiviral transfection process; the final concentration of the Polybrene is 0.8 μg / mL.

9. Use of the glioblastoma cell line according to any one of claims 1 to 2 in screening specific target genes for IDH1 mutant gliomas.

10. A method for screening candidate drugs for treating glioblastoma, characterized in that: A glioblastoma animal model is constructed using the glioblastoma cell line described in any one of claims 1 to 2; a test drug is administered to the glioblastoma animal model, and the test drug that improves or cures the symptoms of glioblastoma after administration is a candidate drug for treating glioblastoma.

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