Construction method and application of modified HL60 cell

Abstract

The invention belongs to the field of molecular biology, and discloses a construction method and application of a modified HL60 cell. According to the method, the electric revolving cup and the electric revolving needle are repeatedly utilized and matched with the SBB buffer solution, so that an allogenic material can be successfully transfected into the HL60 cell, and parameters most suitable for electrotransfection of the HL60 cell are obtained through exploration. Therefore, the construction method not only solves the problem that HL60 is difficult to transfect, but also is low in cost. According to the construction method, the modified HL60 cell strain based on research on acute myelogenous leukemia can be prepared. According to the invention, the HL60 cell strain with CD44 and / or PSGL-1 knocked out is successfully constructed, and the HL60 cell strain can be used for researching endothelial selectin ligands.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and particularly relates to a construction method for transforming HL60 cells and an application thereof. Background Art

[0002] Acute myeloid leukemia (AML) patients may experience leukocytostasis due to high white blood cell counts, a serious complication caused by the accumulation of leukemic cells in capillaries, which may lead to multi-organ failure and death. At present, the specific mechanism of leukocytostasis is not fully understood. Endothelial selectin (E-selectin) plays a key role in the vascular endothelial recruitment and survival of leukemic cells, and E-selectin has been reported as a potential target for the treatment of AML. However, knowledge about selectin ligands expressed by AML is incomplete, so identifying selectin ligands is crucial for the development of new therapies against AML. HL60 cells are a human promyelocytic leukemia cell line derived from patients with acute promyelocytic leukemia and are currently used as a model cell for studying the differentiation of human bone marrow cells. Using HL60 cells to explore the role of E-selectin ligands PSGL-1 (also known as CD162) and CD44 in activating integrins and their signaling pathways is of great significance for providing new methods for the treatment of AML.

[0003] When HL60 cells are transformed, the methods of transfecting exogenous substances include liposome transfection method and electroporation transfection method. Since suspension cells are easy to aggregate and the cell membrane structure is complex, it is not conducive to the attachment of liposomes to the cell membrane, so the transfection efficiency is low. HL60 is a suspension cell that is difficult to transfect. The inventor failed to construct an HL60 cell line with knockout CD44 and / or PSGL-1 using the liposome transfection method. Compared with the liposome transfection method, the electroporation method is conducive to improving the entry of exogenous substances into suspension cells. However, the electroporation method is more harmful to suspension cells, and high-voltage electric pulses can cause cell damage or cell death, which unexpectedly reduces the electroporation efficiency and slows the growth of cells in the later stage. It is particularly critical to explore electroporation parameters that are conducive to transferring exogenous substances into suspension cells and reducing cell damage to successfully transform suspension cells. However, currently commercial electroporation consumables cannot be reused, and important tools such as electroporation cups, electroporation needles, and electroporation fluids cannot be reused multiple times. Commercial companies do not provide services for purchasing supporting experimental tools separately, that is, you need to purchase a complete set of systems to get the supporting tools again. This greatly increases the cost of optimizing suspension cell electroporation parameters and is not conducive to obtaining the target transformed HL60 cells. Summary of the invention

[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a construction method for transforming HL60 cells. The construction method can reuse the consumables of the electroporation system and has the characteristics of low cost.

[0005] Another object of the present invention is to provide an application of the construction method for transforming HL60 cells.

[0006] Another object of the present invention is to provide a HL60 cell line with CD44 and / or PSGL-1 knocked out, and a preparation method and application thereof.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A construction method for transforming HL60 cells comprises the following steps:

[0009] (1) Preparation of electroporation materials: Electroporation materials include SBB buffer and consumables;

[0010] When the consumables are used materials, they are handled as follows:

[0011] A. Electric rotating cup: Wash thoroughly with distilled water, then rinse with 70% to 75% (v / v) ethanol solution, finally air-dry in a sterile laminar flow hood, and place in a cool and dry place for later use; sterilize on a clean bench before use;

[0012] B. Electroporation needle: Use DNA enzyme to remove the residual plasmid DNA on the electroporation needle, then rinse with sterile water and 70% to 75% (v / v) ethanol solution in sequence, and finally air-dry in a sterile laminar flow hood and place in a cool and dry place for later use; sterilize in a clean bench before use;

[0013] The composition of SBB buffer was as follows: MgCl2 1 mM, sucrose 250 mM, and the solvent was DPBS;

[0014] (2) Electroporation: The mixture containing the nucleic acid to be transfected and the HL60 cell solution dispersed with SBB buffer are mixed, sucked into the electroporation needle, and then placed in the electroporation cup. The voltage and pulse time are adjusted to obtain cells electroporated under different parameters;

[0015] (3) Cultivation and detection: The cells obtained by electroporation are cultured and the cell growth status is observed; the cultured cells are then screened and the target gene or protein is detected to obtain optimized electroporation parameters and transform HL60 cells.

[0016] The above-mentioned method for constructing the modified HL60 cells further comprises the following steps:

[0017] (4) Monoclonal cell culture: The transformed HL60 cells were diluted by limiting dilution method and cultured to obtain monoclonal cells;

[0018] (5) Detection: Detect the target gene or protein that has been transformed in the monoclonal cells to obtain transformed HL60 cells.

[0019] The number of flushing in step (1)A is preferably at least 3 times.

[0020] The specific steps of the removal described in step (1) B are preferably as follows: soak the electroporation needle in DNase I solution for 15 to 20 minutes, and repeat the operation at least 3 times.

[0021] The composition of the DNase I solution is as follows: 1 mg / mL DNase I, 5 mM MgCl2, and the solvent is 0.01 M PBS with a pH of 7.4.

[0022] The number of flushing in step (1)B is preferably at least 3 times.

[0023] The voltage described in step (2) is preferably 1200-1400V.

[0024] The pulse time in step (2) is preferably 30 to 40 ms.

[0025] The application of the construction method for obtaining the modified HL60 cells in the study of acute myeloid leukemia.

[0026] A CD44 and / or PSGL-1 knocked-out HL60 cell line is a CD44 gene knocked-out, a PSGL-1 gene knocked-out, or a CD44 and PSGL-1 gene knocked-out HL60 cell line, which is obtained by optimizing electroporation parameters by the above electroporation method.

[0027] The method for preparing the above-mentioned HL60 cell line with CD44 and / or PSGL-1 knockout comprises the following steps:

[0028] 1) Preparation of RNP complex: Mix equal volumes of 1.5 μg / mL Cas9 protein solution and 1 μg / mL sgRNA solution in a sterile EP tube, pipette to mix evenly, and then incubate in a 20-30°C metal bath to fully form an RNP complex; sgRNA is sgRNA targeting CD44 gene and / or sgRNA targeting PSGL-1 gene;

[0029] 2) Preparation of HL60 cell fluid: HL60 cells cultured in complete medium to the logarithmic growth phase were collected, the medium was removed and then washed with PBS, and finally the cells were resuspended in SBB buffer to 2×10 7 / mL, and HL60 cell fluid was obtained;

[0030] 3) Preparation of a mixed system of RNP and cells: The RNP complex and HL60 cell fluid are mixed to obtain a mixed system of RNP and cells, wherein the volume content of the RNP complex in the mixed system of RNP and cells cannot exceed 10%;

[0031] 4) Electroporation: Take 10 μL of the RNP and cell mixture each time and put it into the electroporation needle, then place it in the electroporation cup, use the Neon transfection system to electroporate the cells with 1300V, 35ms pulse, and then transfer to complete medium and culture for 5-7 days;

[0032] 5) Screening: The cells cultured in step 4) were centrifuged and the supernatant was discarded. The cells were then resuspended in PBS containing 2% v / v FBS, and then CD44 antibody and / or CD162 antibody were added and incubated at 4°C. After incubation, the cells were washed to remove the unincubated antibodies, and then the cells were resuspended in PBS containing 2% v / v FBS to 5×10 6 / mL, which are the cells to be sorted; HL60 cells without antibody incubation and electroporation are used as negative control, and HL60 cells incubated with corresponding antibodies but without electroporation are used as positive control. The sorted cells are loaded and negative cells are sorted by flow cytometry;

[0033] 6) Obtaining monoclonal cells: diluting the negative cells obtained by sorting by limiting dilution method to obtain monoclonal cells;

[0034] 7) Verification: After the monoclonal cells obtained in step 6) are expanded and cultured, nucleic acid detection and protein detection of CD44 and / or PSGL-1 are performed to obtain HL60 cell lines with CD44 and / or PSGL-1 knockout.

[0035] The blowing time in step 1) is preferably 20s to 40s; more preferably 30s.

[0036] The temperature of the metal bath in step 1) is preferably 24-26°C; more preferably 25°C.

[0037] The incubation time in step 1) is preferably 15 to 25 minutes, and more preferably 20 minutes.

[0038] The sequence of the sgRNA targeting the CD44 gene described in step 1) is as follows: 5'-CTACAGCATCTCTCGGACGG-3'.

[0039] The sequence of the sgRNA targeting the PSGL-1 gene described in step 1) is as follows: 5'-CTCCGGTCCCGGGCAAGCAG-3'.

[0040] The complete culture medium in step 2) is preferably an RPMI 1640 culture medium containing 10-12% by volume of fetal bovine serum and 1% double antibody (penicillin and streptomycin).

[0041] The DPBS does not contain Ca 2+ Mg 2+ PBS.

[0042] The number of washing in step 2) is preferably 2 to 3 times.

[0043] The RNP complex and the HL60 cell fluid in step 3) are preferably mixed in a volume ratio of 1:10.

[0044] The complete culture medium in step 4) is preferably RPMI1640 culture medium containing 10-12% by volume of fetal bovine serum.

[0045] The incubation time in step 5) is preferably 20 to 40 minutes, and more preferably 30 minutes.

[0046] The number of washings in step 5) is preferably 2 to 3 times.

[0047] The nucleic acid detection in step 7) is performed by amplifying CD44 and / or PSGL-1 genes through PCR, and the obtained products are subjected to gene sequencing.

[0048] The protein detection in step 7) is Western Blot, wherein the antibodies used are CD44 antibody and / or CD162 antibody.

[0049] Application of the above-mentioned HL60 cell line with CD44 and / or PSGL-1 knockout in studying endothelial-selectin ligands.

[0050] Compared with the prior art, the present invention has the following advantages and effects:

[0051] (1) The present invention first provides a low-cost electroporation method for obtaining modified HL60 cells that is suitable for popularization and application. The electroporation method can reuse the consumables in the electroporation system, and after being matched with SBB buffer, the electroporation effect can be guaranteed, so that HL60 cells can be modified and the therapeutic targets of acute myeloid leukemia can be studied.

[0052] (2) The present invention also provides a HL60 cell line with CD44 and / or PSGL-1 knocked out, which is obtained by optimizing the electroporation parameters by the above electroporation method, has low R&D cost, and is conducive to the study of endothelial selectin ligands.

[0053] (3) The present invention also provides a method for preparing a HL60 cell line with knockout of CD44 and / or PSGL-1. The preparation method uses CRISPR-Cas9 RNP complex electroporation gene editing technology, followed by flow cytometry enrichment, monocloning, DNA sequencing and WB verification, which can ensure that positive monoclonal cells are obtained within one month. - / - HL60、CD44 - / - HL60、CD44 - / - PSGL-1 - / - The highest effective gene editing efficiency of HL60 was 66.7%, 3.57%, and 39.9%, respectively. This shows that the preparation method has the characteristics of high gene editing efficiency, low cost, and specialization, which further verifies the reliability and accuracy of the experimental method. After that, the above experimental scheme can be used for various gene knockouts on HL60 cells. More importantly, double knockout can still maintain a high level of gene editing efficiency, which opens up new ideas and directions for future research. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is the electroporation parameter optimization diagram.

[0055] Figure 2 This is a graph showing the gene editing efficiency results.

[0056] Figure 3 This is a graph of flow cytometry results.

[0057] Figure 4 This is a picture of DNA sequencing results.

[0058] Figure 5 This is the WB verification result diagram. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0060] Example 1

[0061] 1. Experimental process

[0062] (1) Design of sgRNA

[0063] The genomic sequences of CD44 and PSGL-1 genes were searched using the NCBI database, and sgRNAs were designed using the crispor website ( http: / / crispor.gi.ucsc.edu / ) and synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0064] Two sgRNAs targeting CD44 were designed, namely CD44-sgRNA1 and CD44-sgRNA2; two sgRNAs targeting PSGL-1 were designed, namely PSGL-1-sgRNA1 and PSGL-1-sgRNA2.

[0065] CD44-sgRNA1: 5'-CTACAGCATCTCTCGGACGG-3'.

[0066] CD44-sgRNA2: 5'-CTTTGCAGGTGTATTCCACG-3'.

[0067] PSGL-1-sgRNA1: 5'-CTCCGGTCCCGGGCAAGCAG-3'.

[0068] PSGL-1-sgRNA2: 5'-ATCTAGGTACTCATATTCGG-3'.

[0069] (2) Preparation of electroporation materials

[0070] ① Clean the electric rotating cup (Neon TM tube) and electroporation needle (Neon TM tips), first soak it in 70% (v / v) ethanol solution, then air-dry it in a sterile laminar flow hood, and then place it on a clean bench for sterilization.

[0071] ② Prepare SBB solution: first prepare 30mL 100mM MgCl2 solution: 0.609g MgCl2·6H2O (Aladdin) dissolved in 30mL DPBS (Gibco). Then take 0.3mL of the above and weigh 2.567g sucrose and add them to 29.7mL DPBS solution. Finally, filter with a 0.22μm pore size nanofiltration membrane (Sigma-Aldrich) in a clean bench, seal and place at 4℃ for use.

[0072] (3) Extraction of cells

[0073] 1) Add 2 mL of culture medium (containing 12% v / v FBS and 1% dual antibody RPIM1640, Gibco) to a 6-well cell culture plate and place it in an incubator;

[0074] 2) HL60 cells were centrifuged at 1000 rpm for 3 min and then washed with sterile PBS (Gibco, Mg-free). 2+ and Ca 2+ ) were washed twice and the cells were counted during the second wash.

[0075] 3) Remove the supernatant and resuspend the cells to 2×10 7 / mL to obtain a cell suspension.

[0076] (4) Optimization of electroporation parameters: The enhanced green fluorescent protein plasmid (pEGFP-N1) was used to identify the transfection efficiency of different electroporation parameters in the cell suspension obtained in step (3). The pEGFP-N1 plasmid was purified using a midi-purification kit (QIAGEN, Germany) and then added to the resuspended WT HL60 cells at a final concentration of 50 μg / mL. The pulse voltages were 1250 V, 1300 V, and 1350 V, respectively, and the pulse times were 30 ms and 35 ms, respectively. 24 h after transfection, the cells were photographed and observed using an inverted fluorescence microscope (ECLIPSETi2, Nikon). The transfection rate represents the proportion of GFP-positive cells to the total surviving cells.

[0077] (5) Preparation of RNP complex

[0078] 1) Take 1.5 μL of Cas9 protein (Takara, 5 μg / mL) and dilute it to 1.5 μg / mL by gently pipetting with 3.5 μL of nuclease-free water;

[0079] 2) Take 3 μL of sgRNA (3 μg / mL) and dilute it to 1 μg / mL with 6 μL of nuclease-free water by gently pipetting;

[0080] 3) Take the above 1.5 μg / mL Cas9 protein and add it to 1 μg / mL sgRNA in equal volumes in a sterilized EP tube. Mix by pipetting for 30 seconds and incubate in a 25°C metal bath for 20 minutes until the RNP complex is fully formed.

[0081] (6) Electroporation of cells

[0082] Take 100 μL of the cell suspension obtained in step (3) and add it to the above 10 μL RNP complex, mix it by pipetting for about 10 times, then take 10 μL of the above mixed system into the electroporator each time, use the Neon transfection system (Thermo Fisher) to electroporate the cells with a voltage of 1300 and a pulse time of 35 ms, then transfer to the above 6-well plate for culture, and observe the cell status. Continue to culture in RPMI 1640 medium (Gibco) containing 10% FBS (Gibco) for 5-7 days.

[0083] (7) Recycling of electroporation cups and electroporation needles

[0084] ①Electrotransfer cup: First, clean thoroughly with distilled water, then rinse three times with 70% (v / v) ethanol solution, and finally air-dry in a sterile laminar flow hood. Place in a cool and dry place until next use.

[0085] ② Electrotransfer needle: First, remove the residual plasmid DNA by 10μL DNase I solution (1mg / ml DNase I + 5mMMgCl2 dissolved in PBS, pH 7.4) for 3 times, and then incubate at room temperature for 15 to 20min. Next, rinse with 10μL sterile water and then 10μL 70% (v / v) ethanol, and repeat the above 3 times. Finally, place in a sterile laminar flow hood to air dry. Place in a cool and dry place for next use.

[0086] (8) Flow cytometry enrichment and limiting dilution

[0087] 1) Collect the cells cultured for 5-7 days in step (5), centrifuge at 1000 rpm for 3 min, discard the supernatant, and then resuspend in PBS containing 2% v / v FBS to adjust the cell density to 1×10 7 cells / mL.

[0088] 2) Then, for every 100 μL of cells (i.e., 10 6 1 μL of CD44 antibody (Sinobiological) and CD162 antibody (BD) were added to each cell and incubated at 4°C for 30 min. The cells were washed twice to remove unincubated antibodies and then resuspended to 5×10 cells in PBS containing 2% v / v FBS. 6 Transfer to a flow cytometry tube and place on ice for later use.

[0089] 3) WT HL60 cells (WT HL60 refers to HL60 cells that have not been electroporated) without antibody incubation are used as negative controls, and WT HL60 cells incubated with the corresponding antibodies are used as positive controls. The cells are loaded onto a flow cytometer for analysis. The obtained data are processed with FlowJo_V10, and the percentage of CD44-negative and / or PSGL-1-negative cells is used as the effective gene editing efficiency. The sorted negative cells will be collected in a specific receiving tube.

[0090] 4) Obtain negative cells with the highest sorting efficiency (effective gene editing efficiency) and obtain monoclonal cells through limiting dilution.

[0091] (8) Gene sequencing

[0092] 1) After 2-3 weeks, take some cells, extract DNA from the cells using a DNA extraction kit, and then measure the DNA concentration using NanoDrop.

[0093] 2) Design amplification primers for CD44 and PSGL-1 (CD162) gene editing fragments. The sequences are shown below. They were synthesized by Qingke Biotechnology Co., Ltd., prepared into a 100 μM solution using TE buffer, and stored in a -20°C refrigerator for later use.

[0094] CD44 upstream primer: 5′-CCGGCCTTATTTGACTTTTTAAGGAGTCTG-3′;

[0095] CD44 downstream primer: 5′-GTGGGAAAGGAGCCTTCCAGTTCTAAG-3′;

[0096] PSGL-1 upstream primer: 5′-CTTGACATCCCTCCGCCCATTGT-3′;

[0097] PSGL-1 downstream primer: 5′-CCAGTAGAACGCCTTGCAGCAG-3′.

[0098] 3) Prepare a PCR reaction system containing a premixed high-fidelity DNA polymerase ( T6 Super PCR Mix (1.1×, Qingke Bio) 43 μL, upstream primer solution 0.5 μL, downstream primer solution 0.5 μL, template DNA 50 ng, and nuclease-free water were used to make up the final volume to 50 μL. The reaction was carried out using a PCR instrument under the following conditions: 98°C for 2 min; 98°C for 10 s, 62°C for 15 s, 72°C for 40 s, 35 cycles; 72°C for 5 min.

[0099] 4) The PCR products were qualitatively confirmed by agarose gel electrophoresis and then sent for gene sequencing (Qingke Biotechnology Co., Ltd.).

[0100] (9) Protein verification

[0101] 1) The cells were washed twice with 4°C pre-cooled PBS and lysed in RIPA lysis buffer (Beyotime). The lysate was centrifuged at 14,000 × g for 5 min at 4°C to remove cell debris.

[0102] 2) The supernatant containing the total cell lysate was collected and the protein concentration was determined using a BCA protein assay kit (Pierce).

[0103] 3) 50 μg of protein in each sample was separated by SDS-PAGE on a 7.5% polyacrylamide gel and then transferred to a nitrocellulose membrane (GE Healthcare) using a semi-dry transfer system (Bio-Rad).

[0104] 4) Block the membrane with 5% skim milk powder in TBST (1M Tris-HCl, 5M NaCl and 10% Tween-20) for 1 hour at room temperature. Then incubate the membrane overnight at 4°C with primary antibodies: CD44 rabbit monoclonal antibody (1:1000, ABclonal) and / or CD162 / PSGL-1 rabbit monoclonal antibody (1:1000, ABclonal) diluted in TBST containing 3% BSA (Sigma-Aldrich).

[0105] 5) After washing three times with TBST for 10 minutes each time, the membrane was incubated with HRP-conjugated secondary antibody goat anti-rabbit IgG (1:5000, ABclonal) at room temperature for 1 hour. After washing three additional times with TBST, immunoreactive bands were detected using ECL (Thermo Fisher Scientific) and visualized using a chemiluminescent imaging system (CLINX).

[0106] 2. Experimental Results

[0107] (1) The results of electroporation parameter optimization are as follows Figure 1 As shown, the transfection efficiency was the highest at 1300 V and 35 ms, reaching 28.9%.

[0108] (2) Single knockout cell lines were constructed in WT HL60, while double knockout cell lines were constructed in CD44 - / - PSGL-1 was knocked out in HL60 cell line. The results are shown in 2: AB showed that PSGL-1 - / - The effective gene editing efficiencies of the two sgRNAs in HL60 were 59.3% and 53%, respectively; CD showed that CD44 - / - The effective gene editing efficiencies of the two sgRNAs in HL60 were 3.57% and 2.57%, respectively; EF showed that CD44 - / - PSGL-1 - / - The effective gene editing efficiencies of the two sgRNAs of HL60 (i.e., the two sgRNAs of PSGL-1) were 39.4% and 26.4%, respectively.

[0109] (3) Flow cytometry sorting results are as follows Figure 3 As shown, the sgRNA with the highest effective gene editing efficiency was selected, and then the cell population was selected for sorting. Wild-type cells (not incubated with CD44 antibody or PSGL-1 antibody) were used as negative control and positive control (incubated with CD44 antibody or PSGL-1 antibody), while those incubated with CD44 / PSGL-1 antibodies were the experimental group. Figure 3 A and B in are PSGL-1 - / - HL60 cells and CD44 - / -Schematic diagram of the sorting process of HL60 cells. WT HL60 cells were used as negative control (not incubated with CD44 antibody or PSGL-1 antibody) and positive control (incubated with CD44 or PSGL-1 antibody). - / - HL60 cells and CD44 - / - HL60 cells were experimental groups, and the figure shows PSGL-1 - / - The effective gene editing efficiency of HL60 was 59.3% and that of CD44 - / - A cell population with an effective gene editing efficiency of 3.57% for HL60 was successfully isolated; Figure 3 The C in the code stands for CD44 - / - PSGL-1 - / - Schematic diagram of the sorting process of HL60 cells, CD44 - / - HL60 cells were used as negative control (not incubated with PSGL-1 antibody) and positive control (incubated with PSGL-1 antibody), CD44 - / - PSGL-1 - / - HL60 cells are the experimental group, and the figure shows that a cell population with an effective gene editing efficiency of 39.4% was successfully sorted.

[0110] (4) DNA sequencing results Figure 4 As shown, the results indicate that the monoclonal PSGL-1 - / - HL60 cells ( Figure 4 A) and CD44 - / - HL60 cells ( Figure 4 B) The deletion of PSGL-1 and CD44 genes was shown at the gene level, indicating that this method is highly targeted.

[0111] (5) WB test results are as follows Figure 5 As shown, the results indicate that the monoclonal PSGL-1 - / - HL60 cells do not express PSGL-1 protein but express CD44 protein. - / - HL60 cells do not express CD44 protein but express PSGL-1 protein. - / - PSGL-1 - / - HL60 does not express PSGL-1 and CD44 proteins. That is, the knockout of PSGL-1 and / or CD44 alone was successfully verified at the protein level and did not affect the expression of another ligand, indicating that this method can ensure the stable expression of the protein.

[0112] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for transforming HL60 cells, characterized in that The steps include: (1) Preparation of electroporation materials: Electroporation materials include SBB buffer and consumables; When the consumables are used materials, they are handled as follows: A. Electric rotating cup: Wash thoroughly with distilled water, then rinse with 70% to 75% (v / v) ethanol solution, finally air-dry in a sterile laminar flow hood, and place in a cool and dry place for later use; sterilize on a clean bench before use; B. Electroporation needle: Use DNA enzyme to remove the residual plasmid DNA on the electroporation needle, then rinse with sterile water and 70% to 75% (v / v) ethanol solution in sequence, and finally air-dry in a sterile laminar flow hood and place in a cool and dry place for later use; sterilize in a clean bench before use; The composition of SBB buffer was as follows: MgCl2 1 mM, sucrose 250 mM, and the solvent was DPBS; (2) Electroporation: The mixture containing the nucleic acid to be transfected and the HL60 cell solution dispersed with SBB buffer are mixed, sucked into the electroporation needle, and then placed in the electroporation cup. The voltage and pulse time are adjusted to obtain cells electroporated under different parameters; (3) Cultivation and detection: The cells obtained by electroporation are cultured and the cell growth status is observed; the cultured cells are then screened and the target gene or protein is detected to obtain optimized electroporation parameters and transform HL60 cells.

2. The method for constructing modified HL60 cells according to claim 1, characterized in that The following steps are also included: (4) Monoclonal cell culture: The modified HL60 cells were diluted by limiting dilution method and cultured to obtain monoclonal cells; (5) Detection: Detect the target gene or protein that has been transformed in the monoclonal cells to obtain transformed HL60 cells.

3. The method for constructing modified HL60 cells according to claim 1 or 2, characterized in that: The number of flushing steps in step (1)A is at least 3 times; The specific steps of the removal described in step (1) B are as follows: soak the electroporation needle in DNase I solution for 15 to 20 minutes, and repeat the operation at least 3 times; The composition of the DNase I solution is as follows: 1 mg / mL DNase I, 5 mM MgCl2, and the solvent is 0.01 M PBS, pH 7.4; The number of flushing steps in step (1)B is at least 3 times; The voltage described in step (2) is 1200-1400V; The pulse time described in step (2) is 30 to 40 ms.

4. Use of the construction method for obtaining modified HL60 cells according to any one of claims 1 to 3 in the study of acute myeloid leukemia.

5. A HL60 cell line with CD44 and / or PSGL-1 knockout, characterized in that: The cell line is a HL60 cell line in which the CD44 gene is knocked out, the PSGL-1 gene is knocked out, or both the CD44 and PSGL-1 genes are knocked out.

6. The method for preparing the HL60 cell line with CD44 and / or PSGL-1 knockout according to claim 5, characterized in that The steps include: 1) Preparation of RNP complex: Mix equal volumes of 1.5 μg / mL Cas9 protein solution and 1 μg / mL sgRNA solution in a sterile EP tube, pipette to mix evenly, and then incubate in a 20-30°C metal bath to fully form an RNP complex; sgRNA is sgRNA targeting CD44 gene and / or sgRNA targeting PSGL-1 gene; 2) Preparation of HL60 cell fluid: HL60 cells cultured in complete medium to the logarithmic growth phase were collected, the medium was removed and then washed with PBS, and finally the cells were resuspended in SBB buffer to 2×10 7 / mL, and HL60 cell fluid was obtained; 3) Preparation of a mixed system of RNP and cells: The RNP complex and HL60 cell fluid are mixed to obtain a mixed system of RNP and cells, wherein the volume content of the RNP complex in the mixed system of RNP and cells cannot exceed 10%; 4) Electroporation: Take 10 μL of the RNP and cell mixture each time and put it into the electroporation needle, then place it in the electroporation cup, use the Neon transfection system to electroporate the cells with 1300V, 35ms pulse, and then transfer to complete medium and culture for 5-7 days; 5) Screening: The cells cultured in step 4) were centrifuged and the supernatant was discarded. The cells were then resuspended in PBS containing 2% v / v FBS, and then CD44 antibody and / or CD162 antibody were added and incubated at 4°C. After incubation, the cells were washed to remove the unincubated antibodies, and then the cells were resuspended in PBS containing 2% v / v FBS to 5×10 6 / mL, which are the cells to be sorted; HL60 cells without antibody incubation and electroporation are used as negative control, and HL60 cells incubated with corresponding antibodies but without electroporation are used as positive control. The sorted cells are loaded and negative cells are sorted by flow cytometry; 6) Obtaining monoclonal cells: diluting the negative cells obtained by sorting by limiting dilution method to obtain monoclonal cells; 7) Verification: After the monoclonal cells obtained in step 6) are expanded and cultured, nucleic acid detection and protein detection of CD44 and / or PSGL-1 are performed to obtain HL60 cell lines with CD44 and / or PSGL-1 knockout.

7. The method for preparing the HL60 cell line with CD44 and / or PSGL-1 knockout according to claim 6, characterized in that: The sequence of the sgRNA targeting the CD44 gene described in step 1) is as follows: 5'-CTACAGCATCTCTCGGACGG-3'; The sequence of the sgRNA targeting the PSGL-1 gene described in step 1) is as follows: 5'-CTCCGGTCCCGGGCAAGCAG-3'.

8. The method for preparing the HL60 cell line with CD44 and / or PSGL-1 knockout according to claim 6, characterized in that: The blowing time in step 1) is 20s to 40s; The temperature of the metal bath in step 1) is 24-26°C; The incubation time in step 1) is 15 to 25 minutes; The complete culture medium in step 2) is RPMI1640 culture medium containing 10-12% fetal bovine serum and 1% double antibody by volume; The number of washing steps in step 2) is 2 to 3 times; The RNP complex described in step 3) and the HL60 cell solution are mixed at a volume ratio of 1:10; The complete culture medium in step 4) is RPMI 1640 culture medium containing 10-12% fetal bovine serum by volume; The incubation time in step 5) is 20 to 40 minutes; The number of washings in step 5) is 2 to 3 times.

9. The method for preparing the HL60 cell line with CD44 and / or PSGL-1 knockout according to claim 6, characterized in that: The nucleic acid detection in step 7) is to amplify CD44 and / or PSGL-1 genes by PCR, and the obtained products are subjected to gene sequencing; The protein detection in step 7) is Western Blot, wherein the antibodies used are CD44 antibody and / or CD162 antibody.

10. Use of the HL60 cell line knocked out of CD44 and / or PSGL-1 according to claim 5 in studying endothelial selectin ligands.

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