Gene editing method of neutrophil

Abstract

The invention relates to the technical field of biology, in particular to a gene editing method of neutrophil. The method comprises the following steps: carrying out electrotransfection on neutrophils; then resuspending the neutrophils subjected to electrotransfection in a cell culture solution, and culturing in an incubator under a standard condition to realize gene editing. The cell culture solution is prepared by taking a universal caspase inhibitor and a growth factor as main bodies, supplementing a lysosomal membrane permeation inhibitor, a cell oxidation inhibitor and a cell necrosis inhibitor, and matching with an RPMI culture solution and fetal calf serum as solvents. The transfected gene can be effectively expressed in the neutrophil for a long time, the transfection efficiency can reach 78% at most, gene editing of the neutrophil is achieved, and the immunocompetence of the cell can be kept for a long time within 5 days after transfection; the problem of gene editing of the neutrophile granulocytes in the fields of life science and medicine at present is solved, and a basis is provided for further research of the neutrophile granulocytes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for gene editing of neutrophils. Background Art

[0002] Neutrophils account for 50-70% of white blood cells in the blood and are the first white blood cells to be recruited to the site of infection. The local accumulation of neutrophils during infection is essential for the body to clear pathogens, and a reduction in the number of neutrophils in the blood can lead to severe immunodeficiency in humans. Neutrophils are terminally differentiated cells with a very short circulation lifespan. Currently, effective gene editing of neutrophils cannot be achieved, and any gene editing operation will lead to massive cell death, which seriously hinders neutrophil-related research and its clinical application.

[0003] Although some cell lines, such as HL-60, PLB-985, NB4, and Kasumi-1, as well as induced pluripotent stem cells, may serve as neutrophil alternatives and can be genetically edited, they are very different from primary neutrophils. Therefore, most researchers in the field still rely on primary neutrophils freshly isolated from human peripheral blood and mouse bone marrow.

[0004] Therefore, gene editing of primary neutrophils has not yet been achieved, which has hindered the progress of neutrophil research. Currently, in vitro gene editing of neutrophils is a major unresolved problem in the fields of life sciences and medicine. Summary of the invention

[0005] In view of the above-mentioned problems or shortcomings, and to solve the above-mentioned difficulties in neutrophil gene editing, the present invention provides a neutrophil gene editing method.

[0006] A method for gene editing of neutrophils, the specific steps are as follows:

[0007] Step 1: Collect the target primary neutrophils by centrifugation and resuspend them in the electroporation reagent, 1-5 million primary neutrophils / 100 μL electroporation reagent.

[0008] Step 2: Add the target plasmid for gene editing to the cell resuspension obtained in step 1 and mix thoroughly at a ratio of 1-10 μg plasmid / million cells.

[0009] Step 3: Transfer the plasmid / cell / electroporation reagent mixture obtained in step 2 to a dedicated electroporation test tube, place it in an electroporator, and turn on the electroporator to perform intracellular transfer of DNA.

[0010] Step 4: After the electroporation procedure in step 3 is completed, the resulting plasmid / cell / electroporation reagent mixture is transferred to a pre-warmed culture plate / dish / flask containing culture medium.

[0011] Preheat the cell culture medium and culture plates / dishes / flasks in a cell culture incubator at 37°C and 5% CO2.

[0012] The cell culture fluid comprises a pan-caspase inhibitor and a growth factor, and the solvent is RPMI culture fluid and fetal bovine serum, wherein the volume ratio of the fetal bovine serum to the RPMI culture fluid is 5%-99%, or full serum culture is used.

[0013] Step 5: Culture the cells in an incubator at 37°C, 5% CO2 for at least 2 hours.

[0014] Furthermore, the growth factor is granulocyte colony-stimulating factor (G-CSF) with a concentration of 1-100 ng / mL.

[0015] Furthermore, the pan-caspase inhibitor is Q-VD-Oph, Z-VAD-FMK or Emr icasan; when it is Q-VD-Oph, its concentration is 1-100 μM; when it is Z-VAD-FMK, its concentration is 1-250 μM; when it is Emr icasan, its concentration is 1-100 μM.

[0016] Furthermore, the cell culture medium and cell freezing medium also include at least one of a lysosomal membrane permeable LMP inhibitor, a cell oxidation inhibitor and a cell necrosis inhibitor.

[0017] Furthermore, the lysosomal membrane permeable LMP inhibitor is Hsp70 and / or deferoxamine mesylate DFO; the concentration of Hsp70 is 1pM-1μM; the concentration of DFO is 0.1-10μM.

[0018] Furthermore, the cell oxidation inhibitor is N-acetylcysteine ​​(NAC) with a concentration of 1 μM-10 mM.

[0019] Furthermore, the cell necrosis inhibitor is Nec-1 s, and its concentration is 1-100 μM.

[0020] Furthermore, the neutrophils are neutrophils of human and mouse origin, extracted from bone marrow and / or peripheral blood.

[0021] In summary, the present invention electrotransforms neutrophils (1-5 million cells / 100 μL electrotransformation reagent / reaction tube); then the electrotransformed neutrophils are resuspended in a cell culture medium, and cultured in an incubator under standard conditions (37°C, 5% CO2) at a cell density of 0.1-10 million cells / mL to achieve gene editing. The present invention can effectively enable the transfected gene to be expressed in neutrophils for a long time, not only achieving gene editing of neutrophils, but also maintaining the immune activity of cells for a long time within 5 days after transfection, solving the problem in this field and providing a basis for further research on neutrophils. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the transfection efficiency of mouse neutrophils after transfection in the example.

[0023] Figure 2 Schematic diagram of the phagocytic function test results of mouse neutrophils after transfection in the example. DETAILED DESCRIPTION

[0024] In order to make the invention purpose, technical scheme and beneficial technical effect of the present invention clearer, the present invention is further described in detail in conjunction with the embodiments below, and people familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Unless otherwise specified, the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, cell culture and related fields in the technical field.

[0025] Example

[0026] A method for gene editing of neutrophils, comprising the following steps:

[0027] Step 1. In this embodiment, primary neutrophils from mouse bone marrow are isolated. The method for extracting mouse neutrophils is as follows: 1) preparing mouse bone marrow cells into a single cell suspension by physical blowing; 2) mixing the single cell suspension provided in step 1) with layering solutions of different concentrations and centrifuging them to provide mouse neutrophils.

[0028] The obtained mouse neutrophils were resuspended in an electroporation reagent solution (Amaxa Lonza VPA-1007) at 2 million / 100 μL.

[0029] Step 2: Add 4-5 μg of plasmid containing green fluorescent protein GFP for gene editing to the cell resuspension obtained in step 1 and mix thoroughly.

[0030] Step 3: Transfer the plasmid / cell / electroporation reagent mixture obtained in step 2 to a dedicated electroporation tube and place it in an electroporator (Amaxa Nucleofector, Lonza). Turn on the electroporator and use program Y-001 to perform intracellular transfer of DNA by electroporation.

[0031] Step 4: After the electroporation procedure in step 3 is completed, gently add 1 mL of preheated cell culture medium to the electroporation tube, and then transfer the resulting plasmid / cell / electroporation reagent mixture to the preheated culture plate.

[0032] Cell culture medium and culture plates were pre-warmed in a cell culture incubator at 37°C and 5% CO2.

[0033] The cell culture medium includes Q-VD-OPH (50 μmol / L), Hsp70 (10 pmol / L), deferoxamine mesylate DFO (1 μmol / L), N-acetylcysteine ​​NAC (10 μmol / L), cell necrosis inhibitor Nec-1 s (1 mmol / L), and granulocyte colony stimulating factor (G-CSF, 10 ng / mL). The volume ratio of the cell culture medium solvent RPMI culture medium and fetal bovine serum is 5:1.

[0034] Step 5: Cultivate the cells in an incubator at 37° C. and 5% CO 2 for at least 2 hours. The cell culture density is 0.1-10 million cells / mL. The density in this embodiment is 2 million cells / mL.

[0035] After the culture was completed, mouse neutrophils were collected and the GFP fluorescence ratio was detected by flow cytometry. Figure 1 Schematic diagram of the transfection efficiency results of transfected mouse neutrophils after culturing for 5 hours, 1 day, 3 days and 5 days in the example.

[0036] The phagocytic function test of neutrophils is as follows:

[0037] 1) The mouse neutrophils after gene editing in the implementation example were cultured in an incubator at 37°C, 5% CO2 standard conditions for 24 hours: Phrodo™ Green E.coli bioparticles labeled with fluorescein (Invitrogen) were taken out from the refrigerator and conditioned with 12.5% ​​mouse serum at 37°C for 30 minutes.

[0038] 2) Serum-conditioned bioparticles were added to mouse neutrophils at a ratio of 10:1 (bioparticles: mouse neutrophils) and incubated at 37°C for 2 hours.

[0039] 3) Take pictures using a fluorescence microscope and count the biological particles internalized into mouse neutrophils.

[0040] Phagocytosis rate (Phagocytosis index) represents the average number of biological particles in a neutrophil that has engulfed biological particles (at least 100 cells should be counted in each random field of view in each group); phagocytosis efficiency (Phagocytosis efficiency) represents the ratio of the number of neutrophils that have engulfed at least one biological particle to the total number of neutrophils (at least 200 cells should be counted in each random field of view in each group). Figure 2 The figure is a schematic diagram of the phagocytic function test results of transfected neutrophils in the embodiment. Neutrophils cultured in vitro for 24 hours after gene editing can maintain the phagocytic activity equivalent to that of freshly extracted neutrophils without gene editing.

[0041] It can be seen from the above embodiments that the present invention electrotransforms neutrophils (1-5 million cells / 100 μL electrotransformation reagent / reaction tube); then the electrotransformed neutrophils are resuspended in a cell culture medium, and cultured in an incubator under standard conditions at a cell density of 0.1-10 million cells / mL to achieve gene editing; the cell culture medium is mainly composed of pan-caspase inhibitors and growth factors, supplemented with lysosomal membrane permeability inhibitors, cell oxidation inhibitors and cell necrosis inhibitors, and then matched with RPMI culture medium and fetal bovine serum as solvents. The present invention can make the transfected genes effectively expressed in neutrophils for a long time, with a transfection efficiency of up to 78%, not only realizing the gene editing of neutrophils, but also maintaining the immune activity of cells for a long time within 5 days after transfection; it solves the current problem of neutrophil gene editing in the fields of life sciences and medicine, and provides a basis for further research on neutrophils.

Claims

1. A method for gene editing of neutrophils, characterized in that: The following steps are involved: Step 1: Collect the target primary neutrophils by centrifugation and resuspend them in an electroporation reagent, 1-5 million primary neutrophils / 100 μL electroporation reagent; Step 2: Add the target plasmid for gene editing to the cell resuspension obtained in step 1 and mix thoroughly at a ratio of 1-10 μg plasmid / million cells; Step 3, transfer the plasmid / cell / electroporation reagent mixture obtained in step 2 into a dedicated test tube for electroporation, place it in an electroporator, and turn on the electroporator to perform intracellular transfer of DNA; Step 4: After the electroporation procedure in step 3 is completed, the resulting plasmid / cell / electroporation reagent mixture is transferred to a pre-warmed culture plate / dish / flask containing culture medium; Preheat the cell culture medium and culture plates / dishes / flasks in a cell culture incubator at 37°C and 5% CO2. The cell culture medium comprises a pan-caspase inhibitor and a growth factor, the solvent is RPMI culture medium and fetal bovine serum, the volume ratio of fetal bovine serum to RPMI culture medium is 5%-99%, or full serum culture; Step 5: Culture the cells in an incubator at 37°C, 5% CO2 for at least 2 hours.

2. The method for gene editing of neutrophils according to claim 1, characterized in that: The growth factor is granulocyte colony stimulating factor G-CSF, and its concentration is 1-100 ng / mL.

3. The method for gene editing of neutrophils according to claim 1, characterized in that: The pan-caspase inhibitor is Q-VD-Oph, Z-VAD-FMK or Emricasan; when it is Q-VD-Oph, its concentration is 1-100 μM; when it is Z-VAD-FMK, its concentration is 1-250 μM; when it is Emricasan, its concentration is 1-100 μM.

4. The method for freezing and resuscitating neutrophils according to claim 1, characterized in that: The cell culture solution and cell freezing solution also include at least one of a lysosome membrane permeable LMP inhibitor, a cell oxidation inhibitor and a cell necrosis inhibitor.

5. The method for gene editing of neutrophils according to claim 4, characterized in that: The lysosomal membrane permeable LMP inhibitor is Hsp70 and / or deferoxamine mesylate DFO; the concentration of Hsp70 is 1pM-1μM; the concentration of DFO is 0.1-10μM.

6. The method for gene editing of neutrophils according to claim 4, characterized in that: The cell oxidation inhibitor is N-acetylcysteine ​​NAC, and its concentration is 1 μM-10 mM.

7. The method for gene editing of neutrophils according to claim 4, characterized in that: The cell necrosis inhibitor is Nec-ls, and its concentration is 1-100 μM.

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