CN116042713A - Electrotransfection method of NK-92 cells - Google Patents

Abstract

The application relates to the technical field of cell transfection, in particular to an electrotransfection method of NK-92 cells. The electrotransfection method comprises the following steps: mixing exogenous gene vectors, NK-92 cells and an electrotransfection buffer solution to obtain an electrotransfection system; carrying out electric shock on NK-92 cells, wherein the electric shock is single pulse, the voltage of the single pulse is 500-580V, and the time of the single pulse is 15-25 ms; the NK-92 cells after electric shock were allowed to stand still, and then cell culture was performed. The electrotransfection method can maintain the cell activity rate and greatly improve the transfection efficiency.

Description

A kind of electrotransfection method of NK-92 cell

technical field

The present application relates to the technical field of cell transfection, in particular to an electrotransfection method for NK-92 cells.

Background technique

Electroporation refers to the transient and reversible breakdown of the cell membrane induced by applying an external electric field that just exceeds the capacitance of the cell membrane. Nucleic acid and other substances can enter the cell through these temporary holes. Physical transfection method for stable transfection. Furthermore, due to the simplicity and speed of electroporation, once the optimal electroporation conditions are determined, it is possible to transfect a large number of cells in a short period of time. The major disadvantages of electroporation are the massive cell death and only partially successful membrane repair induced by high voltage pulses. Compared with other transfection methods, a larger number of cells needs to be used, and electrotransfection parameters and experimental conditions need to be adjusted according to different cell types.

NK-92 cells, an interleukin-2 (IL-2)-dependent natural killer cell line, were derived from peripheral blood mononuclear cells of a 50-year-old Caucasian male with rapidly progressive non-Hodgkin's lymphoma. NK-92 cells are frequently used in cancer, immunology and toxicology research. This cell line is a suitable transfection host and is cytotoxic to a variety of malignant cells: it kills K562 cells and Daudi cells in a chromium release assay. NK-92 cells (irradiated to prevent proliferation) can be effectively used for immune ex vivo clearance of leukemia in blood without affecting hematopoietic cell function.

At present, the existing NK-92 editing method is based on the CRISPR/Cas9 system. The RNP complex formed by the Cas9 protein with a nuclear localization sequence and the sgRNA corresponding to the target gene is transferred into the cell by physical transfection by electroporation. , and the target gene was knocked out after the PAM sequence in the sgRNA recognized the target site. Commonly used electrotransfection parameters for NK-92 cell line include: 360V, 20ms; 300V, 70ms double pulse, and the interval between two pulses is one minute. Commonly used electrotransfection experimental conditions for the NK-92 cell line include: 2.0-3.0× ¹⁰⁶ cells in a 20 μL electroporation system, 50 pmol NLS-SpCas9-EGFP protein and 100 pmol sgRNA A, and a resting time of 5 minutes after electroporation. When the NK-92 cell line was electrotransfected with the electrotransfection parameters and experimental conditions, the positive rate of electrotransfection was only 10%. Moreover, it has been found through many experiments that the gene knockout ratio is only 30% of the transfection positive rate, which is difficult to obtain a gene knockout monoclonal cell line.

Contents of the invention

In view of this, the present invention provides a method for electrotransfection of NK-92 cells. This method uses fewer NK-92 cells to obtain higher transfection efficiency and editing efficiency while ensuring cell viability after electroporation.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a method for electrotransfection of NK-92 cells, comprising the steps of:

Step (1): mixing the exogenous gene carrier, NK-92 cells and electrotransfection buffer to obtain an electrotransfection system;

Step (2): Shock the NK-92 cells with a single pulse, the voltage of the single pulse is 500-580V, and the time of the single pulse is 15-25ms;

Step (3): The NK-92 cells after electric shock were left still, and then cell culture was carried out.

Preferably, the voltage of a single pulse is 540V, and the time of a single pulse is 20ms.

Preferably, the cell density of NK-92 cells in the electroporation system is (5-7.5)×10 ⁴ cells/μL.

Preferably, the cell density of NK-92 cells in the electroporation system is (5-6)×10 ⁴ cells/μL.

More preferably, the cell density of NK-92 cells in the electroporation system is 5×10 ⁴ cells/μL.

Preferably, the volume of the electroporation system is 15-25 μL.

Preferably, the electroporation system is 20 μL.

Preferably, the number of NK-92 cells in the 20 μL electroporation system is (1.0˜1.5)×10 ⁶ .

Preferably, the number of NK-92 cells in the 20 μL electroporation system is 1.0×10 ⁶ .

In a specific embodiment provided by the present invention, the exogenous gene vector includes a target gene knockout vector and/or a target gene transfer vector.

Preferably, the carrier for knocking out the target gene is Cas9/sgRNA ribonucleoprotein;

Preferably, the vector for transferring the target gene is a plasmid containing the target gene.

As preferably, the exogenous gene carrier is Cas9/sgRNA ribonucleoprotein, and step (1) is:

Cas9 protein, sgRNA and electroporation buffer are made into a mixed solution, and after the first incubation, a mixture containing Cas9/sgRNA ribonucleoprotein is obtained;

Mix NK-92 cells with the mixture containing Cas9/sgRNA ribonucleoprotein, and undergo a second incubation to obtain an electroporation system.

As a preference, in the mixed solution, the Cas9 protein is added in an amount of 2-3 pmol/μL;

Preferably, the amount of Cas9 protein added is 2.5 pmol/μL.

Preferably, in the mixed solution, the amount of sgRNA added is 6-9 pmol/μL;

Preferably, the amount of sgRNA added is 7.5 pmol/μL.

Preferably, the temperature of the first incubation is 15-30° C., and the time is 2-10 minutes.

Preferably, the temperature of the first incubation is room temperature, and the time is 5-10 minutes;

Preferably, the temperature of the second incubation is 15-30° C., and the time is 5-15 minutes.

Preferably, the temperature of the second incubation is room temperature and the time is 10 min.

Preferably, the standing temperature is 36-38° C., and the time is 5-10 minutes.

Preferably, the standing temperature is 37° C. and the time is 5 minutes.

Compared with prior art, the beneficial effect that the present invention has is:

1. It can improve the survival rate of NK-92 cell line after electroporation transfection:

The present invention found that parameters such as the number of electric shock pulses, voltage, pulse time, and resting time in electroporation (electrotransfection) have a greater impact on the viability of NK-92 cells after being shocked. Time, resting time and other parameters not only shorten the electric shock time, but also increase the survival rate of NK-92 cell line after electroporation transfection, thereby reducing the number of cells required for electrotransfection.

The existing conventional electrotransfection protocol for NK-92 cell line requires 2.0-3.0× ¹⁰⁶ cells in a 20 μL electroporation system, and the cell viability reaches about 80% on the day after electroporation, and the cell viability 24 hours after electroporation transfection After 48 hours of electrotransfection, the cell viability increased, and the highest recovery rate was about 95%.

The NK-92 cell line electrotransfection scheme of the present invention only needs 1.0-1.5×10 ⁶ cells in a 20 μL electrotransfer system, using a single pulse voltage of 540V, a pulse time of 20ms, and a resting time of 5-10 minutes after electrotransfer. The cell viability reached about 85%. The cell viability decreased significantly 24 hours after electroporation and transfection. After 48 hours of electrotransfection, the cell viability increased, and the highest recovery was 90% (Fig. 6 or Fig. 14).

It can be seen that compared with the conventional electrotransfection method, the electrotransfection method of the present invention can effectively maintain the survival rate of cells after electric shock.

2. It can improve the electrotransfection positive rate of NK-92 cell line based on CRISPR/Cas9 editing system:

Under the premise that the NK-92 cell line can survive after electroporation, the present invention improves the efficiency of the NK-92 cell line based on the CRISPR/Cas9 editing system by optimizing the electroporation transfection parameters and the ratio and addition amount of CRISPR/Cas9 and sgRNA. Electrotransfection positive rate of cell lines, and gene editing efficiency under different conditions.

The existing conventional electrotransfection protocol for NK-92 cell line requires 2.0-3.0× ¹⁰⁶ cells in a 20 μL electroporation system. After 24 hours of electroporation and transfection, the transfection efficiency (EGFP positive rate) detected by flow cytometry is only 10%.

The NK-92 cell line electrotransfection scheme of the present invention only needs 1.0-1.5× ¹⁰⁶ cells in a 20 μL electroporation system, and after 24 hours of electroporation transfection, the transfection efficiency (EGFP positive rate) can reach 47.39% by flow cytometry .

It can be seen that compared with the conventional electrotransfection method, the electrotransfection method of the present invention can greatly improve the transfection efficiency while maintaining the cell viability.

Description of drawings

Figure 1 Effects of different cell additions and different resting times after electroporation on cell viability in the electroporation system;

Figure 2 Effects of different cell additions and different resting times after electroporation on cell density in the electroporation system;

Figure 3 Effects of different resting times after electroporation and different cell additions in the electroporation system on cell viability;

Figure 4 Effects of different resting times and different cell additions in the electroporation system on cell density after electroporation;

Figure 5 Effects of different pulse voltages on cell density during electroporation;

Figure 6 Effects of different pulse voltages on cell viability during electroporation;

Figure 7: Effects of different pulse voltages on cell viability and transfection efficiency during electroporation;

Figure 8 GFP signal flow detection diagram;

Figure 9: Effects of different pulse times on cell density during electroporation;

Figure 10 Influence of different pulse times on cell viability during electroporation;

Figure 11 Influence of different pulse times on cell viability and transfection efficiency during electroporation;

Figure 12 GFP signal flow detection diagram;

The influence of different sgRNA:Cas9-EGFP molar ratio on cell density in the process of preparing RNP in Fig. 13;

Figure 14 The influence of different sgRNA:Cas9-EGFP molar ratios on cell viability in the process of preparing RNP;

The influence of different sgRNA:Cas9-EGFP molar ratio on cell viability and transfection efficiency in the process of preparing RNP in Fig. 15;

Figure 16 GFP signal flow detection diagram;

The effect of different Cas9 protein additions on cell density during the preparation of RNP in Figure 17;

The effect of different Cas9 protein additions on cell viability during the preparation of RNP in Figure 18;

Figure 19 Effects of different Cas9 protein additions on cell viability and transfection efficiency during the preparation of RNP;

Figure 20 GFP signal flow detection diagram.

Detailed ways

The invention discloses a method for electrotransfection of NK-92 cells. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.

Explanation of terms:

NLS-SpCas9-EGFP: It is the wild-type Cas9 protein from Streptococcus pyogenes recombinantly expressed in E. coli. The NLS signal peptide was added to the N-terminus of the protein, and the EGFP fluorescent tag was added to the C-terminus of the protein. It can be used for cutting in vitro, and can also be used for cell gene editing. The protein has all the activities of the Cas9 protein, and at the same time, the successfully transfected cells can be screened by fluorescence.

PD-1: Programmed death receptor 1, also known as CD279, is an important immunosuppressive molecule.

Ribonucleoprotein: The English name ribonucleoprotein, referred to as nucleoprotein or RNP, refers to a nucleoprotein that contains RNA, a form that combines nucleic acid and protein. Ribonucleoproteins include ribosomes, telomerase, and small nuclear RNPs (snRNPs).

In the following examples, the calculation method of the knockout efficiency (electrotransfer editing efficiency) of the PD-1 gene is as follows:

The PD-1 fragment was amplified by PCR, connected to a blunt-ended cloning vector, and then transformed into E. coli competent cells, spread on a solid LB medium plate with kanamycin resistance, and placed in a 37°C incubator Overnight, pick 10-20 monoclonal colonies for sequencing the next day. The sequencing results were compared with the WT PD-1 gene fragments, and the deletion of the gene fragments indicated that the PD-1 knockout was successful. The ratio of the number of single clones successfully knocked out of PD-1 to the total number of single clones is the knockout efficiency or electroporation editing efficiency of the PD-1 gene.

The reagents, instruments, cells or other biological materials used in the present invention can be obtained through commercial channels. Wherein, in the following examples, the electroporation instrument and electroporation buffer used are Celetrix electroporation instrument and supporting electroporation buffer (Celetrix electroporation buffer A solution and B solution).

Below in conjunction with embodiment, further set forth the present invention:

Experimental example 1

1. Preparation before the experiment

1.1 Add 3 mL recovery medium to each group in the six-well plate in advance, preheat in a 5% CO ₂ constant temperature (37°C) incubator for more than 20 minutes, take out Celetrix electroporation buffer A and B from the 4°C refrigerator and place it at room temperature and mix equal volumes of electroporation buffer A and B, and mark it as electroporation buffer C solution (for 20 μL electroporation system, take 15 μL solution of LA + 15 μL solution of B for each group to prepare solution C), and store it at room temperature in the dark.

1.2 The number of cells to be electroporated is 6.0-10.0×10 ⁶ [calculation method: number of cells=density×volume. Specifically, determine the density of cells to be transfected as C1 (cells/mL), draw V1 mL [V1=(6.0～10.0×10 ⁶ )/C1] of NK-92 cells into a 50 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard clear;

1.3 Add 20mL PBS to resuspend the cell pellet, centrifuge at 1000rpm for 5min, and discard the supernatant;

1.4 Add 1-1.5mL PBS to resuspend the cell pellet, pipette evenly, count the cells, and record the cell density as C2 (cells/mL);

2. RNP preparation, namely the preparation of NLS-SpCas9-EGFP/sgRNA:

Take 50pmol NLS-SpCas9-EGFP (about 9.5μg) and 150pmol PD-1sgRNA (the molar ratio of sgRNA to NLS-SpCas9-EGFP is 3:1) into a 0.2mL PCR tube, mix gently and incubate at room temperature for 5min, then add Transfer buffer C to a volume of 20 μL, mix gently and then incubate at room temperature for 5 minutes;

3. Cell preparation

3.1 Take the number as 1.0～1.5×10 ⁶ {calculation method: cell number=density×volume. Specifically, pipette V2mL cells [V2=(1.0～1.5×10 ⁶ )/C2]} to transfer the cells to a 1.5mL centrifuge tube, centrifuge at 1000rpm for 5min, and discard the supernatant;

3.2 Gently mix the cells in 3.1 with the RNP solution in 2.1, and incubate at room temperature for 10 min.

4. Electric transfer

4.1 Electroporation conditions: Celetrix electroporation instrument, 360V/20ms, 300V/70ms double pulse, and the interval between two pulses is one minute;

4.2 Immediately after the electroporation, place the electroporation cup in a 37°C incubator and let it stand for 0-20min, then transfer all the samples to a six-well plate containing preheated medium, in a 5% _CO2 constant temperature (37°C) incubator cultivated in.

The specific number of cells in step 3.2 and the specific resting time in step 4.2 are shown in Table 1:

Table 1

5. Detection of transfection efficiency

5.1 Take 700 μL of cells at 0h, 24h, 48h, and 72h after electroporation into a 1.5mL EP tube, centrifuge at 400g for 5 minutes, and discard the supernatant.

5.2 Add 1mL 1×PBS to resuspend the cells, centrifuge again at 400g for 5 minutes, and discard the supernatant.

5.3 Add 40 μL of 1×PBS to resuspend the cells, use NK-92 cells without electroporation as the negative control, select the FITC channel to set the gate, and detect the viability, density, and GFP positive rate of the cells after electroporation in different electroporation systems. Reflect the positive rate of electrotransfection.

6. Experimental results

The results shown in Figure 1-4 show that the minimum cell number required for the electroporation system of the 20 μL system is 1×10 ⁶ cells, the optimal cell number is 1.0-1.5×10 ⁶ cells, and the optimal resting time after electroporation is 5-10 minutes .

When the number of cells is 5E5, 1E6, 1.5E6, and 2E6, the viability of the cells varies with the resting time after electroporation. When the resting time was 5 or 10 minutes, the recovery of cell viability from 0h to 72h after electroporation was the best.

When the resting time was 0 minutes, 5 minutes, 10 minutes, and 20 minutes, the cell numbers during electroporation were 5E5, 1E6, 1.5E6, and 2E6, and the corresponding cell viability after electroporation was also different. By comparison, when the number of cells was 5E5, the cell viability rates under the four standing times were the worst; the cell viability rates corresponding to the three electroporated cell numbers of 1E6, 1.5E6, and 2E6 were at 0 minutes, 5 minutes, Not much difference at 10 minutes.

In summary, the minimum cell number required for the 20 μL electroporation system is 1×10 ⁶ cells, the preferred cell number is 1.0–1.5×10 ⁶ cells, and the optimal resting time after electroporation is 5–10 minutes.

Experimental example 2

1. Preparation before the experiment

Same as experimental example 1.

2. RNP preparation, namely the preparation of NLS-SpCas9-EGFP/sgRNA:

Take 50pmol NLS-SpCas9-EGFP (about 9.5μg) and 100pmol PD-1sgRNA (the molar ratio of sgRNA to NLS-SpCas9-EGFP is 2:1) into a 0.2mL PCR tube, mix gently and incubate at room temperature for 5min, then add Transfer buffer C to a volume of 20 μL, mix gently and then incubate at room temperature for 5 minutes;

3. Cell preparation

3.1 Take the number as 1.0～1.5×10 ⁶ {calculation method: cell number=density×volume. Specifically, pipette V2mL cells [V2=(1.0～1.5×10 ⁶ )/C2]} to transfer the cells to a 1.5mL centrifuge tube, centrifuge at 1000rpm for 5min, and discard the supernatant;

3.2 Gently mix 1.0×10 ⁶ cells in 3.1 with the RNP solution in 2.1, and incubate at room temperature for 10 min.

4. Electric transfer

4.1 Electroporation conditions: Celetrix electroporation instrument, electroshock conditions are shown in Table 2;

Table 2

4.2 Immediately after the electroporation, place the electroporation cup in a 37°C incubator for 5 minutes, then transfer all samples to a six-well plate containing preheated medium, and culture in a 5% CO ₂ constant temperature (37°C) incubator .

5. Detection of transfection efficiency

Same as experimental example 1.

6. Experimental results

The results in Figure 5-8 show that with the increase of the electroporation voltage, the cell transfection efficiency increased and the cell viability decreased 24 hours after electroporation; when the voltage reached 600V, the transfection efficiency was up to 67.62%, but the cell viability was only 22% %, and the cell viability was only 32% at 48h after electroporation. Considering comprehensively, when the pulse time is 20ms, the optimal single pulse voltage of electrotransfer is 540V. Under the condition of 540V, the knockout efficiency of PD-1 gene was 10%.

Experimental example 3

1. Preparation before the experiment

Same as experimental example 1.

2. RNP preparation, namely the preparation of NLS-SpCas9-EGFP/sgRNA:

Take 50pmol NLS-SpCas9-EGFP (about 9.5μg) and 150pmol PD-1sgRNA (the molar ratio of sgRNA to NLS-SpCas9-EGFP is 3:1) into a 0.2mL PCR tube, mix gently and incubate at room temperature for 5min, then add Transfer buffer C to a volume of 20 μL, mix gently and then incubate at room temperature for 5 minutes;

3. Cell preparation

3.1 Take the number as 1.0～1.5×10 ⁶ {calculation method: cell number=density×volume. Specifically, pipette V2mL cells [V2=(1.0～1.5×10 ⁶ )/C2]} to transfer the cells to a 1.5mL centrifuge tube, centrifuge at 1000rpm for 5min, and discard the supernatant;

3.2 Gently mix 1.0×10 ⁶ cells in 3.1 with the RNP solution in 2.1, and incubate at room temperature for 10 min.

4. Electric transfer

4.1 Electroporation conditions: Celetrix electroporation instrument, electroshock conditions are shown in Table 3;

table 3

4.2 Immediately after the electroporation, place the electroporation cup in a 37°C incubator for 5 minutes, then transfer all samples to a six-well plate containing preheated medium, and culture in a 5% CO ₂ constant temperature (37°C) incubator .

5. Detection of transfection efficiency

Same as experimental example 1.

6. Experimental results

The results shown in Figures 9-12 show that when the single pulse voltage is 540V, as the electroporation pulse time increases, the cell viability decreases, and the transfection efficiency increases 24 hours after electroporation. When the pulse time was increased to 30ms or 40ms, although the transfection efficiency increased significantly, the recovery of cell viability after electroporation was slow. Considering comprehensively, when the single pulse voltage is 540V, the optimal pulse time of electroporation is set at 20ms. Under this optimal condition, the PD-1 gene knockout efficiency was 25%.

Experimental example 4

1. Preparation before the experiment

Same as experimental example 1.

2. RNP preparation, namely the preparation of NLS-SpCas9-EGFP/sgRNA:

Take NLS-SpCas9-EGFP and PD-1sgRNA (see Table 4 for addition amount and addition ratio) into a 0.2mL PCR tube, mix gently and incubate at room temperature for 5min, then add electroporation buffer C to a volume of 20μL, mix gently and then Incubate at room temperature for 5 minutes;

Table 4

3. Cell preparation

3.1 Take the number as 1.0～1.5×10 ⁶ {calculation method: cell number=density×volume. Specifically, pipette V2mL cells [V2=(1.0～1.5×10 ⁶ )/C2]} to transfer the cells to a 1.5mL centrifuge tube, centrifuge at 1000rpm for 5min, and discard the supernatant;

3.2 Gently mix 1.0×10 ⁶ cells in 3.1 with the RNP solution in 2.1, and incubate at room temperature for 10 min.

4. Electric transfer

4.1 Electroporation conditions: Celetrix electroporation instrument, single pulse, 540V/20ms.

4.2 Immediately after the electroporation, place the electroporation cup in a 37°C incubator for 5 minutes, then transfer all samples to a six-well plate containing preheated medium, and culture in a 5% CO ₂ constant temperature (37°C) incubator .

5. Detection of transfection efficiency

Same as experimental example 1.

6. Experimental results

The results in Figure 13-16 show that in the 20 μL system, as the ratio of CRISPR/Cas9 to the corresponding sgRNA increases, the electroporation efficiency increases. But when the ratio was increased to 4:1, the cell viability decreased slightly. Considering the cell viability and the cost of the experiment, the optimal ratio of CRISPR/Cas9 to the corresponding sgRNA in a 20 μL system is 3:1.

Table 5 shows the electroporation editing efficiency of PD-1 gene locus knockout under different electroporation conditions. It can be concluded from Table 5 that different electroporation conditions have a greater impact on electroporation editing efficiency. From the optimization results of this application, it can be concluded that Under the best electroporation conditions, the electroporation editing efficiency is as high as 33.33%, far higher than the current industry technical level (less than 10%), so the electroporation system of the present application plays an important role in improving the gene editing efficiency.

table 5

Experimental example 5

1. Preparation before the experiment

Same as experimental example 1.

2. RNP preparation, namely the preparation of NLS-SpCas9-EGFP/sgRNA:

Take NLS-SpCas9-EGFP and PD-1sgRNA (see Table 6 for addition amount and addition ratio) into a 0.2mL PCR tube, mix gently and incubate at room temperature for 5min, then add electroporation buffer C to a volume of 20 μL, mix gently and then Incubate at room temperature for 5 minutes;

Table 6

3. Cell preparation

3.1 Take the number as 1.0～1.5×10 ⁶ {calculation method: cell number=density×volume. Specifically, pipette V2mL cells [V2=(1.0～1.5×10 ⁶ )/C2]} to transfer the cells to a 1.5mL centrifuge tube, centrifuge at 1000rpm for 5min, and discard the supernatant;

3.2 Gently mix 1.0×10 ⁶ cells in 3.1 with the RNP solution in 2.1, and incubate at room temperature for 10 min.

4. Electric transfer

4.1 Electroporation conditions: Celetrix electroporation instrument, single pulse, 540V/20ms.

4.2 Immediately after the electroporation, place the electroporation cup in a 37°C incubator for 5 minutes, then transfer all samples to a six-well plate containing preheated medium, and culture in a 5% CO ₂ constant temperature (37°C) incubator .

5. Detection of transfection efficiency

Same as experimental example 1.

6. Experimental results

The results in Figure 17-20 show that when the amount of Cas9 protein increased to 50-100 pmol, the electrotransfer efficiency did not increase significantly, but the cell viability decreased, and it was difficult to recover 48 hours after electrotransfer. Therefore, considering all factors, the optimal amount of Cas9 protein added is 50 pmol.

Examples 1-3

1. Preparation before the experiment

1.1 Add 3 mL recovery medium to each group in the six-well plate in advance, preheat in a 5% CO ₂ constant temperature (37°C) incubator for more than 20 minutes, take out Celetrix electroporation buffer A and B from the 4°C refrigerator and place it at room temperature and mix equal volumes of electroporation buffer A and B, and mark it as electroporation buffer C solution (for 20 μL electroporation system, take 15 μL solution of LA + 15 μL solution of B for each group to prepare solution C), and store it at room temperature in the dark.

1.2 The number of cells to be electroporated is 6.0-10.0×10 ⁶ [calculation method: number of cells=density×volume. Specifically, determine the density of cells to be transfected as C1 (cells/mL), draw V1 mL [V1=(6.0～10.0×10 ⁶ )/C1] of NK-92 cells into a 50 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard clear;

1.3 Add 20mL PBS to resuspend the cell pellet, centrifuge at 1000rpm for 5min, and discard the supernatant;

1.4 Add 1-1.5mL PBS to resuspend the cell pellet, pipette evenly, count the cells, and record the cell density as C2 (cells/mL);

2. RNP preparation, namely the preparation of NLS-SpCas9-EGFP/sgRNA:

Take 40-60pmol NLS-SpCas9-EGFP and 120-180pmol PD-1sgRNA into a 0.2mL PCR tube, mix gently and incubate at room temperature for 5min, then add electroporation buffer C to a volume of 20μL, mix gently and then incubate at room temperature for 5min;

3. Cell preparation

3.1 Take the number as 1.0～1.5×10 ⁶ {calculation method: cell number=density×volume. Specifically, pipette V2mL cells [V2=(1.0～1.5×10 ⁶ )/C2]} to transfer the cells to a 1.5mL centrifuge tube, centrifuge at 1000rpm for 5min, and discard the supernatant;

3.2 Gently mix 1.0-1.5×10 ⁶ cells in 3.1 with the RNP solution in 2.1, and incubate at room temperature for 10 min.

4. Electric transfer

4.1 Electroporation conditions: Celetrix electroporation instrument, single pulse, 500-580V/15-25ms.

4.2 Immediately after the electroporation, place the electroporation cup in a 37°C incubator and let it stand for 5-10 minutes, then transfer all the samples to a six-well plate containing preheated medium, in a 5% CO ₂ constant temperature (37°C) incubator cultivated in.

5. Detection of transfection efficiency

5.1 Take 700 μL of cells 24 hours after electroporation into a 1.5 mL EP tube, centrifuge at 400 g for 5 minutes, and discard the supernatant.

5.2 Add 1mL 1×PBS to resuspend the cells, centrifuge again at 400g for 5 minutes, and discard the supernatant.

5.3 Add 40 μL of 1×PBS to resuspend the cells, use NK-92 cells without electroporation as the negative control, select the FITC channel to set the gate, and detect the viability, density, and GFP positive rate of the cells after electroporation in different electroporation systems. Reflect the positive rate of electrotransfection.

The experimental conditions corresponding to the specific embodiments are shown in Table 7, and the experimental results are shown in Table 8.

Table 7

Table 8 Cell viability, density and GFP positive rate 24h after electroporation

serial number Cell viability (%) Cell density (cell/mL) GFP positive rate (%) Example 1 71.88% 2.13E5 37.67% Example 2 65.28% 1.78E5 48.35% Example 3 55.03% 1.46E5 51.26%

The results show that the methods of Examples 1-3 can all obtain higher electroporation efficiency and cell viability, especially the method of Example 2 is better.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (10)

1. an electrotransfection method of NK-92 cells, is characterized in that, comprises the steps: Step (1): mixing the exogenous gene carrier, NK-92 cells and electrotransfection buffer to obtain an electrotransfection system; Step (2): shocking NK-92 cells, the electric shock is a single pulse, the voltage of the single pulse is 500-580V, and the time of the single pulse is 15-25ms; Step (3): The NK-92 cells after electric shock were left still, and then cell culture was carried out. 2. The electrotransfection method according to claim 1, wherein the voltage of the single pulse is 540V, and the time of the single pulse is 20ms. 3. The electrotransfection method according to claim 1, wherein the cell density of the NK-92 cells in the electrotransfection system is (5-7.5)×10 ⁴ cells/μL; Preferably, the cell density of the NK-92 cells in the electroporation system is (5-6)×10 ⁴ cells/μL. 4 . The electrotransfection method according to claim 1 , wherein the volume of the electrotransfection system is 15-25 μL. 5. The electrotransfection method according to claim 1, wherein the exogenous gene carrier includes a carrier for knocking out the gene of interest and/or a carrier for transferring the gene of interest; The carrier for knocking out the target gene is Cas9/sgRNA ribonucleoprotein; The carrier for transferring the target gene is a plasmid containing the target gene. 6. electrotransfection method according to claim 5, is characterized in that, described exogenous gene carrier is Cas9/sgRNA ribonucleoprotein, and step (1) is: Cas9 protein, sgRNA and electroporation buffer are made into a mixed solution, and after the first incubation, a mixture containing Cas9/sgRNA ribonucleoprotein is obtained; The NK-92 cells were mixed with the Cas9/sgRNA ribonucleoprotein-containing mixture, and subjected to a second incubation to obtain an electroporation system. 7. The electrotransfection method according to claim 6, characterized in that, in the mixed solution, the amount of the Cas9 protein added is 2 to 3 pmol/μL; Preferably, the Cas9 protein is added in an amount of 2.5 pmol/μL. 8. The electrotransfection method according to claim 6, characterized in that, in the mixed solution, the added amount of the sgRNA is 6-9 pmol/μL; Preferably, the added amount of the sgRNA is 7.5 pmol/μL. 9. The electrotransfection method according to claim 6, characterized in that, the temperature of the first incubation is 15-30° C., and the time is 2-10 minutes; The temperature of the second incubation is 15-30° C., and the time is 5-15 minutes. 10. The electrotransfection method according to any one of claims 1-9, characterized in that the standing temperature is 36-38° C. and the time is 5-10 min.

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