MDA-MB-231 cell strain for expressing moesin-EGFP and construction method of MDA-MB-231 cell strain

Abstract

The invention provides a method for constructing an MDA-MB-231 cell strain for expressing moesin-EGFP (Enhanced Green Fluorescent Protein). According to the method, sgRNA of a targeted moesin gene terminator region is designed, a Donor plasmid containing LHA, Linker, EGFP and RHA sequences is constructed, and a connecting sequence is added between a moesin protein and an EGFP protein by the plasmid, so that the influence of the EGFP on the function of the moesin protein is avoided. The sgRNA, the Cas9 protein and the linearized Donor plasmid are jointly transferred into the MDA-MB-231 cell by utilizing a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) gene editing technology. According to the method, homozygote cells for expressing moesin-EGFP (enhanced green fluorescent protein) are sorted through flow cytometry, and gene editing and protein expression conditions are verified through PCR (polymerase chain reaction) and Western blot. According to the method, stable expression of the moesin-EGFP fusion protein is achieved, and the purpose of monitoring endogenous moesin expression in cells in real time is achieved. The cell strain can be used for researching the function of moesin in cell migration and signal transduction, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for constructing an MDA-MB-231 cell line expressing moesin-EGFP by utilizing CRISPR-Cas9 gene editing technology. Background Art

[0002] Moesin is a member of the ezrin-radixin-moesin (ERM) family and is widely present in smooth muscle cells and microfilament-rich cell surface structures. Studies have shown that moesin plays a key role in cancer cells, mainly through cytoskeleton remodeling, epithelial-mesenchymal transition (EMT), invadopodia formation and promotion of lymph node metastasis, enhancing the invasion and metastasis of tumor cells. Moesin promotes cytoskeleton reorganization, regulates the expression of cell adhesion molecules, and destroys cell-cell adhesion by binding to F-actin. In addition, the N-terminal FERM structure of moesin can bind to a variety of molecules, such as CD44, MT1-MMP, ICAM-1 and other molecules that are highly related to cancer, and move under the traction of F-actin, thereby bringing these molecules to specific locations on the cell membrane, promoting the function of these molecules, and improving the migration and invasion ability of tumor cells.

[0003] Therefore, researchers hope to understand the distribution of moesin in cells and the process of its recruitment of molecules, so as to make prognostic judgments for cancer patients and prevent cancer metastasis by targeting moesin and its related molecules. Some laboratories use the method of knocking out moesin and then transferring the moesin-EGFP expression plasmid to construct cells expressing moeisn-EGFP, and analyze the interaction of moeisn protein with other proteins by immunofluorescence and fluorescence microscopy.

[0004] Although this method of knocking out the target gene first and then introducing the expression plasmid can study the function of moeisn in cells to a certain extent, the high-expression plasmid and exogenous moesin are not subject to intracellular regulation, which will lead to the expression of a large amount of moeisn protein in the cells, far exceeding the expression level of normal cancer cells. It is impossible to quantitatively analyze the moesin protein, nor can it reflect the normal expression and distribution of moesin in cells. Summary of the invention

[0005] In view of the above reasons, the present invention provides a method for constructing an MDA-MB-231 cell line expressing moesin-EGFP and a method for constructing the same. First, an sgRNA targeting the terminator region of the moesin gene is designed, and a Donor plasmid containing LHA, Linker, EGFP and RHA sequences is constructed. The addition of the Linker sequence avoids the effect of EGFP on the function of the moesin protein. The moesin gene of MDA-MB-231 cells was edited using CRISPR-Cas9 gene editing technology. Then, a flow cytometer was used to screen out homozygous cells expressing moesin-EGFP, and sequencing and Western Blot detection were used to confirm the successful fusion expression of moesin and EGFP. This method achieves the purpose of real-time monitoring of endogenous moesin expression in cells, and in subsequent studies, the expression and recruitment of moesin protein in cells can be grasped in real time by fluorescence microscopy.

[0006] The method for constructing the moesin-EGFP-expressing MDA-MB-231 cell line comprises the following steps:

[0007] 1) Design of sgRNA targeting the terminator region of the moesin gene:

[0008] The genomic sequence of the moesin gene was found using the NCBI database, and the sgRNA was designed using the crispor website.

[0009] 2) Construction of a donor targeting the moesin gene:

[0010] According to the found moesin gene sequence, the corresponding Donor plasmid sequence is designed, which includes LHA, Linker, EGFP and RHA. The function of LHA and RHA is to complement the DNA in the cell, and the function of Linker is to add a connecting sequence between the moesin protein and the EGFP protein to prevent EGFP from affecting the function of the moesin protein.

[0011] 3) Amplification and extraction of plasmid

[0012] Prepare LB medium, sterilize at 121℃ for 20-30 minutes, use a sterile tube to take 3mL LB medium, add ampicillin (Amp), add a small amount of bacteria into the medium, and shake at 37℃ 200rpm for 12 hours; then take 50mL LB medium, add Amp, and then take 50μL bacterial solution, and shake at 37℃ 200rpm for 12 hours; use Adelaide plasmid midi extraction kit to extract Donor plasmid from the cultured bacteria.

[0013] 4) Donor plasmid linearization

[0014] The Donor plasmid was linearized using ScaI-HF enzyme. The reaction system included DNA Donor plasmid, NEBuffer and ScaI-HF enzyme. After enzyme digestion in a 37°C metal bath and enzyme inactivation in an 80°C metal bath, double-stranded DNA was separated and purified by agarose gel electrophoresis. DNA was recovered using a DNA gel recovery kit to finally obtain a linearized DNA Donor plasmid.

[0015] 5) Cell preparation

[0016] In terms of cell culture, 90% DMEM and 10% FBS were used to prepare the culture medium and culture MDA-MB-231 cells to ensure that the cells were in the logarithmic growth phase. When the cell confluence reached 80%, the cells were processed by first aspirating the culture medium and then washing twice with PBS. Trypsin was added to digest the cells. After 2 minutes, the digestion was terminated and centrifuged. The washed cells were resuspended and finally resuspended to the appropriate concentration using SBB electroporation solution.

[0017] 6) Establishment of cell lines expressing moesin-EGFP

[0018] The sgRNA and Cas9 protein were mixed in proportion and incubated in a 25°C metal bath. After being fully mixed with the linearized DNADonor plasmid and cell suspension, electroporation was performed; the electroporation voltage was set to 1400V, the electroporation interval was 100ms, and the number of electroporations was 4; the electroporated cells were cultured in the pre-prepared culture medium, and flow cytometry was used for sorting 7*24 hours after electroporation to obtain homozygous cells expressing moesin-EGFP; after the cells were stabilized, the cells were monocloned by limiting dilution method.

[0019] 7) Verification of moeisn-EGFP at the gene and protein levels

[0020] The sorted cells were verified by PCR and Western blot. The PCR reaction system included premixed high-fidelity reverse transcriptase, upstream primers, downstream primers, template DNA, and nuclease-free water for denaturation, annealing, and extension reactions. In Western blot, cell proteins were extracted, protein concentrations were determined by BCA, proteins were separated using polyacrylamide gels, and proteins were transferred to nitrocellulose membranes and detected using moesin or EGFP antibodies.

[0021] This method obtained the MDA-MB-231 cell line that stably expressed the moesin-EGFP fusion protein. This cell line can be used to study the function of moesin in cell migration and signal transduction, and has broad application prospects in exploring cancer metastasis and the recruitment of related molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a schematic diagram of the constructed Donor plasmid;

[0023] Figure 2 It is a schematic diagram of flow cytometry sorting results;

[0024] Figure 3 is a schematic diagram of sequencing results;

[0025] Figure 4 This is a schematic diagram of Western blot results. DETAILED DESCRIPTION

[0026] 1. Design and Synthesis of sgRNA

[0027] The genomic sequence of the moesin gene was searched on NCBI, and the small guide RNA (sgRNA) was designed using the crispor website (http: / / crispor.gi.ucsc.edu / ), and the sgRNA was designed at the terminator of the moesin gene. The nucleotide sequence of the sgRNA is shown in SEQ ID NO: 1, and the sgRNA was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and a total of 48.55 μg of dry powder was obtained.

[0028] 2. Design and Construction of Donor Plasmid

[0029] The corresponding Donor plasmid was designed using the genomic sequence of the moesin gene found on NCBI. The sequence of Donor was designed as LHA+Linker+EGFP+RHA. The nucleotide sequence of LHA is shown in SEQ ID NO:2, the nucleotide sequence of Linker is shown in SEQ ID NO:3, the nucleotide sequence of EGFP is shown in SEQ ID NO:4, and the nucleotide sequence of RHA is shown in SEQ ID NO:5. The functions of LHA and RHA are to complement the DNA in the cell. The function of Linker is to add a connecting sequence between the moesin protein and the EGFP protein to prevent EGFP from affecting the function of the moesin protein. The Linker sequence consists of 4 glycines + serines. This sequence was synthesized by Qingke Biotechnology Co., Ltd. and inserted into the pUC57 plasmid to obtain a tube of puncture bacteria. The structure of the plasmid is as follows: Figure 1 shown.

[0030] 3. Amplification and Extraction of Plasmid

[0031] 1) Prepare LB medium: weigh 10 g tryptone, 5 g yeast extract, 10 g NaCl, add water to make up to 1 L, and sterilize at 121°C for 20-30 minutes.

[0032] 2) Use a sterile tube to take 3 mL of LB medium, add 6 μL of 500 μg / mL ampicillin (Amp), use a 200 μL pipette tip to pick up bacteria, put them into the medium, and shake at 37°C, 200 rpm for 12 hours.

[0033] 3) Take another 50 mL of LB medium, add 100 μL Amp, and then take 50 μL of bacterial solution, and shake the bacteria at 37°C and 200 rpm for 12 hours.

[0034] 4) Use the Adelaide Plasmid Midi Extraction Kit to extract the Donor plasmid from the cultured bacteria, and finally use sterile ultrapure water to dissolve the plasmid to 1.1 μg / μL.

[0035] 4. Linearization of Donor Plasmid

[0036] The Donor plasmid was linearized using ScaI-HF enzyme. The reaction system used was: 5 μL DNA Donor plasmid, 5 μL 10× NEBuffer, 3 μL ScaI-HF enzyme, and 37 μL ultrapure water were mixed in order, and the DNA Donor plasmid was digested in a 37°C metal bath for 25 minutes, and then the enzyme was inactivated in an 80°C metal bath. The linearization step was repeated 4 times, and then the double-stranded DNA was separated and purified by agarose gel electrophoresis, and the DNA was recovered using a DNA gel recovery kit. Finally, the DNA was dissolved in ultrapure water to obtain 15 μL 0.6 μg / μL linear DNA Donor plasmid.

[0037] 5. Cell Culture and Electroporation

[0038] 1) Use 90% DMEM and 10% FBS to prepare medium for culture of MDA-MB-231 cells, select cells in the logarithmic growth phase, and the cell confluence is 80%. Aspirate the medium, then wash twice with PBS, add 1mL trypsin, digest at 37℃ for 2 minutes, add 5mL medium containing 10% fetal bovine serum to terminate digestion, centrifuge at 1000rpm for 3 minutes, completely discard the supernatant, resuspend and wash the cells with 5mL PBS, centrifuge at 1000rpm for 3 minutes, completely discard the supernatant, and resuspend the cells with SBB electrotransfer solution to 2×10^7 cells / mL; SBB electrotransfer solution preparation method: 2.56g sucrose + 0.3mL 100mM MgCl2 + 29.7mL DPBS.

[0039] 6. sgRNA and Cas9 protein binding and electroporation

[0040] 1) Dissolve sgRNA in 48 μL of nuclease-free water, take 1.5 μL of sgRNA and 4 μL of 3 μg / μL Cas9 protein and mix them in a clean sterilized EP tube, blow evenly, and place in a metal bath at 25°C for 20 minutes to allow sgRNA and Cas9 protein to fully combine.

[0041] 2) Add 30 μL of cell suspension and 6 μL of linearized DNADonor plasmid into the EP tube and mix thoroughly.

[0042] 3) Use a 10 μL electroporation pipette tip to absorb the mixed solution, electroporation voltage: 1400 V, electroporation interval: 100 ms, electroporation times: 4 times.

[0043] 4) The electroporated cells were inoculated into the prepared culture medium (88% DMEM + 12% FBS).

[0044] 7. Flow cytometry and single clone selection

[0045] 1) 7*24 hours after electroporation, flow cytometry was performed: the culture medium was aspirated, and then washed twice with PBS, and the cells were dissociated with dissociation solution. The flow cytometry results showed three peaks, and the cells with the strongest fluorescence intensity accounted for 4.23%. Figure 2 shown.

[0046] 2) The sorted homozygous cells were inoculated into the prepared culture medium (89% DMEM + 10% FBS + 1% triple antibody), cultured for 7*24 hours, and then monoclonal cells were selected.

[0047] 3) Aspirate the culture medium, then wash twice with PBS, add 1 mL of trypsin, digest at 37°C for 2 minutes, add 5 mL of culture medium containing 10% fetal bovine serum to terminate the digestion, centrifuge at 1000 rpm for 3 minutes, and completely discard the supernatant.

[0048] 4) Use the limiting dilution method to select monoclonal cells, dilute the cells to 1 / 100μL, inoculate in a 96-well plate, 100μL of cell suspension in each well, observe the plate under a 100× optical microscope, select the wells with only one cell, and mark them. After 14*24 hours, the cells in some wells can be transferred to a 10cm culture dish for culture.

[0049] 8. Gene Editing Validation

[0050] 1) After 7 to 24 hours of transfer to a 10 cm culture dish, take some cells and use a DNA extraction kit to extract the cell DNA and measure the DNA concentration.

[0051] 2) Design amplification primers for the moesin-EGFP gene editing fragment. The upstream primer sequence is 5'-tgagctgaagactgccatgag-3', and the downstream primer sequence is 5'-ggcagatcccacgaagtgac-3'. The primers were synthesized by Qingke Biotechnology Co., Ltd., prepared into a 100 μM solution using TE buffer, and stored in a -20°C refrigerator for use.

[0052] 3) Prepare a PCR reaction system containing a premixed high-fidelity DNA polymerase ( T6Super 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: (1) 98°C × 2 min pre-denaturation; (2) (98°C × 10 s denaturation, 62°C × 15 s annealing, 72°C × 40 s extension) × 35 cycles; (3) 72°C × 5 min final extension.

[0053] 4) After the PCR product was qualitatively confirmed by agarose gel electrophoresis, it was sent for gene sequencing (Qingke Biotechnology Co., Ltd.). The sequencing results were as follows: Figure 3 As shown, the results indicate that gene editing was successful and the EGFP gene was successfully connected behind the moeisn gene.

[0054] 9. Protein Expression Verification

[0055] 1) After culturing cells for 3 to 24 hours, a portion of the cells were taken and lysed using RIPA lysis buffer to extract proteins, and the protein concentration was determined by BCA.

[0056] 2) Use polyacrylamide gel to separate proteins, then transfer the separated proteins to nitrocellulose membrane, block with 5% skim milk powder, then incubate with moesin or EGFP antibody, wash the antibody after 1 hour, incubate with secondary antibody, wash the antibody after 2 hours, and use ECL chemiluminescent solution to display antibody bands. Western blot results are as follows Figure 4 As shown, the results indicate that cells expressing moesin-EGFP protein and not expressing moesin protein have been successfully screened.

[0057] The MDA-MB-231 cell line expressing moesin-EGFP was obtained according to the construction method.

[0058] Advantages of the present invention include:

[0059] Efficient gene editing: The sgRNA designed using CRISPR / Cas9 technology can accurately target the terminator region of the moesin gene, ensuring efficient gene editing efficiency.

[0060] Optimized plasmid design: The Donor plasmid contains LHA, Linker, EGFP and RHA sequences, which can insert a linker sequence between the moesin protein and the EGFP protein, effectively avoiding the effect of EGFP on the function of the moesin protein.

[0061] Stable protein expression: sgRNA, Cas9 protein and linearized Donor plasmid were co-transfected into MDA-MB-231 cells by electroporation, homozygous cells were sorted by flow cytometry, and monoclonal cells were selected by limiting dilution. PCR and Western blot methods were used to verify gene editing and protein expression, ensuring the stable expression of moesin-EGFP fusion protein.

[0062] Broad application prospects: This cell line can be used to study the function of moesin in cell migration and signal transduction, providing an effective experimental tool to help further explore the biological function and mechanism of moesin.

[0063] Through the above advantages, the present invention provides significant technical advantages and practical value in gene editing, protein expression, cell function research, etc., and provides strong technical support and tools for scientific researchers in related fields.

Claims

1. A sgRNA for constructing an MDA-MB-231 cell line expressing moesin-EGFP, characterized in that: The nucleotide sequence of the sgRNA is shown in SEQ ID NO: 1, which is used to target the terminator region of the moesin gene.

2. A Donor plasmid for constructing a MDA-MB-231 cell line expressing moesin-EGFP, characterized in that: The structure of the Donor plasmid is LHA+Linker+EGFP+RHA, wherein: 1) The nucleotide sequence of LHA is shown in SEQ ID NO: 2, 2) The nucleotide sequence of Linker is shown in SEQ ID NO: 3, 3) The nucleotide sequence of EGFP is shown in SEQ ID NO: 4, 4) The nucleotide sequence of RHA is shown in SEQ ID NO:

5.

3. The Donor plasmid according to claim 2, characterized in that The linker sequence consists of 4 glycines and 1 serine.

4. A method for constructing a MDA-MB-231 cell line expressing moesin-EGFP, characterized in that: The following steps are involved: 1) Prepare LB medium, culture bacteria and extract plasmids; 2) Linearize the plasmid using ScaI-HF enzyme and separate and purify the double-stranded DNA by agarose gel electrophoresis; 3) MDA-MB-231 cells were cultured using a medium prepared with 90% DMEM and 10% FBS; 4) Resuspend the cells to 2×10 using SBB electroporation buffer. 7 / mL, and mix sgRNA and Cas9 protein for electroporation; 5) Inoculate the electroporated cells into the prepared culture medium for culture; 6) Homozygous cells expressing moesin-EGFP were sorted by flow cytometry and single clones were selected; 7) Use PCR and Western blot to verify gene editing and protein expression.

5. The SBB electroporation solution according to claim 4, characterized in that: The formula is: 2.56g sucrose + 0.3mL 100mM MgCl2 + 29.7mL DPBS.

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