ATM gene knockout THP-1 cell line as well as construction method and application thereof

Abstract

The invention discloses an ATM gene knockout THP-1 cell line as well as a construction method and application thereof, and belongs to the technical field of biology. The gRNA for targeted knockout of the ATM gene is provided, the ATM gene of the THP-1 cell line is knocked out in combination with a CRISPR / Cas9 gene editing technology, and the THP-1 cell line with the knocked-out ATM gene is successfully obtained. The cell line stably lacks an ATM gene, can be normally differentiated into macrophages, and can be further polarized into M1 and M2 macrophages. The THP-1 cell line with the ATM gene knocked out, which is constructed by the invention, can be widely applied to research on in-vitro and macrophage physiological and biochemical functions of the ATM gene and immune response drug targets.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the ATM gene knockout THP-1 cell line, its construction method, and its application. Background Technology

[0002] The atax-Telangiectasia mutated (ATM) gene belongs to the phosphoinositol 3-kinase (PI3K)-associated protein kinase (PIKK) family. It encodes a 350 kDa protein composed of 3056 amino acids (Uziel et al., 1996). Inactivation of the ATM gene product results in a complex disease with a highly complex phenotype, termed ataxotelangiectasia (AT) (Rothblum-Oviatt et al., 2016).

[0003] Of the more than 700 proteins, over 900 regulated phosphorylation sites have been identified as potential targets of ATM (Matsuoka et al., 2007). ATM is an important cell cycle checkpoint kinase. When DNA damage occurs, ATM is activated, regulating cell cycle checkpoints by phosphorylating a series of downstream proteins, thereby affecting the cell cycle (Erttmannet et al., 2016). ATM repairs DNA damage and maintains genome stability by phosphorylating various downstream proteins, including tumor suppressor proteins p53 and BRCA1 (Cheng and Chen, 2010), checkpoint kinase CHK2 (Melchionna et al., 2000), checkpoint proteins Rad17 and Rad9, and DNA repair protein NBS1. V(D)J recombination is an important mechanism for the generation of diverse antigen receptors during lymphocyte development, and the normal function of ATM ensures the accurate execution of this process, thereby ensuring that the immune system can recognize multiple antigens (Bassing et al., 2002). Meanwhile, ATM regulates mitochondrial function through ribonucleotide reductase (Eaton et al., 2007).

[0004] The monocyte-like THP-1 cell line was derived from peripheral blood monocytes of a one-year-old infant with acute monocytic leukemia (Bosshart and Heinzelmann, 2016). Different reagents can be used to induce the differentiation and polarization of THP-1 cells. Treatment with 12-myristate-13-acetate (PMA) resulted in differentiation into mature macrophages (M0) (Lund et al., 2016), and M0 macrophages could polarize into M1 macrophages in a medium containing bacterial lipopolysaccharide (LPS) and interferon-γ (IFN-γ), or into M2 macrophages in a medium containing interleukin IL-4 and IL-13 (Baxter et al., 2020). Due to its high plasticity, THP-1 cells have been widely used in research on mechanisms, signaling pathways, and immune responses associated with monocytes and macrophages. Macrophages (abbreviated as MΦ or MP) are white blood cells of the innate immune system that engulf and fight pathogens, protecting the host from infection and damage (Sica et al., 2015). Macrophages have multiple functions: (1) engulfing pathogens, infected cells, debris, and dead cells; (2) processing and presenting antigens through histocompatibility complex (MHC) molecules; and (3) producing different types of cytokines, including interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α) (Wynn et al., 2013). In addition, macrophages play an important role in the progression of inflammatory diseases, including diabetes (Meshkani and Vakili, 2016), cancer (Ruffell and Coussens, 2015), and atherosclerosis (Lu, 2016).

[0005] The ATM gene knockout THP-1 cell line can serve as an experimental tool for accurately simulating the loss of gene function in vitro. It is a core tool in elucidating the molecular mechanisms of ATM in macrophage immune regulation, providing crucial support for revealing ATM's regulation of macrophage polarization, functional execution, and intercellular interactions, as well as for research on the mechanisms of immune-related diseases and target discovery. Currently, there are no reports on the construction of an ATM gene-deficient THP-1 cell line. Summary of the Invention

[0006] In view of this, the purpose of this invention is to overcome the shortcomings of the prior art and provide an ATM gene knockout THP-1 cell line, its construction method and application.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a specific gRNA for targeting and knocking out the ATM gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] In a second aspect, the present invention provides the application of the gRNA described in the first aspect in knocking out the ATM gene in the THP-1 cell line.

[0009] Thirdly, the present invention provides a method for constructing an ATM gene knockout THP-1 cell line, the method comprising: knocking out the ATM gene of the THP-1 cell line using a CRISPR / Cas9 system, wherein the gRNA used in the CRISPR / Cas9 system is the gRNA described in the first aspect.

[0010] Fourthly, the present invention provides an ATM gene knockout THP-1 cell line, which is constructed by the method described in the third aspect.

[0011] Fifthly, the present invention provides the application of the ATM gene knockout THP-1 cell line described in the fourth aspect in the preparation of an ATM gene knockout in vitro cell model.

[0012] In a sixth aspect, the present invention provides the application of the ATM gene knockout THP-1 cell line described in the fourth aspect in the study of the physiological and biochemical functional mechanisms of macrophages and the target of immune response drugs.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention successfully obtained the ATM gene knockout THP-1 cell line using CRISP-Cas9 technology. This cell line stably lacks the ATM gene and can differentiate normally into macrophages, and can further polarize into M1 and M2 macrophages.

[0014] The ATM gene knockout THP-1 cell line of this invention can serve as an experimental tool for accurately simulating the state of gene function loss in vitro, allowing for targeted research on the regulatory mechanisms of ATM on macrophage differentiation and polarization. Simultaneously, it can be used to study the impact of ATM on the core functions of macrophages, elucidating the molecular mechanisms of ATM in phagocytosis and clearance (regulating proteins such as Rac1 / Cdc42), antigen presentation (affecting MHC molecule expression), and inflammatory factor secretion (regulating the release of IL-1β, IL-10, etc.), providing a basis for screening therapeutic targets for related diseases. Furthermore, this model can explore the role of ATM in the interaction between macrophages and other immune cells. Through co-culture and other experiments, its function in the synergistic regulation of immune responses by macrophages, T cells, NK cells, and neutrophils can be clarified, providing a basis for research on the mechanisms of tumor immune escape and chronic infection, as well as the design of combination therapy strategies. Attached Figure Description

[0015] Figure 1 A schematic diagram of gRNA site selection for CRISP-Cas9-mediated knockout of the ATM gene.

[0016] Figure 2 The image shows the morphological changes and identification results of THP-1 cells after the ATM gene was knocked out.

[0017] Figure 3 This image shows the results of differentiating THP-1 cells into M0 macrophages after PMA treatment. Detailed Implementation

[0018] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0019] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0020] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods. Example

[0021] 1. Obtaining THP-1 cells THP-1 cells, a commonly used human monocytic leukemia cell line, originated from the American Culture Collection.

[0022] 2. gRNA design First, the gene target was identified, and effective transcripts were screened. Multiple transcripts of the ATM gene exist; key transcripts with complete coding regions were prioritized, and ATM-222 was selected as the target transcript. Based on exon position, size, and gRNA scoring, exon 4 was ultimately identified as the target region. The average GC content of this region was detected to be 35.18%, suitable for subsequent PCR screening and sequencing verification. Next, the gRNA sequence was designed, and based on scoring from databases such as Crispor, the gRNA sequence (5'-TTGTTTCAGGATCTCGAATCAGG-3') was determined.

[0023] 3. Construction of ATM gene knockout THP-1 cell line Using CRISPER-Cas9 technology ( Figure 1We designed and synthesized gRNA (sequence: 5'-TTGTTTCAGGATCTCGAATCAGG-3') and expressed Cas9 to cooperate in achieving gRNA targeting and recognizing the specific sequence TGG, resulting in cleavage, and targeting and recognizing exon 4 region of the ATM gene. The specific method is as follows: 3 × 10⁶ THP-1 cells in logarithmic growth phase were used. 5Centrifuge cells at 300g for 3 minutes in a sterile tube, discard the supernatant, and resuspend the cells in 20μL of Buffer R reagent. Add 12-50 pmol of sgRNA and 1-3ug of EZ-editor™ Cas9-EGFP protein and mix thoroughly. Using a 10μL pipette tip, aspirate the mixture into an electroporator and perform electroporation (1230V, 10 ms, 2 pulses). After electroporation, seed the cells into 12-well plates with preheated culture medium and continue culturing. Seed the remaining mixture into fresh 12-well plates as a control. 24 hours after transfection, take a portion of the cell suspension, dilute using limiting dilution, and seed into 96-well plates. Incubate the cells statically at 37°C in a 5% CO2 incubator. Genomic DNA was extracted for the remaining cells using a cell DNA extraction kit (TIANGEN, product number: DP304). PCR amplification was performed using target primers (upstream primer: 5'-GCCTCTAATTGTACAGTTAAATCTAAC-3', downstream primer: 5'-CCTCTAATCTGAAGTCTTGTGAAC-3'). The reaction mixture consisted of 2 × Taq Master Mix 25 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, ddH2O, and a template of 2 μL. PCR conditions were: 94 ℃ for 10 min, followed by 30 cycles of 94 ℃ for 30 s, 60 ℃ for 30 s, 72 ℃ for 1 min, and a final cycle of 72 ℃ for 10 min). The PCR products were sequenced to analyze the cleavage efficiency. After approximately one week of culture in 96-well plates, clonal growth was observed, and wells containing single-clonal cell clusters were labeled. Approximately two weeks later, the grown single clones were digested, divided in half, and cultured in two 96-well plates. When the cell confluence reached 40%-60%, one plate was removed for single clone identification. The culture medium in the plate was discarded, and 100 μL of the EZ-editor™ Single Clon Identification Kit (extraction-free) was added to obtain genomic DNA. PCR amplification reaction system: (2 × Taq Master Mix 25 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, ddH2O, Template 2 μL; PCR reaction conditions: 94 ℃ 10 min, 30 cycles of 94 ℃ 30 s, 60 ℃ 30 s, 72 ℃ 1 min, and a final 72 ℃ 10 min) knockout region. The PCR products were sequenced to analyze the knockout results. Positive clones identified as successfully knocked out were further passaged and amplified. When the cell number was sufficient, they were cryopreserved for backup.

[0024] 4. Identification of ATM gene knockout THP-1 cell lines Morphologically, the ATM-deficient THP-1 cell line (KO-ATM) is essentially identical to the wild-type cell line (WT). Figure 2 A), ATM deletion does not affect cell morphology. Total RNA was extracted from cells and reverse transcribed (RT-PCR). PCR primers were designed (upstream primer: 5'-GCGGTTGATACTACTTTGACCTTC-3', downstream primer: 5'-CCAAACTACTGATTTCCTGCATCT-3') to amplify the fragment with the missing base in the gene using polymerase chain reaction (PCR reaction conditions: 94 ℃ for 10 min, 35 cycles of 94 ℃ for 30 s, 53 ℃ for 30 s, 72 ℃ for 1 min, and a final 72 ℃ for 10 min). Results were obtained by 1.0% agarose gel electrophoresis and first-generation Sanger sequencing (…). Figure 2 B) It was found that the mutant cells lacked a single base "T" in the sequence compared to the wild type (GenBank ID: U33841.1, T at position 290), resulting in a frameshift mutation at amino acid position 291. Western blotting was performed using an anti-ATM specific antibody (anti-ATM, Proteintech, product number: 27156-1-AP, dilution ratio: 1:500) to detect ATM expression at the protein level. Figure 2 C). Wild-type cells express ATM protein normally, while cells lacking ATM do not express it.

[0025] 5. Verification of macrophage differentiation capacity THP-1 cell lines were treated with phorbol 1,2-tetradecanoate 1,3-acetate (PMA) at a concentration of 150 nM for 24 hours. After treatment, images were taken using a 10X microscope. Morphologically, both wild-type cells and cells lacking the ATM gene exhibited macrophage morphology. Figure 3 A), and at the protein level, the characteristic proteins of M0 macrophages, CD68, CD71, CD36, and CD14, are all expressed. Figure 3 B), and the expression level of ATM-deficient cells is significantly increased. This indicates that THP-1 cells without the ATM gene can differentiate normally into M0 macrophages.

[0026] 6. Culture of ATM gene knockout THP-1 cell lines (1) Cell culture: THP-1 cells were cultured in a constant temperature incubator. RPMI 1640 medium (containing 10% fetal bovine serum) was used, and the culture conditions were 37°C, 95% humidity and 5% carbon dioxide.

[0027] Based on cell growth status and density, the cell density was controlled at 2*10 during passage. 5 -4*10 5Cells / mL, passaged at a ratio of 1:3.

[0028] (2) Cell differentiation and polarization: THP-1 cells were treated with 150 nM 12-hydroxyphorbol ester (PMA, Sigma-Aldrich) for 24 hours to induce cell differentiation into M0 macrophages.

[0029] After M0 macrophages differentiated and adhered, the culture medium was discarded, and the cells were co-cultured with 20 ng / mL IFN-γ (Beyotime, product number: P5664-100μg) and 10 pg / mL LPS (Beyotime, product number: P5664-20μg) to polarize M0 macrophages into M1 macrophages. Alternatively, M0 macrophages were polarized into M2 macrophages by adding 20 ng / mL IL-13 (MCE, product number: HY-P70568) and 20 ng / mL IL-4 (MCE, product number: HY-P70445) to the culture medium.

[0030] (3) Cell cryopreservation: Select a THP-1 cell suspension that is in good growth condition and free from contamination, gently pipette to mix, transfer to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Adjust the cell concentration to ensure that the cell concentration is not less than 5 × 10⁻⁶ cells / mL. 6 Add cells / mL to pre-chilled cryopreservation buffer (Epizyme, product number: BY0002P), gently pipette until cells are completely dispersed, and adjust the cell concentration to at least 2 × 10⁻⁶ cells / mL. 6 -5×10 6 Cells / mL. Aliquot the cell suspension into cryovials, 1 mL per tube, tighten the cap, and label with cell name, passage number, and date. Store at -80°C or in liquid nitrogen for long-term storage.

[0031] The above description is only for better explaining the embodiments of the present invention and is not intended to limit them. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention shall fall within the scope of the present invention.

[0032] SEQ ID NO.1 gRNA: 5'-TTGTTTCAGGATCTCGAATCAGG-3' SEQ ID NO.2 Upstream primer for target: 5'-GCCTCTAATTGTACAGTTAAATCTAAC-3' SEQ ID NO.3 Downstream primer for target: 5'-CCTCTAATCTGAAGTCTTGTGAAC-3' SEQ ID NO.4 PCR upstream primer: 5'-GCGGTTGATACTACTTTGACCTTC-3' SEQ ID NO.5 PCR downstream primer: 5'-CCAAACTACTGATTTCCTGCATCT-3'.

Claims

1. A specific gRNA targeting knock-out of ATM gene, characterized in that, The nucleotide sequence of the gRNA is shown as SEQ ID NO.

1.

2. The gRNA of claim 1 is used in knocking out ATM gene in THP-1 cell line.

3. A method of constructing an ATM-knockout THP-1 cell line, characterized by, The construction method comprises: knocking out ATM gene of THP-1 cell line by using CRISPR / Cas9 system, wherein the gRNA used by the CRISPR / Cas9 system is the gRNA of claim 1.

4. An ATM knockout THP-1 cell line, characterized in that, The THP-1 cell line is constructed by the method of claim 3.

5. The ATM gene knockout THP-1 cell line of claim 4 is used in preparing ATM gene knockout in vitro cell model.

6. The ATM gene knockout THP-1 cell line of claim 4 is used in researching physiological and biochemical functions of macrophages and drug target points of immune response.

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