Construction method and application of a motor neuron MNX1 reporter gene cell line
By using donor vectors designed with highly active sgRNA and homologous arms in the CRISPR/Cas9 system, a stable MNX1 reporter gene cell line was constructed, which solved the problems of low editing efficiency and genome instability in existing technologies, and achieved efficient and precise gene editing and reporter labeling.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI YANGZHI REHABILITATION HOSPITAL
- Filing Date
- 2026-03-23
- Publication Date
- 2026-06-05
AI Technical Summary
In existing technologies, the CRISPR/Cas9 system suffers from problems such as low editing efficiency, off-target risk, and genome instability caused by secondary cutting when editing motor neurons.
Using highly active sgRNA (such as sgMNX1-2) and donor vectors with designed upstream and downstream homologous arms, a MNX1 reporter gene cell line was constructed. The line was precisely edited using the CRISPR/Cas9 system, introduced into human pluripotent stem cells via electroporation, and labeled with the fluorescent reporter gene EGFP.
Efficient and precise MNX1 gene editing was achieved, and a stable motor neuron reporter cell line was constructed for research and drug screening, reducing the risk of off-target effects and secondary cleavage.
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Figure CN122146784A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for constructing and applying a motor neuron MNX1 reporter gene cell line, belonging to the field of gene editing and stem cell technology. Background Technology
[0002] In the central nervous system, the MNX1 gene is specifically expressed in motor neurons, and the protein it encodes is involved in the development and functional maintenance of motor neurons. Motor neurons control muscle activity and are closely related to the occurrence and development of diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Therefore, establishing reporter cell lines of fluorescently labeled motor neurons will be beneficial to related research on human motor neurons. Among existing technologies, the CRISPR / Cas9 system has been used for gene editing, but due to the efficiency differences in sgRNA design, conventional methods face the following problems: (1) low editing efficiency; (2) off-target risk: non-specific cutting affects the precise insertion of reporter genes; (3) secondary cutting: continuous cutting of Cas9 protein leads to genomic instability. Summary of the Invention
[0003] To address the aforementioned issues, this invention provides an efficient, precise, and stable method for constructing MNX1 gene reporter cell lines by screening for highly active sgRNAs, thereby meeting the high demands of gene editing technology in fields such as motor neuron research, drug screening, and disease model construction.
[0004] The first objective of this invention is to provide a gene vector system for constructing a motor neuron MNX1 reporter cell line, the vector system comprising: A guide vector containing sgRNA with a nucleotide sequence as shown in SEQ ID NO.1; The donor vector contains upstream and downstream homologous arms designed based on the stop codon of the motor neuron MNX1 gene and a specific length sequence preceding it, as well as a reporter gene located between the upstream and downstream homologous arms.
[0005] Furthermore, the reporter gene includes, but is not limited to, a fluorescent gene.
[0006] Furthermore, the backbone of the guide vector includes, but is not limited to, pX459 plasmid, pX458 plasmid, pX330 plasmid, etc.; when the backbone is pX459 plasmid, the sequence of the guide vector is as shown in SEQ ID NO.2.
[0007] Furthermore, the sequences of the upstream and downstream homologous arms in the donor carrier are shown in SEQ ID NO.4-5.
[0008] Furthermore, the sequence of the donor vector is shown in SEQ ID NO.6.
[0009] A second objective of this invention is to provide a CRISPR gene editing system containing the aforementioned vector system.
[0010] Furthermore, the CRISPR gene editing system also contains a Cas9 expression vector.
[0011] A third objective of this invention is to provide recombinant cells (processed with the gene editing system) containing the vector system or the CRISPR gene editing system.
[0012] Furthermore, after editing, the recombinant cells contain linked MNX1 and reporter genes, which are connected via a 2A peptide.
[0013] Furthermore, the host cell of the recombinant cells is a human cell, preferably a human pluripotent stem cell.
[0014] It should be noted here that: Human pluripotent stem cells are cells with self-renewal capacity and multipotent differentiation potential, mainly including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Human pluripotent stem cells can differentiate into almost all cell types in the human body under appropriate conditions, making them extremely promising for basic research and clinical applications. To avoid ethical concerns, the human pluripotent stem cells described in this invention are embryonic stem cells, which are ethically approved cells that do not have the potential to develop into a complete new individual, such as the E14 ES cell line and the commercially available H1 cell line used in the following examples.
[0015] A fourth objective of this invention is to provide the application of the vector system, the CRISPR gene editing system, or the recombinant cells in the preparation of motor neuron MNX1 gene-related (cell) models.
[0016] A fifth objective of this invention is to provide a method for constructing a motor neuron MNX1 reporter gene cell line, comprising the step of introducing the vector system or the CRISPR gene editing system into the cell line.
[0017] Furthermore, when the cell line is a stem cell, the method further includes a step of differentiating and culturing the gene-edited stem cells.
[0018] Furthermore, the preferred method for introducing the carrier system is electrotransfer.
[0019] A sixth object of the present invention is to provide the application of the vector system, the CRISPR gene editing system, or the recombinant cells in the medical field, including any one of the following: (1) Application in biomedical research on muscle activity-related diseases; (2) Applications in research on human motor neurons; (3) Application in the preparation of animal models.
[0020] The beneficial effects of this invention are: This invention, through a comparison of the editing efficiencies of sgMNX1-1 / 2 / 3, found that sgMNX1-2 effectively avoids secondary cleavage of the Cas9 protein while exhibiting superior editing efficiency. The excellent performance of sgMNX1-2 enables its successful application in MNX1 gene editing, such as the construction of fluorescent reporter cell lines, providing a precise and efficient tool for motor neuron-related research. Subsequently, based on this tool, the construction of a human pluripotent stem cell line using the MNX1 reporter gene was achieved. Attached Figure Description
[0021] Figure 1 Illustration of a reporter gene tool vector.
[0022] Figure 2 Illustration of the MNX1 reporter gene vector.
[0023] Figure 3 A diagram illustrating the construction of the MNX1 reporter gene human pluripotent stem cell line.
[0024] Figure 4 PCR amplification diagram for cell line identification.
[0025] Figure 5 This is a colocalization map of motor neurons and green fluorescence.
[0026] Figure 6 This is a diagram of spontaneous action potentials in motor neurons.
[0027] Figure 7 Na for motor neurons + Current diagram. Detailed Implementation
[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.
[0029] The technical solution involved in this invention is as follows: On one hand, the present invention provides a vector system for expressing the motor neuron MNX1 reporter gene, the vector system including but not limited to: a guide vector that targets a single-stranded guide RNA near the stop codon of the human motor neuron MNX1 gene, and a donor vector carrying a homologous arm near the stop codon of the human motor neuron MNX1 gene.
[0030] In this invention, the sgRNA recognition sequence spans the fluorescent insertion sequence, and the sgRNA sequence is destroyed by homologous recombination to prevent secondary cleavage.
[0031] Single-stranded guide RNA near the stop codon of the MNX1 gene refers to the single-stranded guide RNA within 100 bp upstream and downstream of the stop codon of the MNX1 gene. Homologous arms near the stop codon of the MNX1 gene refer to the single-stranded guide RNA within approximately 1500 bp upstream and downstream of the stop codon.
[0032] On the other hand, the present invention provides a method for constructing a stable human pluripotent stem cell line based on the MNX1 reporter gene of motor neurons, comprising the following steps: (1) Construct a guide vector PX459-sgMNX1 carrying a single-stranded guide RNA targeting the stop codon of the MNX1 gene; We obtained sgRNA targeting the stop codon of the human MNX1 gene, synthesized two oligonucleotide chains, annealed and phosphorylated them, and inserted them into the BbSI site of PX459. The recombinant vector was transformed into E. coli, and single clones were selected for sequencing to verify the successful recombination of plasmid PX459-sgMNX1. (2) Construct the donor vector MNX1-T2A-EGFP for the MNX1 reporter gene; The left and right homologous arms of MNX1, 1.5kb before and after the stop codon, MNX1-HAL and MNX1-HAR, were amplified from the human genome. HAL and HAR were inserted into the multiple cloning sites on the left and right sides of the tool vector pUC19-T2A-EGFP-CMV-PuroR, respectively, to obtain the MNX1 reporter gene donor vector MNX1-T2A-EGFP. (3) Construction of MNX1 reporter gene human pluripotent stem cell line; Guide vector PX459-sgMNX1 and donor vector MNX1-T2A-EGFP were electroporated into human pluripotent stem cells H1. After selection with puromycin, resistant clones were obtained. The cell genome was extracted, and the inserted fragment was identified by PCR amplification and sequencing. Finally, a stable human pluripotent stem cell line with the EGFP-labeled reporter motor neuron MNX1 gene was obtained.
[0033] In one embodiment of the present invention, the tool carrier for constructing the guide carrier in step 1) includes, but is not limited to, one or more of PX459, PX458 or PX330.
[0034] In one embodiment of the present invention, the reporter gene element used to construct the targeting vector tool vector in step 2) includes, but is not limited to, one or more of tdTomato, YFP, RFP or EGFP.
[0035] In one embodiment of the present invention, the resistance screening elements used in step 2) for constructing the target carrier tool carrier include, but are not limited to, PuroR and NeoR.
[0036] In one embodiment of the present invention, step 2) involves inserting the left and right homologous arms of the MNX1 gene from the tool vector pUC19-T2A-EGFP-CMV-PuroR.
[0037] In one embodiment of the present invention, the human pluripotent stem cell line electroporated in step 3) includes, but is not limited to, human embryonic stem cell lines or induced pluripotent stem cell lines (e.g., H9, RUES1, RUES2 and pluripotent stem cells derived from human cell reprogramming).
[0038] In one embodiment of the present invention, the method for constructing a stable cell line of the human motor neuron MNX1 reporter gene is as follows: 1.1 Construct vectors carrying a single guide RNA sgMNX1-2 targeting the stop codon of the MNX1 gene (e.g., PX459-sgMNX1-2, PX458-sgMNX1-2, or PX330-sgMNX1-2); 1.2 Construct a donor vector (MNX1-T2A-EGFP) carrying a homologous arm near the stop codon of the MNX1 gene; 1.3 The PX459-sgMNX1-2 and MNX1-T2A-EGFP vectors were electroporated into human pluripotent stem cell lines, and stable human pluripotent stem cell lines with the EGFP-labeled MNX1 gene were obtained after puromycin resistance selection.
[0039] In the above method for constructing a stable cell line of human motor neuron MNX1 reporter gene, the sgRNA in step 1.1 includes sgMNX1-1 / 2 / 3, preferably sgMNX1-2.
[0040] In the above method for constructing a stable cell line for the human motor neuron MNX1 reporter gene, the targeting vector carrying sgRNA in step 1.1 includes PX459 / PX458 / PX330, with PX459 being preferred. After the oligonucleotides targeting the MNX1 stop codon anneal to form a double strand, they are inserted into the BbSI site of the PX459 vector by enzyme digestion and ligation. The recombinant vector is then amplified by E. coli to obtain the endotoxin-free PX459-sgMNX1-2 plasmid.
[0041] In step 1.2, the reporter gene of the donor vector (MNX1-T2A-EGFP) can be mCherry, RFP, GFP, tdTomato, etc., and EGFP is selected in this invention.
[0042] Homologous recombination of approximately 1.5 kb before and after the stop codon of MNX1 amplified from the human genome was performed into the multiple cloning site of the tool vector pUC19-T2A-EGFP-CMV-PuroR sequence. The recombinant vector was amplified by E. coli to obtain an endotoxin-free MNX1-T2A-EGFP plasmid, the sequence of which is shown in Table 5 (SEQ ID NO 21).
[0043] In another aspect, the present invention provides a motor neuron MNX1 reporter gene human pluripotent stem cell line obtained using the above-described method for constructing the motor neuron MNX1 reporter gene human pluripotent stem cell line.
[0044] Preferably, the motor neuron MNX1 reporter gene human pluripotent stem cell line is derived from H1.
[0045] Furthermore, the present invention also provides applications of the aforementioned motor neuron MNX1 reporter gene pluripotent stem cell line, including its applications in the preparation of targeted differentiation drugs for motor neurons, morphological and functional studies of motor neurons, and screening of targeted differentiation drugs for motor neurons.
[0046] In another preferred embodiment of the present invention, the method for determining the homologous recombination efficiency of sgMNX1 is to count the number of clones that successfully undergo homologous recombination per million electroporated cells.
[0047] In another preferred embodiment of the present invention, the method for identifying the human motor neuron MNX1 reporter gene pluripotent stem cell line is as follows: Motor neurons were obtained by in vitro induction of MNX1 gene reporter human pluripotent stem cell lines. Green fluorescence expression was visible under a fluorescence microscope, allowing for direct observation of differentiation efficiency. Cellular immunofluorescence assay showed that red fluorescently labeled motor neuron-specific markers co-localized with green EGFP fluorescent cells. Recording the electrophysiology of motor neurons labeled with green fluorescent material improves the efficiency and accuracy of motor neuron electrophysiological detection.
[0048] This invention discloses an efficient, precise, and stable sgRNA targeting the MNX1 gene of human motor neurons and its applications. This facilitates gene editing following the MNX1 sequence, promoting research on human motor neurons in living cells.
[0049] The sequences involved in the embodiments of this invention are: sgMNX1-2, SEQ ID NO.1; pX459-sgMNX1-2 plasmid, SEQ ID NO.2; pUC19-T2A-EGFP-CMV-PuroR, SEQ ID NO.3; left homologous arm sequence (HAL), SEQ ID NO.4; right homologous arm sequence (HAR), SEQ ID NO.5; MNX1-T2A-EGFP, SEQ ID NO.6.
[0050] Example 1: The left and right homologous arms of the MNX1 gene were inserted into the reporter gene tool vector pUC19-T2A-EGFP-CMV-PuroR to construct the MNX1 reporter gene donor vector. (1) Genomic DNA was extracted from H1 cells and used as a template to amplify and purify the left and right homologous arm fragments before and after the MNX1 stop codon.
[0051] Left homologous arm amplification primers: Forward primer: 5'- gaccatgattacgccaagcttAACAGGGACACTCAGACGTCTCCTA -3' Reverse primer: 5'- acttcctctgccctcCTGGGGCGCGGGCT -3' Right homologous arm amplification primers: Forward primer: 5'-ctatggcttaagcgcTAGGAGCCCCACGGACCAGCAGGTG -3' Reverse primer: 5'- agtgaattcgagctcggtaccGCACAGAGCCAAGAAGACCGATCC -3' (2) pUC19-T2A-EGFP-CMV-PuroR (see graph) Figure 1 Purification was achieved using HindIII and KpnI enzyme digestion.
[0052] (3) The left and right homologous arms were recombined into the HindIII and KpnI spaces of pUC19-T2A-EGFP-CMV-PuroR, respectively. DH5α competent cells were transformed, and single colonies were picked. Sequencing was used to verify the successful recombination of the donor vector MNX1-T2A-EGFP (see figure). Figure 2 ).
[0053] Example 2: Construction of a targeting vector PX459-sgMNX1 targeting the vicinity of the MNX1 stop codon (1) A series of candidate sgRNA sequences were obtained using the sgRNA design website (https: / / benchling.com). The sgRNAs with high scores were selected. The specific sequences are as follows: sgMNX1-1-F: 5'-caccGCCGCGCCCCAGTAGGAGCCCG-3' sgMNX1-1-R: 5'-aaacCGGGGCTCCTACTGGGGCGCGGC-3' sgMNX1-2-F: 5'-caccGGGCCGCGGGGCTCCTACTG-3' sgMNX1-2-R: 5'-aaacCAGTAGGAGCCCCGCGGCCC-3' sgMNX1-3-F: 5'-caccGGGGGCGGCGAGTCGTCCTCCG-3' sgMNX1-3-F: 5'-aaacCGGAGGACGACTCGCCGCCCCC-3' The lowercase letter sequence represents the complementary sticky ends after BbSI site cleavage, and the underlined sequence is the stop codon. (2) Oligochain annealing and phosphorylation to form double-chain sgMNX1.
[0054] (3) The double-stranded sgMNX1 with exposed sticky ends was inserted into the BbSI restriction site of PX459 (addgene #62988) by enzyme digestion and ligation. Competent cells were transformed, single colonies were picked, and the PX459-sgMNX1 vector was obtained by sequencing.
[0055] Example 3: Construction of MNX1-EGFP human pluripotent stem cell line (1) Human pluripotent stem cells (H1 cells) were digested into single cells using TrypLE. (2) 1×10 6 Resuspend the cells in 400 μL HEPES electroporation buffer, add 5 μg PX459-sgMNX1 and 10 μg MNX1-T2A-EGFP, transfer to an electroporation cuvette 4 mm wide, and place it into a Biorad GenePulser Xcell electroporator. (3) Set the power transfer parameters to: exponential wave, voltage 250 V, capacitance 500 μF, and execute the power transfer program. (4) According to 8×10 3 / cm 2 Density, seeding cells into six-well plates pre-coated with X-ray-treated mouse fibroblasts. (5) After electroporation for 24 hours, the sample was screened with 0.4 μg / ml puromycin for one week. (6) Select monoclonal cells 10-17 days after electroporation for expansion. (7) Extract genomic DNA from cells and perform PCR amplification to identify the cell line of MNX1-EGFP reporter motor neurons that were successfully targeted.
[0056] (8) Schematic diagram of the identification principle as follows Figure 3 As shown, if 5TP, 3TP, and TN have amplification products, and TN has only a single 3392bp amplification product, then the individual is homozygous; if 5TP, 3TP, and TN have amplification products, and TN has two amplification products (3392bp and 858bp respectively), then the insertion occurs on only one chromosome, indicating heterozygosity; if 5TP and 3TP have no amplification products, but TN has an 858bp amplification product, then no insertion has occurred; if only one of 5TP and 3TP has an amplification product, then it is a partial insertion. The MNX1 reporter gene cell line is homozygous, as identified by the following method: Figure 4 As shown in the figure, sgMNX1-2 yielded the highest proportion of homozygotes.
[0057] (9) The identification sequence is as follows: The 5TP sequence amplification sequence length is 1956 bp; 5TP-F: 5'-TCCCTGGTGAATACACAGACTTG-3' 5TP-R:5'-CCGCATGTTAGCAGACTTCCTCT-3' The 3TP sequence amplification sequence is 2616 bp in length; 3TP-F: 5'-GCTAACTAGAGAACCCACTGCTTACTG-3' 3TP-R: 5'-ATGAGTTGCCAGAGAGGATATTAATCTCC-3' The TN sequence amplification sequence length is 3392 bp or 858 bp; TN-F: 5'-AACCGGCGGATGAAATGGAA-3' TN-R: 5'-GCAGTTTGAACGCTCGTGAC-3'.
[0058] The results of the identification are as follows: 1. Comparison of editing efficiency of sgMNX1-1 / 2 / 3 The PX459-sgMNX1-1, PX459-sgMNX1-2, and PX459-sgMNX1-3 vectors were obtained according to the method described in Example 2.
[0059] The MNX1-EGFP reporter motor neuron cell line was constructed and identified according to the method described in Example 3. Ultimately, sgMNX1-2 had the highest number of clones successfully undergoing homologous recombination per 100,000 electroporated cells, with an average clone number of 30 per 100,000 cells; sgMNX1-1 was second highest with an average clone number of 7 per 100,000 cells; and sgMNX1-3 had the lowest average clone number of 0.5 per 100,000 cells.
[0060] 2. Secondary cutting verification (1) The MNX1 reporter gene donor vector MNX1-T2A-EGFP was obtained according to the method described in Example 1.
[0061] (2) PX459-sgMNX1-1, PX459-sgMNX1-2, and PX459-sgMNX1-3 vectors were obtained according to the method described in Example 2.
[0062] (3) The MNX1-EGFP reporter motor neuron cell line was obtained and identified according to the method described in Example 3. Sequencing results showed that although the reporter gene was successfully inserted upstream of the MNX1 stop codon, the sgRNA-3 re-recognized the target sequence, guiding the Cas9 protein to perform a secondary cleavage, resulting in a base deletion of the MNX1 gene at that site. The sgMNX1-2 recognition sequence spans the Cas9 protein cleavage site, and the original sgRNA recognition sequence is destroyed after the reporter gene is inserted, thus preventing secondary cleavage. Although sgMNX1-1 can also prevent secondary cleavage, and its website prediction score is comparable to Sg2, the number of clones formed after actual transfection was significantly lower than that of sgMNX1-2.
[0063] Example 4: Optimization of the method for constructing the MNX1-EGFP human pluripotent stem cell line The method described in Example 3 is the optimal route, but we have optimized the steps prior to this, specifically involving: 1. Optimization of transfection methods Common methods for introducing plasmids into cells include liposome transfection, PEI transfection, and electroporation. We tested and compared the transfection efficiencies of liposome transfection, PEI transfection, and electroporation, finding that electroporation was more efficient (reaching up to 15%, while liposome transfection and PEI transfection efficiencies were both below 1 / 1000). The electroporation process was further optimized, and the specific implementation method is as follows: (1) Obtain electroporated cells according to steps (1)-(4) of Example 3, and perform electroporation at a rate of 2×10 3 / cm 2 8×10 3 / cm 2 3.2×10 4 / cm 2 The density was seeded into a six-well plate containing X-ray-treated mouse fibroblasts.
[0064] (2) Perform drug screening according to step (6) of Example 3, 10-17 days after electroporation, 2×10 3 / cm 2 The number of surviving clones was small, 3.2 × 10⁻⁶. 4 / cm 2 The inoculation density results in a high final clone density, making it inconvenient to pick individual clones; 8×10 3 / cm 2 The optimal inoculation density is [not specified].
[0065] 2. Optimization of puromycin drug screening concentration (1) Cells obtained by electroporation according to steps (1)-(4) of Example 3 were screened with 0.2 μg / ml, 0.3 μg / ml, 0.4 μg / ml and 0.5 μg / ml puromycin for one week after electroporation for 24 hours.
[0066] (2) According to steps (6)-(9) of Example 3, clones were picked for verification. The probability of positive clones in the 0.4 μg / ml puromycin treatment group was the highest. (After puromycin treatment, non-edited cells survived to a certain extent at concentrations of 0.2 μg / ml and 0.3 μg / ml, resulting in a large number of clones in the culture wells. The adjacent clones healed together quickly, making it difficult to pick single clones. However, after treatment with 0.4 μg / ml puromycin, the number of clones was particularly small, which was conducive to picking single clones.)
[0067] Example 5: Directed Differentiation of MNX1 Reporter Gene-Induced Human Pluripotent Stem Cell Line into Motor Neurons The MNX1 reporter gene pluripotent stem cells constructed using the above optimal method were processed at a rate of 3-5 × 10⁻⁵. 4 / cm 2The samples were seeded into Matrigel (Corning, catalog number 354277) coated six-well plates and cultured using TeSR™-E8™ (Stem Cell technology, catalog number 05990). Two to four days after cell seeding, when the cell density reaches approximately 80%, the cells are switched to neural induction medium (DMEM / F12 (Gibco, catalog number 11330-032): neurobasal (Gibco, catalog number 21103049) (1:1), 0.5 × N2 (Gibco, catalog number 17502048), 0.5 × B27 (Gibco, catalog number 12587010), 1 × GlutaMAX (Gibco, catalog number 35050061), 0.1 mM vitamin C (sigma-Ardrich, catalog number A4403), 2 µM SB431542 (TargetMol, catalog number T1726), 2 µM DMH1 (TargetMol, catalog number T1942), 3 μM CHIR99021 (TargetMol, catalog number T2310)) and cultured for one week. In the second week, the medium was transferred to a new six-well plate at a 1:3 ratio, with 0.1 mM RA (Sigma-Ardrich, catalog number R2625) and 0.5 mM Purmorphamine (TargetMol, catalog number T1810) added to the culture medium from the first week. At the end of the second week, cells were digested into single cells using TrypLE (Gibco, catalog number 12604021), passaged 1:3 into uncoated six-well plates, and cultured in suspension. The next day, the medium was changed to neural differentiation medium (DMEM / F12 (Gibco, catalog number 11330-032): neurobasal (Gibco, catalog number 21103049) (1:1), 0.5 × N2 (Gibco, catalog number 17502048), 0.5 × B27 (Gibco, catalog number 12587010), 1 × GlutaMAX (Gibco, catalog number 35050061), 0.1 mM vitamin C (Sigma-Ardrich, catalog number A4403), 0.5 mM RA (Sigma Ardrich, catalog number R2625) and 0.1 mM Purmorphamine (TargetMol, catalog number T1810).
[0068] At the end of the fourth week, cells were digested into single cells using Accutase (STEMCELL Technologies, catalog number 07920). 10,000 motor neuron precursor cells were seeded on each slide (pre-coated with PO (Sigma-Ardrich, catalog number P3655)-laminin (Thermo Fisher, catalog number 23017015)). On the second day of passage, the culture medium was changed to neural maturation medium (DMEM / F12:neurobasal (1:1), 0.5 × N2, 0.5 × B27, 1 × GlutaMAX, 0.2 mM vitamin C, 0.5 mM RA and 0.1 mM Purmorphamine, 2.5 µM DAPT (TargetMol, catalog number T6202), 10 ng / ml CNTF (TargetMol, catalog number TMPJ-00081), 10 ng / ml BDNF (PeproTech, catalog number 450-02), 10 ng / ml IGF1 (PeproTech, catalog number 100-11).
[0069] Two weeks after culturing neurons in differentiation medium, the expression of MNX1 and green fluorescent protein was detected by immunofluorescence. Figure 5 Green fluorescence (EGFP) and red fluorescence (MNX1) co-localized. Electrophysiological detection results of spontaneous neuronal action potentials and sodium currents are as follows: Figure 6 , Figure 7 As shown.
[0070] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A vector system for constructing a motor neuron MNX1 reporter gene cell line, characterized in that, The carrier system includes: A guide vector containing sgRNA with a nucleotide sequence as shown in SEQ ID NO.1; The donor vector contains upstream and downstream homologous arms designed based on the stop codon of the motor neuron MNX1 gene and its upstream and downstream sequences, as well as a reporter gene located between the upstream and downstream homologous arms.
2. The carrier system according to claim 1, characterized in that, It must contain at least one of the following characteristics: (1) The reporter gene includes a fluorescent gene; (2) The skeleton of the guide vector includes pX459 plasmid, pX458 plasmid or pX330 plasmid; When the backbone is pX459 plasmid, the sequence of the guide vector is shown in SEQ ID NO.2; (3) The sequences of the upstream and downstream homologous arms in the donor vector are shown in SEQ ID NO.4-5; (4) The sequence of the donor vector is shown in SEQ ID NO.
6.
3. A CRISPR gene editing system containing the vector system described in claim 1 or 2.
4. The CRISPR gene editing system according to claim 3, characterized in that, The CRISPR gene editing system also contains a Cas9 expression vector.
5. Recombinant cells containing the vector system of claim 1 or 2 or the CRISPR gene editing system of claim 3 or 4.
6. The use of the vector system of claim 1 or 2, the CRISPR gene editing system of claim 3 or 4, or the recombinant cells of claim 5 in the preparation of a motor neuron MNX1 gene-related model.
7. A method for constructing a motor neuron MNX1 reporter gene cell line, characterized in that, The method includes the step of introducing the vector system of claim 1 or 2 or the CRISPR gene editing system of claim 3 or 4 into a cell line.
8. The construction method according to claim 7, characterized in that, The cell line includes stem cells; And / or, the vector system or CRISPR gene editing system is introduced via electroporation.
9. The construction method according to claim 8, characterized in that, When the cell line is a stem cell, the method further includes the step of differentiating and culturing the gene-edited stem cells.
10. The application of the vector system of claim 1 or 2, the CRISPR gene editing system of claim 3 or 4, the recombinant cell of claim 5, or the cell line obtained by the construction method of any one of claims 7-9 in the pharmaceutical field, characterized in that, Including any of the following: (1) For research in biomedicine related to muscle activity; (2) Related research on motor neurons; (3) Used to prepare cell models or animal models.