Construction and screening identification technology of immortalized muscle stem cell line

Abstract

According to the invention, Tibetan pig satellite cells are selected, and overexpression of different genes is realized through genetic engineering so as to mediate acquisition of immortalized cells. According to the present invention, the gene modification cell strain capable of achieving the simultaneous overexpression of the hTERT and the CDK4 and the simultaneous overexpression of the hTERT, the CDK4 and the survivin gene is harvested; the two cells both show an immortalization trend and still maintain differentiation ability relative to primary cells, and the latter cell has a more superior proliferation characteristic compared with the former. The gene modification result proves the feasibility of the immortalization method of the muscle stem cell, and provides thoughts and research directions for carrying out related researches.

Description

Technical Field

[0003] This technology belongs to the field of cell engineering, and specifically involves achieving immortalization of muscle stem cells through genetic modification. Background Art

[0004] As key cells for repairing damage in muscle tissue, muscle stem cells play an important role in the development of the whole body and are important materials for studying the construction of in vitro models of muscle damage. Muscle stem cells have a certain proliferation ability, the ability to differentiate into muscle fibers, and a tendency to easily differentiate in vitro, which has great application prospects in the field of medical treatment and future cultured meat production. However, the proliferation ability of primary isolated muscle stem cells is extremely limited when cultured in vitro, and it is costly to improve the culture conditions, such as adding corresponding growth factors or promoting proliferation chemical molecules, which is an important obstacle to the research of muscle stem cells. At present, the application of muscle stem cells has certain requirements for their proliferation ability in the construction of disease models, but it cannot be guaranteed that they can be used as needed, and re-acquisition from primary separation is time-consuming, labor-intensive and costly. In the most popular cultured meat research, muscle stem cells also play an extremely important role in the whole process. In the whole process with the goal of commercializing the product, the acquisition and proliferation maintenance of primary muscle stem cells are limiting factors. There have been related studies that have used hTERT and CDK4 genes to achieve the immortalization of human satellite cells or myoblasts and construct corresponding disease models. There are also related patents abroad that realize the immortalization of chicken satellite cells, aiming to promote the development of cultured meat. On this basis, combined with related research, a method for immortalizing cell transformation has been developed. Summary of the invention

[0005] Based on the generation limitation problem existing in the current research on muscle stem cells, the present invention increases the generation of muscle stem cells by gene transformation, so as to realize the immortality of muscle stem cells and maintain their ability to differentiate into muscle cells.

[0006] The present invention selects Tibetan pig muscle stem cells as experimental objects, performs gene transformation and identification.

[0007] The present invention selects lentivirus as a gene conversion vector, constructs a lentivirus vector containing a target gene and a screening gene, and then packages the lentivirus vector into lentivirus particles to infect primary Tibetan pig muscle stem cells to screen immortalized cell strains.

[0008] The present invention constructs immortalized cell lines containing two genes and three genes respectively, and performs characterization and verification respectively.

[0009] The present invention is based on the successful separation of Tibetan pig muscle stem cells and carries out immortalization transformation to create an immortalization method, which is a method for immortalizing muscle stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of recombinant lentiviral plasmids, including lentiviral vectors containing hTERT, CDK4 and survivin genes respectively.

[0011] Figure 2 This is a diagram of the construction process of the lentiviral plasmid containing the CDK4 gene and the survivin gene. Figure 2 A is a schematic diagram of the constructed plasmid. Figure 2 B is the electrophoresis diagram of nucleic acid during the construction process.

[0012] Figure 3 To collect the HEK293T cell morphology of three gene lentiviruses, it was proved that the packaging cells still maintained a good growth state during the lentivirus packaging process.

[0013] Figure 4 These are the morphological images of the transformed Tibetan pig satellite cells after screening, which are the morphological images of the cell lines that simultaneously overexpress hTERT and CDK4 before and after screening, and the morphological images of the cell lines that simultaneously overexpress hTERT, CDK4 and survivin genes.

[0014] Figure 5 To detect the gene insertion schematic diagram and the PCR electrophoresis diagram and sequencing results of the genome, Figures A and B are verification schematic diagrams, Figure C is a nucleic acid electrophoresis diagram for verifying the insertion of the hTERT gene expression frame, and the results show that there are bands of the same size compared to the primary cells, and Figure D is a nucleic acid electrophoresis diagram for verifying the insertion of the CDK4 gene, and the results show that there are bands of the same size compared to the primary cells. Figures E and F are the sequencing results of the fragments expanded from the genome of the transformed cells from reference Figures A and B, respectively, which are completely consistent with the target gene, proving that the gene has been successfully inserted.

[0015] Figure 6 These are immunofluorescence images of the three cell types using Desmin to verify their myogenicity. The results show that the cells maintained their myogenic characteristics before and after genetic modification.

[0016] Figure 7 The immunofluorescence results of the proliferation marker validation of the three cells using Ki67 demonstrated that the modified cells had a significant proliferation advantage over the primary cells, and that the three-gene cell line had a proliferation advantage over the two-gene cell line.

[0017] Figure 8 The immunofluorescence results of the differentiation ability of the three cells using MyHC differentiation markers show that the transformed cell lines still maintain myogenic differentiation ability.

[0018] Fig. 9The growth and proliferation curves of the primary and two types of cells show that the order of proliferation ability is three-gene cells, two-gene cells and primary cells.

[0019] Fig.10 To verify the results of cell aging of three types of cells, the primary cells were all stained blue, and a small amount of the two modified cell lines were stained, showing anti-aging activity, which further indirectly proved the immortality of the modified cells. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally carried out according to conventional conditions, such as the conditions described in the "Molecular Cloning Experiment Guide" compiled by J. Sambrook (Joseph Sambrook) et al., or according to the conditions recommended by the manufacturer.

[0021] Example 1: Construction of lentiviral management and acquisition of lentiviral packaging

[0022] The lentiviral plasmid pLV-hTERT-hygr containing the hTERT gene was purchased from a commercial source (addgene-). The plasmid map is shown in Figure 1 A. The plasmid map of the lentiviral plasmid pLV-CDK4-puro containing the CDK4 gene is as follows Figure 1 B. The plasmid map of the lentiviral plasmid pLV-NGES containing the survivin gene is as follows Figure 1 C.

[0023] Reagents and kits: Premix Taq DNApolymerase, Pyrobest DNApolymerase, restriction endonuclease (EcoR I, BamH I), PEI, Premix Taq DNApolymerase TaKaRa, rTaq, T4 DNAligase, ClonExpress IIOne Step Cloning Kit, DNA marker, GelRed nucleic acid stain, plasmid extraction kit, DNA gel recovery kit, blood / cell / tissue genomic DNA extraction kit.

[0024] Strains and plasmids: E. coli DH5α was used as the plasmid cloning strain, plasmid pLV-hTERT-hygr was purchased from commercial sources, and recombinant plasmids pLV-CDK4-puro and pLV-NGES were both constructed based on pLV-mcheery-eGFP plasmid.

[0025] The remaining screening genes and plasmid requirements are all referenced from the website addgene.

[0026] Cell line and cell culture medium: The primary cells are Tibetan pig satellite cells, the basal culture medium is DMEM / F12, and the formula is 15% FBS+84% DMEM / F12+1% triple antibody (penicillin-streptomycin-amphotericin)+10ng / mL bFGF. The virus packaging cell line is HEK293T, the basal culture medium is RPM1640, and the formula is 10% FBS+89% RPM1640+1% triple antibody. The drugs used for screening are neomycin, puromycin, and hygromycin.

[0027] Antibodies used: Anti-Pax7; Anti-Desmin; GoatAnti-Mouse IgGAF 594; Goat Anti-Rbbit IgGAF 594; GoatAnti-Rbbit IgG H&L (Alexa 488);

[0028] Anti-MyHC; Anti-Ki67.

[0029] Ampicillin (50 mg / mL): weigh 1 g and dissolve in 20 mL ddHO 2 O, sterilize by filtration using a 0.22 μm sterile filter membrane, aliquot, and store at -20°C. When culturing E. coli, add 1 μL of ampicillin solution (1:1000) to each 1 mL of LB medium.

[0030] LB liquid culture medium: weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride, dilute to 1 L with deionized water, sterilize at 121°C for 20 min, and store at room temperature.

[0031] Construction of recombinant plasmid

[0032] Primer design

[0033] Table 1.1 Primer design specific sequences

[0034]

[0035] PCR reaction conditions and system are shown in Table 1.2.

[0036] Table 1.2 PCR reaction system

[0037]

[0038]

[0039] Table 1.3 PCR reaction conditions

[0040]

[0041] Table 1.4 Double enzyme digestion system

[0042]

[0043] Table 1.5 System for connecting target gene and vector

[0044]

[0045]

[0046] After the ligation reaction is completed, centrifuge the PCR reaction tube and place the centrifuge tube on ice for subsequent transformation experiments. The next day, pick a single clone colony and culture it in 5 mL of LB liquid culture medium containing kanamycin for 16 hours, then take 1 mL of bacterial solution in a sterile EP tube and send it to Shanghai Ruimian Biotechnology Co., Ltd. for sequencing. The bacterial solution PCR and sequencing results are shown in Figure 2. Figure 2 A.

[0047] Regarding the construction of pLV-NGES plasmid, homologous recombination was used for construction.

[0048] The PCR system refers to Table 1.2, with pcDNA3.1-NGES plasmid and pLV-mcheery-eGFP plasmid as template or target fragment and linear vector respectively, and the reaction conditions are as shown in Table 1.6.

[0049] Table 1.6 PCR reaction conditions

[0050]

[0051] After the PCR reaction, the fragments are collected by gel recovery, and then the next step of homologous recombination reaction is carried out. The homologous recombination system is shown in Table 1.7.

[0052] Table 1.7 Homologous recombination system

[0053]

[0054] The reaction conditions were 37°C for 30 min.

[0055] After the ligation reaction is completed, centrifuge the PCR reaction tube and place the centrifuge tube on ice for subsequent transformation experiments. The next day, pick a single clone colony and culture it in 5 mL of LB liquid culture medium containing kanamycin for 16 hours, then take 1 mL of bacterial solution in a sterile EP tube and send it to Shanghai Ruimian Biotechnology Co., Ltd. for sequencing. The bacterial solution PCR and sequencing results are shown in Figure 2. Figure 2 B.

[0056] Collection and concentration of lentiviral particles

[0057] HEK293T cell culture

[0058] (1) Prepare complete culture medium as shown in the culture medium formula.

[0059] (2) Take out the HEK293T cells from the liquid nitrogen tank and quickly thaw them in 37°C warm water.

[0060] (3) Transfer the cryopreserved solution to a new EP tube, centrifuge at 1000 r / min for 5 min, and discard the supernatant.

[0061] (4) Resuspend in complete medium and transfer to a culture flask in 5% CO 2 , cultured at 37°C, and the medium was changed every 2 days.

[0062] Preparation of hTERT, CDK4 and NGES lentivirus

[0063] Transformation and extraction of PMD2.0 and PSPAX2 packaging plasmids. Specific operations are as per the Molecular Cloning Experiment Guide. The concentration of the extracted plasmids was measured using a micro-UV spectrophotometer.

[0064] (1) Preparation and concentration of hTERT-hygr lentiviral particles

[0065] Table 1.8 Lentiviral packaging system

[0066]

[0067] The steps for transfecting HEK293T cells with PEI are as follows:

[0068] ①Preparation of PEI-DNA mixture

[0069] The total reaction volume of the 6 cm culture dish is 420ul, and the specific reaction system is shown in Table 1.9.

[0070] Table 1.9 Lentivirus plasmid transfection system

[0071]

[0072]

[0073] The specific order of addition is to mix the plasmid DNA according to the volumes in the table above. First, add the plasmid DNA to 240 μL of basal culture medium and mix well. Then add PEI to the mixture at a specification of 1.16 μL / μg, and make up to 420 μL with basal culture medium. After thorough mixing, incubate at room temperature for 20-30 minutes.

[0074] ②Take out the HEK293T cells, remove the cell culture medium, and add preheated serum-free culture medium. The amount of culture medium added should be based on the amount of culture medium added during subculturing.

[0075] ③ Add the mixture drop by drop into the culture dish and shake gently to make the mixture evenly distributed in the culture medium.

[0076] ④ In 5% CO 2 After culturing at 37°C for 4 h, the cell culture medium was replaced with complete culture medium.

[0077] ⑤ Collect the culture fluid after 48h and 72h of culture respectively, and filter through a 0.45μm filter for later use. After collecting the culture fluid after 48h of culture, it can be temporarily stored at 4℃, and then re-added with culture medium and cultured for another 24h before collecting again. The two collections are mixed.

[0078] ⑥ Collection and concentration of virus particles. The collected virus stock solution is first filtered through a 0.45 μm filter membrane, and then concentrated by centrifugal ultrafiltration using a 100 KDa concentration column. 500 μL of concentrated solution is collected for every 10 mL of crude virus solution and stored in aliquots.

[0079] (2) Preparation and concentration of CDK4 lentiviral particles

[0080] Table 1.10 Lentivirus packaging system

[0081]

[0082] The above plasmids were transfected into HEK293T cells using PEI to prepare viral particles.

[0083] ① Preparation of PEI-DNA mixture

[0084] The total reaction volume of the 6 cm culture dish is 420 μL, and the specific reaction system is as follows.

[0085] Table 1.11 Lentiviral plasmid transfection system

[0086]

[0087]

[0088] The transfection operation and the collection and concentration of lentivirus were consistent with the packaging of hTERT plasmid and stored at -80°C for future use.

[0089] (3) Preparation and concentration of NGES lentivirus

[0090] The lentiviral packaging system is shown in Table 1.12

[0091] Table 1.12 Lentivirus packaging system

[0092]

[0093] The above plasmids were transfected into HEK293T cells using PEI to prepare viral particles.

[0094] ① Preparation of PEI-DNA mixture

[0095] Table 1.13 Lentiviral plasmid transfection system

[0096]

[0097] The transfection procedure and collection and concentration of lentivirus were the same as described above. Figure 3 The lentivirus was stored at -80°C for future use.

[0098] Example 2 Screening and identification of immortalized cell lines

[0099] ① Tibetan pig satellite cells were plated at 2×10 5 The cells were plated at a density of 100 / mL and the final volume in each well was 2 mL.

[0100] ② The concentrated hTERT lentivirus and CDK4 lentivirus were used to infect Tibetan pig satellite cells simultaneously. After 24 hours, the medium was replaced with growth medium. After 72 hours of culture, a screening medium containing hygromycin at a final concentration of 0.2 mg / mL and puromycin at 3 μg / mL was prepared for 10 days of screening, with the medium changed every 2 days.

[0101] ③ Replace the growth medium for the screened cells, culture and expand them, and collect the cells for subsequent testing.

[0102] ④ Then use the collected NGES lentiviral particles to infect the cells obtained after co-screening, and observe the green fluorescence for judgment. Then, screen for 10 days. Similar to the above steps, use 0.2 mg / mL G418 screening concentration for screening. The obtained cells are as follows Figure 4 shown.

[0103] Identification of immortalized cell lines

[0104] Stable cell lines were tested, and the gene level of the hTERT+CDK4 dual-gene stable cells was determined to determine whether the two genes were inserted into the genome of satellite cells.

[0105] ① Collect the obtained hTERT+CDK4 gene inserted cells and extract the genome. The genome extraction method refers to the description of the kit, extract the genome and measure the concentration.

[0106] Primers are designed according to the expression frame inserted into the genome. Figure 5 (A)(B), design primers F1, R1, F2, R2. The specific sequences of the primers are shown in Table 2.1.

[0107] Table 2.1 Sequences of identification primers

[0108]

[0109] Table 2.2 PCR reaction system for hTERT gene verification

[0110]

[0111]

[0112] Table 2.3 PCR reaction procedure for hTERT gene verification

[0113]

[0114] Table 2.4 CDK4 gene verification PCR reaction system

[0115]

[0116] Table 2.5 CDK4 gene verification PCR reaction procedure

[0117]

[0118] ② Carry out PCR reaction according to Table 2.2, Table 2.3, Table 2.4, and Table 2.5 respectively. Verify the obtained PCR products by nucleic acid electrophoresis and observe whether the band size is consistent with the expected one. Figure 5 (C)(D).

[0119] ③Then the obtained product was sent for sequencing, and the sequencing results were completely consistent. The results are as follows Figure 5 (E)(F).

[0120] After identifying the successful insertion of the two genes, the insertion of the survivin gene was judged based on fluorescence to verify the complete insertion of the three genes.

[0121] Example 3 Characterization and Identification of Immortalized Cell Lines

[0122] Myogenic identification

[0123] Myogenicity was identified using the myogenic marker Desmin and detected using immunofluorescence technology. The specific steps are as follows:

[0124] (1) Fixation: At the scheduled treatment time, the cells were washed twice with pre-cooled PBS and then fixed with pre-cooled 4% paraformaldehyde at room temperature for 15 min.

[0125] (2) Permeabilization: Wash the cells twice with pre-cooled PBS and treat with pre-cooled permeabilization solution at room temperature for 10 min. Wash the cells 3 times with PBS and incubate on a shaker during the washing process, each time for 5 min.

[0126] (3) Blocking: Add an appropriate amount of blocking solution to the culture dish, incubate at room temperature for 2 h, discard the blocking solution and wash the cells three times with pre-cooled PBS, each time for 5 min, incubating on a shaker during the washing process;

[0127] (4) Primary antibody incubation: dilute the Anti-Pax7 primary antibody to the working concentration using antibody diluent, refer to Table 3.5, incubate overnight at 4°C and recover the primary antibody, then wash three times with pre-cooled PBS, 5 min each time;

[0128] (5) Secondary antibody incubation: dilute the secondary antibody labeled with 488 nm fluorescence to the working concentration using secondary antibody diluent, refer to Table 3.5, and incubate at room temperature for 1 h. The incubation process is carried out on a shaker and protected from light with tin foil;

[0129] (6) Nuclear staining: discard the secondary antibody, wash with pre-cooled PBS on a shaker in the dark for 3 times, 5 min each time. Add working concentration of DAPI and stain for 10 min at room temperature in the dark.

[0130] (7) Photographing: Discard DAPI, wash with PBS three times, 5 min each time, then add 1 mL of PBS, and observe and photograph under a confocal microscope. Use a Leica confocal microscope to observe and photograph the immunofluorescence results, such as Figure 6 .

[0131] Differential analysis of proliferation markers

[0132] Ki67 was used as a cell proliferation marker to identify the proliferation markers of the two gene-edited cells in the logarithmic phase. Immunofluorescence detection was also used for detection. The specific method is as follows:

[0133] (1) Cell plating: Select two gene-inserted cell lines for plating at a rate of 2 × 10 cells per well. 4 The cells were plated at a density of 100 / well and immunofluorescence identification was performed after 48 h of growth.

[0134] (2) The specific process of the immunofluorescence detection method is shown in Example 3.

[0135] (3) Take photos and analyze them using a fluorescent inverted microscope, and process the data using Image J. The results are as follows: Figure 7 .

[0136] Induce myogenic differentiation and verify

[0137] (1) Inducing myogenic differentiation of the two gene-edited cells obtained, first preparing a differentiation medium composed of 2% HBS, 97% DMEM (HG), and 1% triple antibody;

[0138] (2) Cell plating. For TC and TCS cell lines, 1x10 5 The cells were plated at a density of 100 μL / mL in a 24-well plate in a total volume of 500 μL to achieve a cell confluence of 70%-80%;

[0139] (3) Replace the culture medium and induce differentiation. On the second day after plating, replace the proliferation medium with differentiation medium. Change the medium every 2 days and observe the cell morphology changes under a microscope.

[0140] (4) After 7 days of differentiation induction, immunofluorescence detection of differentiation marker MyHC was performed. The specific method and process were as shown in Example 3. Figure 8 .

[0141] Proliferation curve drawing

[0142] Proliferation curves were drawn for the obtained gene-inserted cell lines to determine the cell growth cycle and doubling time, and the MTT method was used for detection.

[0143] (1) Cell plating. The two gene-edited cells were plated at a density of 2,000 cells per well, with 30 wells for each type of cell.

[0144] (2) For the specific MTT method, refer to Chapter 2, 2.2.7. Measure the absorbance value for 6 consecutive days and use Origin to analyze and draw a growth curve. The results are as follows: Fig. 9 .

[0145] Cell senescence verification

[0146] Select primary satellite cells after multiple passages and two gene-edited cells for simultaneous plating to determine the proportion and number of senescent cells. Refer to the instructions of the Bio-Tech Cell Senescence β-Galactosidase Staining Kit for operation. The specific steps are as follows:

[0147] a. For cells cultured in 6-well plates, remove the cell culture medium, wash once with PBS or HBSS, add 1 ml of β-galactosidase staining fixative, and fix for 15 minutes at room temperature. For other types of culture plates, refer to this ratio for the amount of fixative and subsequent solutions.

[0148] b. Aspirate the cell fixative and wash the cells 3 times with PBS or HBSS, 3 minutes each time.

[0149] c. Remove PBS or HBSS and add 1 ml of staining solution to each well. For the preparation of staining solution, refer to Table 3.1.

[0150] Table 3.1 Configuration of dyeing working solution

[0151]

[0152] d. Incubate at 37℃ overnight. You can seal the 6-well plate with parafilm or plastic wrap to prevent evaporation. Note: Incubation at 37℃ cannot be performed in a carbon dioxide incubator.

[0153] e. Observe under an ordinary optical microscope. If it is not possible to observe and count in time, remove the staining solution, add 2 ml PBS, and store at 4℃ for several days; or add sealing solution to seal the slides, and store at 4℃ for a longer time. Note: If crystals are formed, please refer to the recommendations in the precautions and use 70% ethanol for washing. Fig.10 .

[0154] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A muscle stem cell immortalization method, which obtains different immortalized cell lines by overexpressing different gene combinations. Two genetically modified cell lines are obtained by simultaneously overexpressing hTERT and CDK4, and by simultaneously overexpressing hTERT, CDK4 and survivin genes.

2. For the Tibetan pig muscle stem cell transformation as described in claim 1, two immortalized cell lines were harvested, and there was a comparative optimization between the two.

3. According to claim 1, the method for achieving gene insertion and overexpression is to use a lentiviral vector, and construct a related vector through corresponding genetic engineering to achieve lentiviral packaging and subsequent infection and screening.

4. As described in claim 2, the Tibetan pig muscle stem cells are characterized in that they can be propagated for more than 40 generations after genetic modification, and their differentiation ability can be maintained.

5. The method of claim 3, wherein each of the hTERT, CDK4 and survivin genes is individually linked to a resistance gene, comprises three lentiviral vectors, and the screening drugs include hygromycin, puromycin and neomycin.

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