参见作者原研究论文

本实验方案简略版
Aug 2016

本文章节


 

Sleeping Beauty Transposon-based System for Rapid Generation of HBV-replicating Stable Cell Lines
使用基于睡美人转座子的系统快速生成HBV复制稳定细胞系    

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

The stable HBV-transfected cell lines, which based on stable integration of replication-competent HBV genome into hepatic cells, are widely used in basic research and antiviral drug evaluation against HBV. However, previous reported strategies to generate HBV-replicating cell lines, which primarily rely on random integration of exogenous DNA by plasmid transfection, are inefficient and time-consuming. We newly developed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable HBV-replicating cell lines of different genotype. The pTSMP-HBV vector contains HBV 1.3-copy genome and dual selection markers (mCherry and puromycin resistance gene), allowing rapid enrichment of stably-transfected cells via red fluorescence-activated cell sorting and puromycin antibiotic selection. In this protocol, we described the detailed procedure for constructing the HBV-replicating stable cells and systematically evaluating HBV replication and viral protein expression profiles of these cells.

Keywords: HBV (HBV), HBV-replicating cell lines (HBV复制细胞系), Sleeping Beauty transposon system (睡美人转座子系统), HBV 1.3-copy genome (HBV 1.3拷贝基因组), HepG2 (HepG2)

Background

Chronic hepatitis B virus (HBV) infection is currently a major public health burden, affecting over 240 million individuals globally (Witt-Kehati et al., 2016). Patients with chronic HBV have an elevated risk of chronic active hepatitis, cirrhosis, or primary hepatocellular carcinoma (HCC) (Schweitzer et al., 2015). Current treatments with interferon-α or nucleoside analogs do not eradicate the virus, and their effects on clearing hepatitis B surface antigen (HBsAg) are limited (Lucifora and Protzer, 2016; Soriano et al., 2017). Therefore, there is an urgent need for the development of novel antiviral inhibitors (Nassal, 2015).

A cell culture model for evaluating the activity of new agents against HBV is an important tool for new drug development. The stable HBV-replicating cell lines, which carry replication-competent HBV genome stably integrated into the genome of human hepatoma cell lines (Huh7 and/or HepG2), are widely used to evaluate the effects of antiviral agents (Witt-Kehati et al., 2016). The stable HBV-producing human hepatoma cell lines (HepG2.2.15 and HepaAD38) integrated the D-genotype HBV genome, which are widely used in antiviral research (Chang et al., 1987; Ladner et al., 1997). However, stable HBV-producing cell lines of genotypes A, B, and C are not commonly used in the research field. Therefore, there is a need to develop cell lines of HBV genotypes A-C for drug development.

The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans (Structure of SB can be seen in Figure 1A). Sleeping Beauty transposition is a cut-and-paste process, during which the element ‘jumps’ from one DNA molecule to another (Figure 1B) (Ivics and Izsvak, 2011). Since its reconstruction in 1997 from the salmonid fish genome (Ivics et al., 1997), the SB system has been undergoing several modifications to improve its efficacy (Geurts et al., 2003; Baus et al., 2005; Score et al., 2006). The development of the hyperactive transposase SB100X has increased approximately 100-fold of efficiency compared with the first-generation transposase (Mátés et al., 2009), which is expected to facilitate widespread applications in functional genomics and gene therapy (Izsvak and Ivics, 2004).


Figure 1. The Sleeping Beauty transposable element and its transposition. A. The Sleeping Beauty (SB) system. The transposase gene (yellow rectangle) is flanked by terminal inverted repeats (IR/DRs, blue arrows), each containing two binding sites for the transposase (small green arrows). The transposase consists of an N-terminal, DNA-binding domain (PAI and RED), a nuclear localization signal (NLS), a C-terminal and catalytic domain (DDE). B. Transposition. The transposase gene within the element can be replaced by a therapeutic gene, and the resultant transposon can be maintained in a simple plasmid vector. The transposase is supplied in trans. The transposase binds to its binding sites within the IR/DR repeats and, together with host factors such as HMGB1, forms a synaptic complex, in which the ends of the transposon are paired. The transposon is excised from the donor molecule and integrates into a new location.

Materials and Reagents

  1. Pipette tips, 10 μl (Haimen plastic, 20111088)
  2. Pipette tips, 200 μl (Corning, Axygen®, catalog number: T-200-Y-R )
  3. Pipette tips, 1 ml (Haimen plastic, 20111011)
  4. Cell culture plate (100 mm) (Corning, catalog number: 430167 )
  5. Cell culture plate (60 mm) (Thermo Fisher Scientific, catalog number: 150288 )
  6. 15 ml tube (Thermo Fisher Scientific, catalog number: 339651 )
  7. 70 μm cell strainer (Fisher Scientific, Fisherbrand, catalog number: 22-363-548 )
  8. Cell culture plate (6 well) (Corning, catalog number: 3516 )
  9. Cell culture plate (24 well) (Corning, catalog number: 3524 )
  10. Cell Imaging Plate, 24 well, glass bottom (Eppendorf, catalog number: 0030741021 )
  11. Nylon membranes (Roche Diagnostics, catalog number: 11417240001 )
  12. Human hepatoma HepG2 cells (Originally from the China Centre for Type Culture Collection, Wuhan, China)
  13. Minimum essential medium (MEM, powder) (Thermo Fisher Scientific, GibcoTM, catalog number: 41500-083 )
  14. Fetal bovine serum, Qualified, Australia Origin (Thermo Fisher Scientific, catalog number: 10099141 )
  15. X-tremeGENE HP DNA Transfection Reagent (Roche Diagnostics, catalog number: 06366236001 )
  16. Opti-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 31985070 )
  17. 0.25% Trypsin-EDTA (1x), Phenol Red (Thermo Fisher Scientific, GibcoTM, catalog number: 25200-114 )
  18. Puromycin (Thermo Fisher Scientific, InvitrogenTM, catalog number: A1113803 )
  19. Mouse anti-HBcAg (Innodx Biotechnology, catalog number: 2A7-21 [it's new anti-HBc mAb, available on request])
  20. Alexa Fluor® 488 Donkey Anti-Mouse IgG (H+L) (Thermo Fisher Scientific, catalog number: A-21202 )
  21. DAPI (Thermo Fisher Scientific, InvitrogenTM, catalog number: D1306 )
  22. Micrococcal nuclease (Takara Bio, catalog number: D2910 )
  23. Proteinase K (Takara Bio, catalog number: D9033 )
  24. Ethanol, C2H6O, AR (Xilong Scientific, catalog number: 1030029AR )
  25. Oligonucleotides

  26. dNTP Mixture 2.5 μM (Takara Bio, catalog number: D4030A )
  27. DIG Easy Hyb Granules (Roche Diagnostics, catalog number: 11796895001 )
  28. PrimeSTAR GXL DNA Polymerase (Takara Bio, catalog number: DR050A )
  29. Premix Ex TaqTM (Probe qPCR) (Takara Bio, catalog number: RR390A )
  30. Casein 10x blocking buffer (Sigma-Aldrich, catalog number: B6429-500ML )
  31. Anti-DIG (AP) antibody (Roche Diagnostics, catalog number: 11093274910 )
  32. CDP-Star AP substrate (Roche Diagnostics, catalog number: 12041677001 )
  33. Universal DNA Purification Kit (TIANGEN Biotech, catalog number: DP214-03 )
  34. Diagnostic kit for Hepatitis B surface antigen (CLEIA) (Wantai Biological Pharmacy, catalog number: HBV-1396 )
  35. Diagnostic kit for Hepatitis B e-antigen (ELISA) (Wantai Biological Pharmacy, catalog number: HBV-0396 )
  36. ED-11 (Innovax Biotechnology)
  37. Virus DNA/RNA Extraction kit (GenMag Biotechnology, catalog number: NA007 )
  38. Mycoplasma-free neonatal bovine serum (Tianhang Biotechnology, catalog number: 11011-8615 )
  39. Sodium chloride (NaCl, AR) (Xilong Scientific, catalog number: 1001012AR )
  40. Potassium chloride (KCl, cell culture) (Sigma-Aldrich, catalog number: P5405 )
  41. Hydrochloric acid (HCl, AR) (Xilong Scientific, catalog number: 1029013AR )
  42. Sodium hydroxide (NaOH, AR) (Xilong Scientific, catalog number: 1001037AR )
  43. Disodium hydrogen phosphate (Na2HPO4·12H2O, AR) (Xilong Scientific, catalog number: 1001067AR )
  44. Potassium dihydrogen phosphate (KH2PO4, AR) (Xilong Scientific, catalog number: 1002048AR500 )
  45. Sodium bicarbonate (NaHCO3, AR) (Xilong Scientific, catalog number: 44558 )
  46. Paraformaldehyde (Sigma-Aldrich, catalog number: 16005-1KG-R )
  47. Triton X-100 (AMRESCO, catalog number: 0694 )
  48. Bovine albumin (Low endotoxin) (ICPbio International, catalog number: ABRE-1KG )
  49. Tris base (SEEBIO BIOTECH, catalog number: 183995 )
  50. Tris-saturated phenol (Solarbio, catalog number: T0250 )
  51. Ethylenediaminetetraacetic acid (EDTA, AR) (Xilong Scientific)
  52. NONIDET® P-40 Substitute (AMRESCO, catalog number: M158-500ML )
  53. Calcium chloride (CaCl2, AR) (Xilong Scientific, catalog number: U1000566-500g )
  54. Sodium dodecyl sulfate (SDS) (Merck, catalog number: 428015 )
  55. Chloroform (AR) (Xilong Scientific, catalog number: 1039013AR500 )
  56. Isoamyl alcohol (AR) (Xilong Scientific, catalog number: U1001975-500ml )
  57. Maleic acid (Sigma-Aldrich, catalog number: M0375 )
  58. Tween-20 (BBI Solutions, catalog number: TB0560-500ml )
  59. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-Na2·2H2O, AR) (Xilong Scientific, catalog number: 100186 )
  60. Acetate (CH3COOH, AR) (Xilong Scientific, catalog number: 1029047AR )
  61. Sodium citrate (C6H5Na3O7, AR) (Xilong Scientific, catalog number: 1001059AR )
  62. Verson buffer (see Recipes)
  63. PBS buffer (see Recipes)
  64. 4% paraformaldehyde (see Recipes)
  65. 0.2% Triton X-100 (see Recipes)
  66. 3% BSA (see Recipes)
  67. NET buffer (see Recipes)
  68. 1.2 M CaCl2 (see Recipes)
  69. 0.5 M EDTA (see Recipes)
  70. 10% SDS (see Recipes)
  71. Phenol/chloroform/isoamyl alcohol (25:24:1) (see Recipes)
  72. Maleic acid buffer (see Recipes)
  73. Washing buffer (see Recipes)
  74. Detection buffer (see Recipes)
  75. 50x TAE buffer (see Recipes)
  76. Depurination buffer (see Recipes)
  77. Denaturation buffer (see Recipes)
  78. Neutralization buffer (see Recipes)
  79. 10x SSC (see Recipes)
  80. 2x SSC/0.1% SDS (see Recipes)
  81. 0.5x SSC/0.1% SDS (see Recipes)

Equipment

  1. Pipettes (Mettler-Toledo International, RAININ, model: Pipet-Lite )
  2. CO2 Incubator (Thermo Fisher Scientific, catalog number: 3111 )
  3. Centrifuge (Thermo Fisher Scientific, model: HeraeusTM PicoTM 17 )
  4. Sorvall refrigeration Centrifuge (Thermo Fisher Scientific, model: SorvallTM ST 16R )
  5. BD FACS Aria III (BD, model: FACSAriaTM III )
  6. High Content Screening System (PerkinElmer, model: Opera PhenixTM )
  7. Water bath (Grant Instruments, model: GD100 )
  8. UV cross-linking instrument (Shanghai SIGMA High-tech, model: SH4 )
  9. Bio-Rad vacuum blotter (Bio-Rad Laboratories, model: Model 785 )
  10. Multifunctional molecular hybridization oven (UVP, model: HM-4000 )
  11. ImageQuant LAS4000 mini (GE Healthcare, model: ImageQuant LAS 4000 mini )
  12. Plastic film sealing machine (Shanghai Mingwei, model: F-400 )
  13. Biomek NXP (Beckman Coulter, model: Biomek NXP )
  14. Microplate reader (Autobio, model: PHOmo )
  15. Orion II Microplate Lumimometer (Titertek-Berthold, model: Orion II )
  16. Electrophoresis apparatus (Bio-Rad Laboratories, catalog number: 1645050 )
  17. LightCycler® 96 (Roche Molecular Systems, model: LightCycler® 96 )

Procedure

  1. Generation of HBV-replicating stable cell lines
    1. Transfection of HepG2 cells
      1. Grow HepG2 cells in MEM/10% FBS at a 37 °C, CO2 incubator. Split the cells to a 10-cm plate 24 h before transfection; the cell density should reach 70-90% at the time of DNA transfection.
      2. Dilute 20 μg each of pTSMP-HBV1.3 plasmids of genotype Ae (AY707087), Ba (GU357842), Ce (GU357845) and D1 (GU357846) with 2 ml opti-MEM serum-free medium, respectively. Mix gently.
        Note: The map of pTSMP-HBV1.3 plasmid and the genome organization of HBV1.3 used in this study are shown in Figures 2A and 2B, respectively. The construction of pTSMP-HBV1.3 plasmids can be referred to (Wu et al., 2016).


      3. Figure 2. Schematic representation of the pTsmP vector (A) and HBV 1.3 genome construction (B)

      1. Pipet the X-tremeGENE HP DNA Transfection Reagent (60 µl) directly into the medium containing the diluted DNA without contact with the walls of the plastic tubes. Mix gently.
      2. Incubate the transfection reagent: DNA complex for 15 min at room temperature.
      3. Add the transfection complex to the cells in a dropwise manner.
      4. Following transfection, incubate cells for 48 h at 37 °C in a 5% CO2 incubator; successful transfected cells with activated red fluorescence (mCherry positive) can be visualized by fluorescence microscopy and sorted by flow cytometry.
    2. Flow cytometric sorting of successful transfected cells
      1. Wash the cells with 5 ml Verson buffer (see Recipes) and discard the supernatant.
      2. Add 500 µl 0.25% trypsin-EDTA to the cells and incubate at 37 °C for 3 min.
      3. Add 5 ml MEM/10% FBS to stop digestion.
      4. Transfer 2 x 107 cells into a 15 ml tube.
      5. Centrifuge cell 1,000 x g for 3 min, and then discard supernatant.
      6. Wash with 5 ml PBS buffer (see Recipes).
      7. Centrifuge cells at 1,000 x g for 3 min, and then discard supernatant.
      8. Resuspend cells in 7 ml PBS buffer, and then filter through a 70 μm cell strainer.
      9. Run samples on BD FACS Aria III machine, the excitation light required is 561 nm (Figure 3).


      10. Figure 3. The first round of mCherry-activated FACS sorting. Negative cell with unexpressed mCherry is HepG2 (blue peak), positive cell with expressed mCherry is HepG2-pTsmp-1.3HBV-S11 (red peak).

      1. The sorted mCherry-expressing cells are cultured in a 6-well culture plate for expansion in the presence of puromycin selection (2 μg/ml). Refresh Culture medium containing puromycin every 2 days.
      2. On the following day, when cell confluence is 70-80%, the cells are subjected to the second round of mCherry fluorescence-activated FACS sorting and further puromycin selection.
      3. After about a 4-round of mCherry fluorescence-activated FACS sorting and puromycin-resistant cell selection, over 90% cells are puromycin resistant and stably expressed mCherry.
      4. These cells are further propagated. After 10 passages, nearly 100% of cells of the 4 HBV cell lines persistently express mCherry and grow well in the presence of puromycin.

  2. Characterization of HBV-replicating stable cell lines
    1. Immunofluorescence of hepatitis B virus core antibody
      1. Cell preparation
        Seed 4 x 105 pTSMP-HBV1.3 cells and 3.5 x 105 parental HepG2 cells (serve as a negative control) on the cell imaging plate with a density of 70%. Incubate the cells at 37 °C and 5% CO2 in an incubator for 24 h.
      2. Cell washing
        Discard the medium and wash once with 500 μl PBS.
      3. Fixation
        Add 250 μl per well of 4% formaldehyde (see Recipes) along the side of cell plate. Allow cells to fix in the dark for 15 min at room temperature (RT). Rinse three times in 1 ml 1x PBS for 3 min each.
      4. Permeabilization
        Incubate the samples for 5 min in 250 μl 0.2% Triton X-100 (see Recipes) per well. Rinse three times in 1 ml 1x PBS for 3 min each.
      5. Blocking
        Incubate cells with 250 μl 3% BSA (see Recipes) in PBS at RT for 1 h to block potential non-specific binding of the antibody, then discard the supernatants.
      6. Primary antibody
        Incubate the cells with 250 μl anti-HBcAg antibody (1:1,000 dilute in 3% BSA) for 1 h at RT (or overnight at 4 °C) in the dark. Next, remove the solution and wash cells 3 times with 1 ml/well PBS for 3 min each.
      7. Secondary antibody
        Incubate the cells with 250 μl secondary antibody (Alexa Fluor® 488 Donkey Anti-Mouse IgG (H+L), 1:1,000 dilute in 3% BSA) for 30 min at RT in the dark. Remove the solution and wash 3 times with 1 ml/well PBS for 3 min each in the dark.
      8. Counterstaining
        Incubate the cells with 250 μl DAPI (1:2,000 dilute in 3% BSA) for 5 min at RT in the dark. Discard the solution and wash 3 times with 1 ml/well PBS for 3 min each.
      9. Photographs
        Photograph in High Content Screening System (Figure 4). The setting used in the imager for imaging (Table 1).

        Table 1. The setting used in the High Content Screening System for imaging



      10. Figure 4. Immunofluorescence for HBcAg of HBV cell lines (Confocal mode, 40x water lens)

    2. Southern blot
      1. Cell: Plate 4 x 106 cells in the 6 cm dish with 60-70% confluent. Culture cells for 2 days and proceed to the next experiment.
      2. Extraction of HBV total DNA
        1. Discard the supernatant of cells and wash 2 times with cold PBS.
        2. Add 600 μl NET buffer (see Recipes) in cell pellet and incubate on ice (or 4 °C) for 1 h.
          Note: We usually add 200-300 μl in one well of a 6-well plate; or 400-600 μl a 6 cm dish; or 1.2-1.8 ml in a 10 cm dish.
        3. Centrifuge the lysates at 13,400 x g for 20 min at 4 °C and transfer the supernatant (600 μl) into a new 1.5 ml EP tube.
        4. Add 2 μl Micrococcal nuclease (10 mg/ml) and 3 µl 1.2 M CaCl2 (final concentration of 6 mM) (see Recipes) and incubate for 30 min in a 37 °C water bath.
        5. Add 30 μl 0.5 M EDTA (final concentration of 25 mM) (see Recipes) and incubate for 15 min in a 65 °C water bath.
        6. Add 6.68 μl Proteinase K (19 mg/ml) and 32.1μl 10% SDS (see Recipes) and incubate overnight (At least 12 h but no more than 18 h) in a 50 °C water bath (final concentration of 200 μg/μl proteinase K and 0.5% SDS).
        7. Add an equal volume of phenol/chloroform/isoamylalcohol (approx. 600 μl), mix thoroughly by inverting the tube several times and incubate at room temperature (15-25 °C) for 5 min. Centrifuge at 14,000 x g for 10 min and transfer the aqueous phase into a new 1.5 ml EP tube.
        8. Repeat the above step (Step B2b) vii twice.
        9. Precipitate DNA by adding twice the volume of absolute ethanol and 1/10 volume of sodium acetate and then placing at -80 °C for 2 h. Centrifuge at 14,000 x g for 10 min at 4 °C. Carefully decant supernatant.
        10. Wash the DNA pellet with 500 μl 75% ethanol and centrifuge at 14,000 x g for 5 min. Carefully decant supernatant.
        11. Air-dry pellet for 5-10 min and re-dissolve DNA in 35 μl distilled deionized water. Store HBV DNA at -80 °C until use.
      3. The construction of the HBV probe
        1. Prepare PCR reaction solution according to the following components:
          5x PrimeSTAR GXL buffer
          5 μl
          HBx-probe-F (10 μM)
          1 μl
          HBx-probe-R (10 μM)
          1 μl
          dNTP Mix (2.5 μM)
          2 μl
          DIG-dUTP
          2 μl
          pGEM-1.3 HBV
          1 μl
          PrimeSTAR GXL DNA polymerase
          1 μl
          ddH2O
          37 μl
        2. PCR reaction

        3. Purify the HBV probe by using Universal DNA Purification Kit.
      4. Evaluate the effect of the probe
        1. The probe is diluted to 2 ng/μl as the first gradient and 10-fold down-diluted to a total of 5 gradients with a minimum concentration of 0.2 pg/μl.
        2. Drop the diluted probe on a piece of nylon membrane via pipette, 5 μl each (final loading: 10,000 pg, 1,000 pg, 100 pg, 10 pg, 1 pg). Keep a certain distance between each point to avoid contamination.
        3. UV irradiation (1.5 J/cm2) for 3 min.
        4. Block for 30 min with 1x blocking buffer (Casein blocking buffer [10x] diluted in Maleic acid buffer [see Recipes]).
        5. Incubate for 40 min with anti-DIG (AP) antibody (1:3,000 diluted in 1x blocking buffer).
        6. Wash nylon membrane 3 times with washing buffer (see Recipes) for 10 min each.
        7. Rinse with detection buffer (see Recipes) for 2 min.
        8. Add the AP substrates to the membrane to completely infiltrate it.
        9. Exposure by using ImageQuant LAS4000 mini. The intensity of your experimental probe is required to make the point of 2 pg/μl or below visible to yield satisfactory hybridization results. If the probe signal is weaker than this, it will be difficult to detect signals from low abundance species (Figure 5).


        10. Figure 5. Southern blotting analysis for the effect of probe

        1. Southern blot analysis
          1. Separate the DNA samples by electrophoresis through 1.2% agarose gel at 80 V for 2 h in 1x TAE buffer (see Recipes).
          2. After electrophoresis is completed, gel is treated as followed (The following steps are operated on a shaker):
            1)
            Transfer the gel to a tray and add depurination buffer (see Recipes) to soak the gel, incubate for 15 min on the shaker at RT and shake gently (Breaking the DNA into smaller pieces, thus allowing more efficient transferring from the gel to membrane).
            2)
            Remove the depurination buffer. Rinse the gel twice with ddH2O for 1 min each.
            3)
            Rinse twice with denaturation buffer (see Recipes) for 15 min each.
            4)
            Rinse the gel twice with ddH2O for 1 min each.
            5)
            Rinse with neutralization buffer (see Recipes) for 10 min.
          3. Transfer DNA from the gel to nylon membrane with 10x SSC for 1.5 h by vacuum blotter (Figure 6).
            Note: Wet the precut nylon membrane in double distilled water by slowly lowering the membrane at a 45-degree angle to the water. Then, wet the membrane and the filter paper in 10x SSC; Place the gel and nylon membrane in the order shown in Figure 6; Remove bubbles between the gel and membrane; Start the vacuum source and slowly turn the bleeder valve clockwise until the gauge reads at 5 inches of Hg.


          4. Figure 6. Scheme of capillary transfer method for Southern blotting

            1. After gel transfer, fix the DNA to the nylon membranes by UV crosslinking at 1.5 J/cm2 for 3 min.
              Note: Irradiation time = irradiation dose (mJ/cm2)/irradiation intensity (mW/cm2), the irradiation intensity of this instrument is 9.0 mW/cm2.
            2. Pre-hybridization step
              Place membrane in hybridization plastic film contained with 10 ml DIG Easy Hyb buffer and incubate the membrane for 2 h in 42 °C hybridization chamber with gentle agitation.
            3. Hybridization
              1)
              Denature the DNA probe at 95 °C for 5 min and immediately place on ice for 2 min.
              2)
              Replace pre-warmed 10 ml of DIG Easy Hyb buffer with 300 ng DIG-dUTP- labeled HBV probe and incubate overnight in a 42 °C hybridization chamber with gentle agitation.
            4. Wash
              1)
              Wash 2 times with 2x SSC/0.1% SDS buffer for 5 min at room temperature.
              2)
              Wash 2 times with pre-warmed 0.5x SSC/0.1% SDS buffer for 5 min at 65 °C.
              Note: Shake the buffer before use.
            5. Detection
              1)
              Rinse with Maleic acid buffer (see Recipes) for 2 min.
              2)
              Block for 30 min with 1x blocking buffer (10x blocking buffer diluted with Maleic acid buffer).
              3)
              Incubate for 40 min with anti-DIG(AP) antibody (1:3,000 diluted in 1x blocking buffer)
              4)
              Wash 3 times with 100 ml washing buffer for 15 min each.
              Note: Washing buffer shake evenly before use.
              5)
              Equilibrate for 3 min in 20 ml detection buffer.
              6)
              Add the membrane with AP substrate in dropwise so that to completely infiltrate.
              7)
              Expose the membrane continuously and take images using ImageQuant LAS4000 mini.
        2. Detection of the levels of viral markers in supernatants
          1. Cell
            1. Seed 3.5 x 105 pTSMP-HBV1.3 cells in the 24-well plates with a density of 60-70%.
            2. Replenish and collect the culture medium for measurement of viral antigens and HBV DNA every 2 days.
            3. Monitor the levels of these viral markers in supernatants for each cell line during 1 month.
          2. Viral antigens
            1. Measure viral antigens (HBsAg and HBeAg) in culture medium by chemiluminescence method using commercial assay kits (Wantai, Beijing, China).
            2. Antigen (HBsAg and HBeAg) standards are standardized patient serum, with the original concentration are 90,000 IU/ml and 10,000 Ncu/ml respectively.
            3. The standard of HBeAg is diluted with 20% NBS into six gradient concentrations, 10, 5, 2.5, 1.25, 0.25, 0.05 Ncu/ml respectively.
            4. The standard of HBsAg is diluted with ED-11 into six gradient concentrations, 45, 9, 1.8, 0.36, 0.072, 0.0144 IU/ml respectively.
          3. HBV total DNA
            1. HBV total DNA extraction
              1)
              Extract the total DNA in culture medium by using virus DNA/RNA extraction kit on Biomek NXP (Beckman, California, USA).
              2)
              The standard of HBV total DNA is standardized patient serum with the original concentration is 1.5 x 108 IU/ml, diluting with 10-fold dilutions in PBS into six gradients.
            2. Prepare PCR reaction solution according to the following components.
              2x Premix Ex Taq (Probe qPCR)
              10 μl
              KHF2 (100 μM)
              0.1 μl
              KHR1 (100 μM)
              0.1 μl
              KHP4 (100 μM)
              0.05 μl
              DEPC water
              4.75 μl
              Template
              5 μl
            3. Real-time PCR reaction is performed with LightCycler 96.

      Notes

      1. The state of the cells is critical to the transfection efficiency and cell state after sorting.
      2. During the sorting process, cells should be sorted at 4 °C, and the flow rate should be controlled below 9 µl/min. This is crucial for the cell state after sorting.
      3. The number of cells after sorting should at least be enough to plate in 48-well plates so that the cells are more likely to survive.

      Recipes

      1. Verson buffer(1 L)
        0.4 g KCl
        0.06 g KH2PO4
        8.0 g NaCl
        0.35 g NaHCO3
        0.08 g Na2HPO4·12H2O
        0.2 g EDTA
        Filter sterilize (0.22 μm) or sterilize by autoclavation
      2. PBS buffer
        145 mmol/L NaCl
        2.68 mmol/L KCl
        10 mmol/L Na2HPO4·12H2O
        1.76 mmol/L KH2PO4
        Adjust to pH 7.4
        Filter sterilize (0.22 μm) or sterilize by autoclavation 
      3. 4% Paraformaldehyde
        4 g Paraformaldehyde
        100 ml PBS
        1-2 drops 10 M NaOH
        Store at 4 °C and protect from light
      4. 0.2% Triton X-100
        200 μl Triton X-100
        100 ml PBS
        Store at 4 °C
      5. 3% BSA
        3 g BSA
        100 ml PBS
        Sub-package and store at -20 °C
      6. NET buffer
        50 mM Tris-base (pH = 8.0)
        1 mM EDTA
        100 mM NaCl
        0.5% NP-40
      7. 1.2 M CaCl2
        13.32 g CaCl2
        100 ml ultra-pure water
        Store at 4 °C
      8. 0.5 M EDTA
        Store at 4 °C
      9. 10% SDS
        100 g electrophoresis grade SDS
        Add ddH2O to 1 L
        Heat to 68 °C
        Adjust to pH 7.2
      10. Phenol/chloroform/isoamyl alcohol (25:24:1)
        Equilibrate phenol, chloroform and isoamyl alcohol in a ratio of 25:24:1, then store in a brown glass bottle and cover with a layer of Tris-Cl, store at 4 °C until use
      11. Maleic acid buffer
        0.1 M Maleic acid
        0.15 M NaCl
        Adjust to pH 7.5
        Sterilize by autoclavation
      12. Washing buffer
        0.1 M Maleic acid
        0.15 M NaCl
        3 ml/L Tween 20
        Adjust to pH 7.5
        Sterilize by autoclavation
      13. Detection buffer
        0.1 M Tris-base
        0.1 M NaCl
        Adjust to pH 9.5
        Sterilize by autoclavation 
      14. 50x TAE buffer
        242 g Tris-base
        100 ml EDTA (0.5 M, pH 8.0)
        57.1 ml acetate
        Add ddH2O to 1 L
        Adjust to pH 8.5
      15. Depurination buffer
        0.2 N HCl
        Sterilize by autoclavation
      16. Denaturation buffer
        0.5 M NaOH
        1.5 M NaCl
        Sterilize by autoclavation
        Neutralization buffer
        1 M Tris-base
        1.5 M NaCl
        Sterilize by autoclavation
      17. 10x SSC
        1.5 M NaCl
        0.15 M Sodium citrate
        Adjust to pH 7.0
        Sterilize by autoclavation
      18. 2x SSC/0.1% SDS
        200 ml 10x SSC
        10 ml 10% SDS
        Add ultra-pure water to 1 L
        Sterilize by autoclavation
      19. 0.5x SSC/0.1% SDS
        50 ml 10x SSC
        10 ml 10% SDS
        Add ultra-pure water to 1 L
        Sterilize by autoclavation

      Acknowledgments

      We would like to acknowledge the following publication on which this protocol is based: Wu et al. (2016). The authors declare no competing financial interests.

      References

      1. Baus, J., Liu, L., Heggestad, A. D., Sanz, S. and Fletcher, B. S. (2005). Hyperactive transposase mutants of the Sleeping Beauty transposon. Mol Ther 12(6): 1148-1156.
      2. Chang, C. M., Jeng, K. S., Hu, C. P., Lo, S. J., Su, T. S., Ting, L. P., Chou, C. K., Han, S. H., Pfaff, E., Salfeld, J. and et al. (1987). Production of hepatitis B virus in vitro by transient expression of cloned HBV DNA in a hepatoma cell line. EMBO J 6(3): 675-680.
      3. Geurts, A. M., Yang, Y., Clark, K. J., Liu, G., Cui, Z., Dupuy, A. J., Bell, J. B., Largaespada, D. A. and Hackett, P. B. (2003). Gene transfer into genomes of human cells by the sleeping beauty transposon system. Mol Ther 8(1): 108-117.
      4. Ivics, Z. and Izsvak, Z. (2011). Nonviral gene delivery with the Sleeping Beauty transposon system. Hum Gene Ther 22(9): 1043-1051.
      5. Ivics, Z., Hackett, P. B., Plasterk, R. H. and Izsvak, Z. (1997). Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91(4): 501-510.
      6. Izsvak, Z. and Ivics, Z. (2004). Sleeping beauty transposition: biology and applications formolecular therapy. Mol Ther 9(2): 147-156.
      7. Ladner, S. K., Otto, M. J., Barker, C. S., Zaifert, K., Wang, G. H., Guo, J. T., Seeger, C. and King, R. W. (1997). Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob Agents Chemother 41(8): 1715-1720. 
      8. Lucifora, J. and Protzer, U. (2016). Attacking hepatitis B virus cccDNA--The holy grail to hepatitis B cure. J Hepatol 64(1 Suppl): S41-S48.
      9. Mátés, L., Chuah, M. K., Belay, E., Jerchow, B., Manoj, N., Acosta-Sanchez, A., Grzela, D. P., Schmitt, A., Becker, K., Matrai, J., Ma, L., Samara-Kuko, E., Gysemans, C., Pryputniewicz, D., Miskey, C., Fletcher, B., VandenDriessche, T., Ivics, Z. and Izsvak, Z. (2009). Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet 41(6): 753-761.
      10. Nassal, M. (2015). HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut 64(12): 1972-1984.
      11. Schweitzer, A., Horn, J., Mikolajczyk, R. T., Krause, G. and Ott, J. J. (2015). Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet 386(10003): 1546-1555.
      12. Score, P. R., Belur, L. R., Frandsen, J. L., Geurts, J. L., Yamaguchi, T., Somia, N. V., Hackett, P. B., Largaespada, D. A. and McIvor, R. S. (2006). Sleeping Beauty-mediated transposition and long-term expression in vivo: use of the LoxP/Cre recombinase system to distinguish transposition-specific expression. Mol Ther 13(3): 617-624.
      13. Soriano, V., Barreiro, P., Benitez, L., Pena, J. M. and de Mendoza, C. (2017). New antivirals for the treatment of chronic hepatitis B. Expert Opin Investig Drugs 26(7): 843-851.
      14. Witt-Kehati, D., Bitton Alaluf, M. and Shlomai, A. (2016). Advances and challenges in studying hepatitis B virus in vitro. Viruses 8(1).
      15. Wu, Y., Zhang, T. Y., Fang, L. L., Chen, Z. X., Song, L. W., Cao, J. L., Yang, L., Yuan, Q. and Xia, N. S. (2016). Sleeping Beauty transposon-based system for rapid generation of HBV-replicating stable cell lines. J Virol Methods 234: 96-100.

简介

稳定的HBV转染细胞系基于将复制能力的HBV基因组稳定整合到肝细胞中,广泛用于基础研究和针对HBV的抗病毒药物评估。然而,先前报道的产生HBV复制细胞系的策略(其主要依赖于通过质粒转染的外源DNA的随机整合)是低效且耗时的。我们新开发了一体化睡眠美容转座子系统(表示为pTSMP-HBV载体),用于稳定产生不同基因型的稳定HBV复制细胞系。 pTSMP-HBV载体含有HBV1.3拷贝基因组和双重选择标记(mCherry和嘌呤霉素抗性基因),允许通过红色荧光激活细胞分选和嘌呤霉素抗生素选择快速富集稳定转染的细胞。在该方案中,我们描述了构建HBV复制稳定细胞和系统评估这些细胞的HBV复制和病毒蛋白表达谱的详细程序。

【背景】慢性乙型肝炎病毒(HBV)感染目前是一个主要的公共卫生负担,影响全球超过2.4亿人(Witt-Kehati et al。,2016)。慢性HBV患者患慢性活动性肝炎,肝硬化或原发性肝细胞癌(HCC)的风险升高(Schweitzer et al。,2015)。目前用干扰素-α或核苷类似物治疗并不能根除病毒,它们对清除乙型肝炎表面抗原(HBsAg)的作用有限(Lucifora和Protzer,2016; Soriano et al。,2017) 。因此,迫切需要开发新的抗病毒抑制剂(Nassal,2015)。

用于评估新药抗HBV活性的细胞培养模型是新药开发的重要工具。稳定的HBV复制细胞系,携带复制能力的HBV基因组稳定整合到人肝癌细胞系(Huh7和/或HepG2)的基因组中,被广泛用于评估抗病毒药物的作用(Witt-Kehati 等人。,2016)。产生稳定HBV的人肝癌细胞系(HepG2.2.15和HepaAD38)整合了D-基因型HBV基因组,广泛用于抗病毒研究(Chang et al。,1987; Ladner 等人,,1997)。然而,基因型A,B和C的稳定的HBV生成细胞系不常用于研究领域。因此,需要开发用于药物开发的HBV基因型A-C的细胞系。

源自硬骨鱼类序列的睡美人(SB)转座子系统在向脊椎动物基因组(包括人类)提供DNA方面非常有效(SB的结构可见于图1A)。睡美人换位是一种剪切和粘贴过程,在此过程中元素从一个DNA分子“跳跃”到另一个DNA分子(图1B)(Ivics和Izsvak,2011)。自1997年从鲑科鱼类基因组重建(Ivics et al。,1997)以来,SB系统经过多次修改以提高其功效(Geurts et al。 ,2003; Baus et al。,2005; Score et al。,2006)。与第一代转座酶(Mátés et al。,2009)相比,高活性转座酶SB100X的开发效率提高了约100倍,有望促进功能基因组学和基因的广泛应用。治疗(Izsvak和Ivics,2004)。


图1. 睡美人转座元素及其转置。 A. 睡美人(SB)系统。转座酶基因(黄色矩形)的侧翼是末端反向重复序列(IR / DR,蓝色箭头),每个重复序列含有转座酶的两个结合位点(小绿色箭头)。转座酶由N-末端DNA结合结构域(PAI和RED),核定位信号(NLS),C-末端和催化结构域(DDE)组成。 B.换位。元件内的转座酶基因可以被治疗基因取代,并且所得的转座子可以保持在简单的质粒载体中。转座酶以反式提供。转座酶与IR / DR重复中的结合位点结合,并与宿主因子如HMGB1一起形成突触复合物,其中转座子的末端配对。转座子从供体分子中切除并整合到新的位置。

关键字:HBV, HBV复制细胞系, 睡美人转座子系统, HBV 1.3拷贝基因组, HepG2

材料和试剂

  1. 移液器吸头,10μl(海门塑料,20111088)
  2. 移液器吸头,200μl(Corning,Axygen ®,目录号:T-200-Y-R)
  3. 移液器吸头,1毫升(海门塑料,20111011)
  4. 细胞培养板(100mm)(Corning,目录号:430167)
  5. 细胞培养板(60mm)(Thermo Fisher Scientific,目录号:150288)
  6. 15毫升管(Thermo Fisher Scientific,目录号:339651)
  7. 70μm细胞过滤器(Fisher Scientific,Fisherbrand,目录号:22-363-548)
  8. 细胞培养板(6孔)(Corning,目录号:3516)
  9. 细胞培养板(24孔)(Corning,目录号:3524)
  10. 细胞成像板,24孔,玻璃底(Eppendorf,目录号:0030741021)
  11. 尼龙膜(罗氏诊断,目录号:11417240001)
  12. 人肝癌HepG2细胞(原来自中国武汉中国典型培养物保藏中心)
  13. 最低必需培养基(MEM,粉末)(Thermo Fisher Scientific,Gibco TM ,目录号:41500-083)
  14. 胎牛血清,Qualified,Australia Origin(赛默飞世尔科技,目录号:10099141)
  15. X-tremeGENE HP DNA Transfection Reagent(罗氏诊断,目录号:06366236001)
  16. Opti-MEM(Thermo Fisher Scientific,Gibco TM ,目录号:31985070)
  17. 0.25%胰蛋白酶-EDTA(1x),酚红(Thermo Fisher Scientific,Gibco TM ,目录号:25200-114)
  18. 嘌呤霉素(Thermo Fisher Scientific,Invitrogen TM ,目录号:A1113803)
  19. 小鼠抗-HBcAg(Innodx Biotechnology,目录号:2A7-21 [它是新的抗HBc单克隆抗体,可根据要求提供])
  20. Alexa Fluor ® 488驴抗小鼠IgG(H + L)(赛默飞世尔科技,目录号:A-21202)
  21. DAPI(Thermo Fisher Scientific,Invitrogen TM ,目录号:D1306)
  22. Micrococcal nuclease(Takara Bio,目录号:D2910)
  23. 蛋白酶K(Takara Bio,目录号:D9033)
  24. 乙醇,C 2 H 6 O,AR(Xilong Scientific,目录号:1030029AR)
  25. 寡核苷酸

  26. dNTP Mixture2.5μM(Takara Bio,目录号:D4030A)
  27. DIG Easy Hyb颗粒(罗氏诊断,目录号:11796895001)
  28. PrimeSTAR GXL DNA聚合酶(Takara Bio,目录号:DR050A)
  29. Premix Ex Taq TM (探针qPCR)(Takara Bio,目录号:RR390A)
  30. 酪蛋白10x封闭缓冲液(Sigma-Aldrich,目录号:B6429-500ML)
  31. 抗DIG(AP)抗体(Roche Diagnostics,目录号:11093274910)
  32. CDP-Star AP底物(Roche Diagnostics,目录号:12041677001)
  33. 通用DNA纯化试剂盒(TIANGEN Biotech,目录号:DP214-03)
  34. 乙型肝炎表面抗原诊断试剂盒(CLEIA)(万泰生物制药,目录号:HBV-1396)
  35. 乙型肝炎e抗原诊断试剂盒(ELISA)(万泰生物制药,目录号:HBV-0396)
  36. ED-11(Innovax Biotechnology)
  37. 病毒DNA / RNA提取试剂盒(GenMag Biotechnology,目录号:NA007)
  38. 无支原体的新生牛血清(天行生物技术,目录号:11011-8615)
  39. 氯化钠(NaCl,AR)(Xilong Scientific,目录号:1001012AR)
  40. 氯化钾(KCl,细胞培养物)(Sigma-Aldrich,目录号:P5405)
  41. 盐酸(HCl,AR)(Xilong Scientific,目录号:1029013AR)
  42. 氢氧化钠(NaOH,AR)(Xilong Scientific,目录号:1001037AR)
  43. 磷酸氢二钠(Na 2 HPO 4 ·12H 2 O,AR)(Xilong Scientific,目录号:1001067AR)
  44. 磷酸二氢钾(KH 2 PO 4 ,AR)(Xilong Scientific,目录号:1002048AR500)
  45. 碳酸氢钠(NaHCO 3 ,AR)(Xilong Scientific,目录号:44558)
  46. 多聚甲醛(Sigma-Aldrich,目录号:16005-1KG-R)
  47. Triton X-100(AMRESCO,目录号:0694)
  48. 牛白蛋白(低内毒素)(ICPbio International,目录号:ABRE-1KG)
  49. Tris base(SEEBIO BIOTECH,目录号:183995)
  50. 三饱和苯酚(Solarbio,目录号:T0250)
  51. 乙二胺四乙酸(EDTA,AR)(Xilong Scientific)
  52. NONIDET ® P-40替代品(AMRESCO,目录号:M158-500ML)
  53. 氯化钙(CaCl 2 ,AR)(Xilong Scientific,目录号:U1000566-500g)
  54. 十二烷基硫酸钠(SDS)(Merck,目录号:428015)
  55. 氯仿(AR)(Xilong Scientific,目录号:1039013AR500)
  56. 异戊醇(AR)(西龙科学,目录号:U1001975-500ml)
  57. 马来酸(Sigma-Aldrich,目录号:M0375)
  58. Tween-20(BBI Solutions,目录号:TB0560-500ml)
  59. 乙二胺四乙酸二钠盐二水合物(EDTA-Na 2 ·2H 2 O,AR)(Xilong Scientific,目录号:100186)
  60. 乙酸盐(CH 3 COOH,AR)(Xilong Scientific,目录号:1029047AR)
  61. 柠檬酸钠(C 6 H 5 Na 3 O 7 ,AR)(Xilong Scientific,目录号:1001059AR )
  62. Verson缓冲区(见食谱)
  63. PBS缓冲液(见食谱)
  64. 4%多聚甲醛(见食谱)
  65. 0.2%Triton X-100(见食谱)
  66. 3%BSA(见食谱)
  67. NET缓冲区(见食谱)
  68. 1.2 M CaCl 2 (见食谱)
  69. 0.5 M EDTA(见食谱)
  70. 10%SDS(见食谱)
  71. 苯酚/氯仿/异戊醇(25:24:1)(见食谱)
  72. 马来酸缓冲液(见食谱)
  73. 洗涤缓冲液(见食谱)
  74. 检测缓冲液(见食谱)
  75. 50x TAE缓冲液(见食谱)
  76. 脱脓缓冲液(见食谱)
  77. 变性缓冲液(见食谱)
  78. 中和缓冲液(见食谱)
  79. 10x SSC(见食谱)
  80. 2x SSC / 0.1%SDS(见食谱)
  81. 0.5x SSC / 0.1%SDS(见食谱)

设备

  1. 移液器(Mettler-Toledo International,RAININ,型号:Pipet-Lite)
  2. CO 2 培养箱(Thermo Fisher Scientific,目录号:3111)
  3. 离心机(Thermo Fisher Scientific,型号:Heraeus TM Pico TM 17)
  4. Sorvall制冷离心机(赛默飞世尔科技,型号:Sorvall TM ST 16R)
  5. BD FACS Aria III(BD,型号:FACSAria TM III)
  6. 高内容筛选系统(PerkinElmer,型号:Opera Phenix TM )
  7. 水浴(Grant Instruments,型号:GD100)
  8. UV交联仪(上海西格玛高科技,型号:SH4)
  9. Bio-Rad真空吸墨纸(Bio-Rad Laboratories,型号:Model 785)
  10. 多功能分子杂交烤箱(UVP,型号:HM-4000)
  11. ImageQuant LAS4000 mini(GE Healthcare,型号:ImageQuant LAS 4000 mini)
  12. 塑料薄膜封口机(上海明威,型号:F-400)
  13. Biomek NX P (Beckman Coulter,型号:Biomek NX P )
  14. 微孔板阅读器(Autobio,型号:PHOmo)
  15. 猎户座II微孔板光度计(Titertek-Berthold,型号:猎户座II)
  16. 电泳仪(Bio-Rad Laboratories,目录号:1645050)
  17. LightCycler ® 96(Roche Molecular Systems,型号:LightCycler ® 96)

程序

  1. 产生HBV复制的稳定细胞系
    1. 转染HepG2细胞
      1. 在37℃,CO 2 培养箱中在MEM / 10%FBS中培养HepG2细胞。在转染前24小时将细胞分裂成10-cm平板; DNA转染时细胞密度应达到70-90%。
      2. 分别用2ml opti-MEM无血清培养基稀释20μg基因型Ae(AY707087),Ba(GU357842),Ce(GU357845)和D1(GU357846)的pTSMP-HBV1.3质粒。轻轻混合。
        注意:本研究中使用的pTSMP-HBV1.3质粒图谱和HBV1.3基因组结构分别如图2A和2B所示。可以参考pTSMP-HBV1.3质粒的构建(Wu等,2016)。


      图2. pTsmP载体(A)和HBV 1.3基因组构建(B)的示意图

      1. 将X-tremeGENE HP DNA转染试剂(60μl)直接吸移到含有稀释DNA的培养基中,不要与塑料管壁接触。轻轻混合。
      2. 将转染试剂:DNA复合物在室温下孵育15分钟。
      3. 以逐滴的方式将转染复合物添加到细胞中。
      4. 转染后,将细胞在37℃,5%CO 2 培养箱中孵育48小时;具有活化红色荧光(mCherry阳性)的成功转染细胞可通过荧光显微镜观察并通过流式细胞术分选。
    2. 流式细胞术分选成功转染的细胞
      1. 用5 ml Verson缓冲液清洗细胞(参见食谱)并弃去上清液。
      2. 向细胞中加入500μl0.25%胰蛋白酶-EDTA,在37℃下孵育3分钟。
      3. 加入5 ml MEM / 10%FBS以阻止消化。
      4. 将2×10 7 细胞转移到15ml管中。
      5. 离心细胞1,000 x g 3分钟,然后弃去上清液。
      6. 用5 ml PBS缓冲液清洗(参见食谱)。
      7. 将细胞以1,000 x g 离心3分钟,然后弃去上清液。
      8. 将细胞重悬于7 ml PBS缓冲液中,然后通过70μm细胞过滤器过滤。
      9. 在BD FACS Aria III机器上运行样品,所需的激发光为561nm(图3)。


      图3.第一轮mCherry激活的FACS分选。具有未表达的mCherry的阴性细胞是HepG2(蓝色峰),表达mCherry的阳性细胞是HepG2-pTsmp-1.3HBV-S11(红色峰) 。

      1. 将分选的表达mCherry的细胞在6孔培养板中培养,在嘌呤霉素选择(2μg/ ml)存在下进行扩增。每2天刷新含有嘌呤霉素的培养基。
      2. 第二天,当细胞汇合度为70-80%时,将细胞进行第二轮mCherry荧光激活FACS分选并进一步选择嘌呤霉素。
      3. 在大约4轮mCherry荧光激活FACS分选和嘌呤霉素抗性细胞选择后,超过90%的细胞是嘌呤霉素抗性并且稳定表达mCherry。
      4. 这些细胞进一步繁殖。传代10次后,4种HBV细胞系中几乎100%的细胞持续表达mCherry并在嘌呤霉素存在下生长良好。

  2. HBV复制稳定细胞系的表征
    1. 乙型肝炎病毒核心抗体的免疫荧光
      1. 细胞制备
        在细胞成像板上种子4×10 5个 5 pTSMP-HBV1.3细胞和3.5×10 5个 5 亲本HepG2细胞(用作阴性对照),密度为70 %。将细胞在37°C和5%CO 2 的培养箱中孵育24小时。
      2. 细胞洗涤
        丢弃培养基,用500μlPBS洗一次。
      3. 固定
        沿细胞板一侧每孔加入250μl4%甲醛(见食谱)。让细胞在室温(RT)下在黑暗中固定15分钟。用1 ml 1x PBS冲洗3次,每次3分钟。
      4. 渗透性
        每孔在250μl0.2%Triton X-100(参见配方)中孵育样品5分钟。用1 ml 1x PBS冲洗3次,每次3分钟。
      5. 封锁
        用PBS中的250μl3%BSA(参见食谱)孵育细胞1小时以阻断抗体的潜在非特异性结合,然后丢弃上清液。
      6. 一抗 将细胞与250μl抗HBcAg抗体(在3%BSA中1:1,000稀释)在室温下(或在4℃下过夜)在黑暗中孵育1小时。接下来,取出溶液,用1 ml /孔PBS洗涤细胞3次,每次3分钟。
      7. 二抗
        将细胞与250μl二抗(Alexa Fluor ® 488驴抗小鼠IgG(H + L),1:1,000稀释于3%BSA中)在室温下在黑暗中孵育30分钟。取出溶液,用1 ml /孔PBS洗涤3次,每次3分钟,黑暗。
      8. 反击
        将细胞与250μlDAPI(在3%BSA中1:2,000稀释)在室温下在黑暗中孵育5分钟。弃去溶液,用1 ml /孔PBS洗涤3次,每次3分钟。
      9. 照片
        照片在高内容筛选系统中(图4)。成像仪中使用的设置用于成像(表1)。

        表1.高内容筛选系统中用于成像的设置



      图4. HBV细胞系HBcAg的免疫荧光(共焦模式,40x水镜片)

    2. Southern印迹
      1. 细胞:在6cm培养皿中铺板4×10 6个细胞,具有60-70%汇合。培养细胞2天,然后进行下一个实验。
      2. 提取HBV总DNA
        1. 弃去细胞上清液,用冷PBS洗涤2次。
        2. 在细胞沉淀中加入600μlNET缓冲液(参见食谱),在冰上(或4°C)孵育1小时。
          注意:我们通常在6孔板的一个孔中加入200-300μl;或400-600μl一个6厘米的碟子;或10厘米培养皿中的1.2-1.8毫升。
        3. 将裂解液在13,400 x g 中于4°C离心20分钟,并将上清液(600μl)转移到新的1.5 ml EP管中。
        4. 加入2μl微球菌核酸酶(10 mg / ml)和3μl1.2M CaCl 2 (最终浓度为6 mM)(参见配方),在37°C水浴中孵育30分钟。 br />
        5. 加入30μl0.5M EDTA(终浓度为25 mM)(参见配方),在65°C水浴中孵育15分钟。
        6. 加入6.68μl蛋白酶K(19 mg / ml)和32.1μl10%SDS(参见食谱),在50°C水浴中孵育过夜(至少12 h但不超过18 h)(终浓度为200μg/ μl蛋白酶K和0.5%SDS)。
        7. 加入等体积的苯酚/氯仿/异戊醇(约600μl),通过倒置管多次彻底混合,并在室温(15-25℃)下孵育5分钟。在14,000 x g 离心10分钟,并将水相转移到新的1.5 ml EP管中。
        8. 重复上述步骤(步骤B2b)vii两次。
        9. 通过加入两倍体积的无水乙醇和1/10体积的乙酸钠沉淀DNA,然后在-80℃下放置2小时。在4℃下以14,000 x g 离心10分钟。小心滗出上清液。
        10. 用500μl75%乙醇洗涤DNA沉淀,并在14,000 x g 离心5分钟。小心滗出上清液。
        11. 风干颗粒5-10分钟,并将DNA重新溶解在35μl蒸馏去离子水中。将HBV DNA保存在-80°C直至使用。
      3. HBV探针的构建
        1. 根据以下组分制备PCR反应溶液:
          5x PrimeSTAR GXL缓冲区
          5μl
          HBx-probe-F(10μM)
          1μl
          HBx-probe-R(10μM)
          1μl
          dNTP Mix(2.5μM)
          2μl
          DIG-dUTP
          2μl
          pGEM-1.3 HBV
          1μl
          PrimeSTAR GXL DNA聚合酶
          1μl
          ddH 2 O
          37μl
        2. PCR反应

        3. 使用Universal DNA Purification Kit纯化HBV探针。
        4. 评估探针的效果
          1. 将探针稀释至2 ng /μl作为第一梯度,10倍稀释至总共5个梯度,最小浓度为0.2 pg /μl。
          2. 通过移液管将稀释的探针放在一片尼龙膜上,每个5μl(最终负载:10,000 pg,1,000 pg,100 pg,10 pg,1 pg)。在每个点之间保持一定的距离,以避免污染。
          3. 紫外线照射(1.5 J / cm 2 )3分钟。
          4. 用1x封闭缓冲液封闭30分钟(在马来酸缓冲液中稀释的酪蛋白封闭缓冲液[10x] [见食谱])。
          5. 用抗DIG(AP)抗体(在1x封闭缓冲液中1:3,000稀释)孵育40分钟。
          6. 用洗涤缓冲液(参见食谱)洗涤尼龙膜3次,每次10分钟。
          7. 用检测缓冲液(参见配方)冲洗2分钟。
          8. 将AP底物添加到膜上以完全渗透它。
          9. 使用ImageQuant LAS4000 mini进行曝光。需要实验探针的强度才能使2 pg /μl或以下的点可见,以产生令人满意的杂交结果。如果探测信号弱于此,则很难检测来自低丰度物种的信号(图5)。


        5. 图5.探针效应的Southern印迹分析

          1. Southern印迹分析
            1. 将DNA样品通过1.2%琼脂糖凝胶在80 V电泳下在1x TAE缓冲液中分离2小时(参见食谱)。
            2. 电泳完成后,如下处理凝胶(以下步骤在振荡器上操作):
              1)
              将凝胶转移到托盘中并加入脱嘌呤缓冲液(参见食谱)以浸泡凝胶,在振荡器上在室温下孵育15分钟并轻轻摇动(将DNA分解成更小的片段,从而允许更有效地从凝胶转移到膜上) 。
              2)
              去除脱嘌呤缓冲液。用ddH 2 O冲洗凝胶两次,每次1分钟。
              3)
              用变性缓冲液(参见食谱)冲洗两次,每次15分钟。
              4)
              用ddH 2 O冲洗凝胶两次,每次1分钟。
              5)
              用中和缓冲液(参见配方)冲洗10分钟。
            3. 用真空吸墨纸将DNA从凝胶转移到尼龙膜上,用10x SSC处理1.5小时(图6)。
              注意:通过将膜以45度角缓慢降低至水中,在双蒸水中润湿预切尼龙膜。然后,用10x SSC润湿膜和滤纸;按图6所示的顺序放置凝胶和尼龙膜;去除凝胶和膜之间的气泡;启动真空源并慢慢地顺时针旋转放气阀,直到仪表读数为5英寸汞柱。


          图6.用于Southern印迹的毛细管转移方法

            1. 凝胶转移后,通过1.5 J / cm 2 的UV交联将DNA固定在尼龙膜上3分钟。
              注:照射时间=照射剂量(mJ / cm 2 )/照射强度(mW / cm 2 ),该仪器的照射强度为9.0 mW / cm 2 。
            2. 预杂交步骤
              将膜置于含有10ml DIG Easy Hyb缓冲液的杂交塑料薄膜中,在42℃杂交室中温育2小时,温和搅拌。
            3. 杂交
              1)
              将DNA探针在95°C下变性5分钟,然后立即置于冰上2分钟。
              2)
              用300 ng DIG-dUTP标记的HBV探针替换预热的10 ml DIG Easy Hyb缓冲液,在42°C杂交室中温和搅拌过夜。
            4. 1)
              在室温下用2x SSC / 0.1%SDS缓冲液洗涤2次,每次5分钟。
              2)
              用预热的0.5x SSC / 0.1%SDS缓冲液在65°C下洗涤2次。
              注意:使用前摇动缓冲区。
            5. 发现
              1)
              用马来酸缓冲液(见食谱)冲洗2分钟。
              2)
              用1x封闭缓冲液(用马来酸缓冲液稀释10倍封闭缓冲液)封闭30分钟。
              3)
              用抗DIG(AP)抗体(在1x封闭缓冲液中1:3,000稀释)孵育40分钟
              4)
              用100ml洗涤缓冲液洗涤3次,每次15分钟。
              注意:洗涤缓冲液在使用前均匀摇匀。
              5)
              在20 ml检测缓冲液中平衡3 min。
              6)
              逐滴添加带有AP底物的膜,以便完全渗透。
              7)
              连续曝光膜并使用ImageQuant LAS4000 mini拍摄图像。
        6. 检测上清液中病毒标志物的水平
          1. 细胞
            1. 在24孔板中种子3.5×10 5 pTSMP-HBV1.3细胞,密度为60-70%。
            2. 每2天补充并收集用于测量病毒抗原和HBV DNA的培养基。
            3. 在1个月内监测每种细胞系的上清液中这些病毒标记物的水平。
          2. 病毒抗原
            1. 使用商业化验试剂盒(万泰,北京,中国)通过化学发光法测定培养基中的病毒抗原(HBsAg和HBeAg)。
            2. 抗原(HBsAg和HBeAg)标准品是标准化患者血清,原始浓度分别为90,000 IU / ml和10,000 Ncu / ml。
            3. HBeAg标准品用20%NBS稀释成六种梯度浓度,分别为10,5,2.5,1.25,0.25,0.05 Ncu / ml。
            4. 将HBsAg的标准品用ED-11稀释成六种梯度浓度,分别为45,9,1.8,0.36,0.072,0.0144IU / ml。
          3. HBV总DNA
            1. HBV总DNA提取
              1)
              在Biomek NX P (Beckman,California,USA)上使用病毒DNA / RNA提取试剂盒提取培养基中的总DNA。
              2)
              HBV总DNA标准品是标准化患者血清,原始浓度为1.5×10×sup <8> IU / ml,用PBS稀释10倍稀释至6个梯度。
            2. 根据以下组分制备PCR反应溶液。
              2x Premix Ex Taq(探针qPCR)
              10μl
              KHF2(100μM)
              0.1μl
              KHR1(100μM)
              0.1μl
              KHP4(100μM)
              0.05μl
              DEPC水
              4.75μl
              模板
              5μl
            3. 用LightCycler 96进行实时PCR反应。
            4. 笔记

              1. 细胞状态对分选后的转染效率和细胞状态至关重要。
              2. 在分选过程中,细胞应在4°C下分选,流速应控制在9μl/ min以下。这对于分选后的细胞状态至关重要。
              3. 分选后的细胞数量应至少足以在48孔板中铺板,以便细胞更有可能存活。

              食谱

              1. Verson缓冲液(1升)
                0.4克KCl
                0.06克KH 2 PO 4
                8.0克NaCl
                0.35g NaHCO 3
                0.08 g Na 2 HPO 4 ·12H 2 O
                0.2克EDTA
                过滤灭菌(0.22μm)或通过高压灭菌消毒
              2. PBS缓冲区
                145 mmol / L NaCl
                2.68 mmol / L KCl
                10 mmol / L Na 2 HPO 4 ·12H 2 O
                1.76mmol / L KH 2 PO 4
                调节至pH 7.4
                过滤灭菌(0.22μm)或通过高压灭菌消毒&nbsp;
              3. 4%多聚甲醛
                4克多聚甲醛
                100毫升PBS
                1-2滴10 M NaOH
                储存在4°C并避光保存
              4. 0.2%Triton X-100
                200μlTritonX-100
                100毫升PBS
                储存在4°C
              5. 3%BSA
                3克BSA
                100毫升PBS
                分包并存放在-20°C
              6. NET缓冲区
                50mM Tris碱(pH = 8.0)
                1 mM EDTA
                100 mM NaCl
                0.5%NP-40
              7. 1.2 M CaCl 2
                13.32克CaCl 2
                100毫升超纯水
                储存在4°C
              8. 0.5 M EDTA
                储存在4°C
              9. 10%SDS
                100克电泳级SDS
                将ddH 2 O添加到1 L
                加热至68°C
                调节至pH 7.2
              10. 苯酚/氯仿/异戊醇(25:24:1)
                以25:24:1的比例平衡苯酚,氯仿和异戊醇,然后储存在棕色玻璃瓶中,盖上一层Tris-Cl,储存在4°C直至使用
              11. 马来酸缓冲液
                0.1 M马来酸
                0.15 M NaCl
                调节至pH 7.5
                通过高压灭菌消毒
              12. 洗涤缓冲区
                0.1 M马来酸
                0.15 M NaCl
                3毫升/升吐温20
                调节至pH 7.5
                通过高压灭菌消毒
              13. 检测缓冲区
                0.1 M Tris-base
                0.1 M NaCl
                调节至pH 9.5
                通过高压灭菌消毒&nbsp;
              14. 50x TAE缓冲区
                242克Tris-base
                100ml EDTA(0.5M,pH8.0)
                57.1毫升醋酸盐
                将ddH 2 O添加到1 L
                调节至pH 8.5
              15. 去除缓冲剂
                0.2 N HCl
                通过高压灭菌消毒
              16. 变性缓冲液
                0.5 M NaOH
                1.5 M NaCl
                通过高压灭菌消毒
                中和缓冲区
                1 M Tris-base
                1.5 M NaCl
                通过高压灭菌消毒
              17. 10x SSC
                1.5 M NaCl
                0.15 M柠檬酸钠
                调节至pH 7.0
                通过高压灭菌消毒
              18. 2x SSC / 0.1%SDS
                200毫升10倍SSC
                10毫升10%SDS
                加入超纯水至1升
                通过高压灭菌消毒
              19. 0.5x SSC / 0.1%SDS
                50毫升10倍SSC
                10毫升10%SDS
                加入超纯水至1升
                通过高压灭菌消毒

              致谢

              我们要感谢本协议所依据的以下出版物:Wu et al。(2016)。作者声明没有竞争性的经济利益。

              参考

              1. Baus,J.,Liu,L.,Heggestad,A。D.,Sanz,S。和Fletcher,B。S.(2005)。 睡美人转座子的过度转座酶突变体。 Mol Ther 12(6):1148-1156。
              2. Chang,CM,Jeng,KS,Hu,CP,Lo,SJ,Su,TS,Ting,LP,Chou,CK,Han,SH,Pfaff,E.,Salfeld,J。和 et al。(1987)。通过瞬时表达克隆的HBV DNA 体外产生乙型肝炎病毒 在肝癌细胞系中。 EMBO J 6(3):675-680。
              3. Geurts,A.M.,Yang,Y.,Clark,K.J.,Liu,G.,Cui,Z.,Dupuy,A.J。,Bell,J.B.,Largaespada,D.A。和Hackett,P.B。(2003)。 睡眠美容转座子系统将基因转移到人体细胞的基因组中。 Mol Ther 8(1):108-117。
              4. Ivics,Z。和Izsvak,Z。(2011)。 使用Sleeping Beauty转座子系统进行非病毒基因传递。 Hum Gene Ther < / em> 22(9):1043-1051。
              5. Ivics,Z.,Hackett,P.B.,Plasterk,R.H。和Izsvak,Z。(1997)。 睡美人的分子重建,来自鱼类的 Tc1 样转座子,及其在人体细胞中的转座。 细胞 91(4):501-510。
              6. Izsvak,Z。和Ivics,Z。(2004)。 睡美人换位:生物学和应用分子疗法。 Mol Ther 9(2):147-156。
              7. Ladner,S.K.,Otto,M.J.,Barker,C.S.,Zaifert,K.,Wang,G.H.,Guo,J.T。,Seeger,C.and King,R.W。(1997)。 人类乙型肝炎病毒(HBV)在稳定转染的肝母细胞瘤细胞中的诱导表达:一种新的筛查系统HBV复制的潜在抑制剂。 Antimicrob Agents Chemother 41(8):1715-1720。&nbsp;
              8. Lucifora,J。和Protzer,U。(2016)。 攻击乙型肝炎病毒cccDNA - 乙型肝炎治愈的圣杯。 J Hepatol 64(1 Suppl):S41-S48。
              9. Mátés,L.,Chuah,MK,Belay,E.,Jerchow,B.,Manoj,N.,Acosta-Sanchez,A.,Grzela,DP,Schmitt,A.,Becker,K.,Matrai,J., Ma,L.,Samara-Kuko,E.,Gysemans,C.,Pryputniewicz,D.,Miskey,C.,Fletcher,B.,VandenDriessche,T.,Ivics,Z。和Izsvak,Z。(2009)。 新型多动睡眠美容转座酶的分子进化能够在脊椎动物中实现稳定的稳定基因转移。 Nat Genet 41(6):753-761。
              10. Nassal,M。(2015)。 HBV cccDNA:病毒持续存储库和治疗慢性乙型肝炎的主要障碍 Gut 64(12):1972-1984。
              11. Schweitzer,A.,Horn,J.,Mikolajczyk,R.T.,Krause,G。和Ott,J.J。(2015)。 估计全球慢性乙型肝炎病毒感染率:对1965年至2013年间公布的数据进行系统评价。 Lancet 386(10003):1546-1555。
              12. Score,P.R.,Belur,L.R.,Frandsen,J.L.,Geurts,J.L.,Yamaguchi,T.,Somia,N.V.,Hackett,P.B.,Largaespada,D.A。和McIvor,R.S。(2006)。 睡美人介导的转座和长期表达体内:使用LoxP / Cre重组酶系统用于区分转座特异性表达。 Mol Ther 13(3):617-624。
              13. Soriano,V.,Barreiro,P.,Benitez,L.,Pena,J。M.和de Mendoza,C。(2017)。 用于治疗慢性乙型肝炎的新型抗病毒药物。 Expert Opin Investig Drugs 26(7):843-851。
              14. Witt-Kehati,D.,Bitton Alaluf,M。和Shlomai,A。(2016)。 体外研究乙型肝炎病毒的进展和挑战。 病毒 8(1)。
              15. Wu,Y.,Zhang,T.Y.,Fang,L.L.,Chen,Z.X.,Song,L.W。,Cao,J.L.,Yang,L.,Yuan,Q。和Xia,N.S。(2016)。 睡美人基于转座子的系统,用于快速生成HBV复制稳定细胞系。 J Virol Methods 234:96-100。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zheng, J., Cao, J. and Yuan, Q. (2018). Sleeping Beauty Transposon-based System for Rapid Generation of HBV-replicating Stable Cell Lines. Bio-protocol 8(13): e2908. DOI: 10.21769/BioProtoc.2908.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。