搜索

Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting
通过Hirt提取法与DNA印迹实验分析腺相关病毒的复制中间体   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).

Keywords: Adeno-associated virus(腺相关病毒), AAV(AAV), DNA replication(DNA复制), Replicative intermediates(复制中间体), Southern blot(DNA印迹)

Background

AAV DNA replication is carried out by a rolling hairpin mechanism in cells co-infected by AAV and helper viruses such as adenovirus or herpes virus (Ward, 2006). The AAV DNA consists of a 4.7 kb linear DNA molecule with inverted terminal repeats (ITRs) that fold back to form T-shaped hairpin structures. The 3’ end hairpin serves as a primer for the replication of the AAV DNA. These hairpin structures are regenerated by the AAV Rep protein, allowing further rounds of replication (Im and Muzyczka, 1990). Both + and - strands of the AAV DNA are packaged and are infectious (Rose et al., 1969). When replicating AAV DNA is analyzed, several replicative intermediates can be detected (Straus et al., 1976). The most abundant replicative intermediate is a linear monomeric duplex molecule, formed by one + and one - strand of the AAV DNA, which is thought to be the immediate precursor of progeny single-stranded molecules that will be packaged in pre-formed capsids (Straus et al., 1976). Dimeric replicative intermediates are also common, and the AAV replication model is compatible with even larger replicative intermediates. The study of AAV replication benefitted from the discovery that AAV plasmids are infectious–the AAV DNA can be fully rescued from a plasmid (in the presence of helper virus) and its replication mimics that of the native virus (Samulski et al., 1982). The method detailed here allows the investigation of the DNA intermediates formed during DNA replication initiated from an AAV plasmid, and was used to compare different mutants of the AAV Rep protein for their ability to support AAV replication. The same method can be used to study other aspects of the AAV life cycle that can affect DNA replication of this virus, such as the effect of helper virus proteins or other factors that restrict/enhance AAV replication.

Materials and Reagents

  1. Transfection of 293T cells and wild type (wt) AAV production
    1. 100 mm dishes (Corning, catalog number: 430293 )
    2. 293T cells (ATCC, catalog number: CRL-11268 )
    3. pAV2 plasmid (Laughlin et al., 1983, available from ATCC, catalog number: 37216 )
    4. Linear polyethylenimine (PEI), MW 25,000, at 1 mg/ml and pH 7.0 (Polysciences, catalog number: 23966-1 )
    5. wt adenovirus serotype 5 (Graham and Prevec, 1991, available from ATCC, catalog number: VR-1516 )
    6. Dulbecco modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, catalog number: 31966021 )
    7. Foetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: 10270106 )
    8. 1x Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190094 )

  2. Extraction of low molecular weight DNA
    1. 15 ml conical tube (Corning, catalog number: 430791 )
    2. 1.5 ml tube
    3. Hirt lysis buffer (see Recipes)
      1. Sodium dodecyl sulfate (SDS) (Thermo Fisher Scientific, AmbionTM, catalog number: AM9822 )
      2. Tris (pH 7.5)
      3. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
    4. Proteinase K solution (20 mg/ml) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 25530049 )
    5. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
    6. Phenol-chloroform-isoamyl alcohol 25:24:1 (Sigma-Aldrich, catalog number: 77617 )
    7. Sodium acetate (CH3COONa) (Sigma-Aldrich, catalog number: 71183 )
    8. 2-propanol (EMD Millipore, catalog number: 109634 )
    9. Ethanol (EMD Millipore, catalog number: 100983 )
    10. ddH2O (Thermo Fisher Scientific, AmbionTM, catalog number: AM9937 )
    11. RNaseA (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EN0531 )

  3. Southern blot assay using Rep and Amp probes
    1. WhatmanTM paper (GE Healthcare, catalog number: 3030-917 )
    2. Amersham Hybond-N+ membrane (GE Healthcare, catalog number: RPN203B )
    3. Hybridisation tubes (Chemglass Life Sciences, catalog number: CG-1140-05 )
    4. Quick Spin Columns for radiolabeled DNA purification (Roche Diagnostics, catalog number: 11273973001 )
    5. DpnI (New England Biolabs, catalog number: R0176S )
    6. UltraPureTM agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500500 )
    7. TAE (Tris-acetate-EDTA) buffer (see Recipes)
      1. Tris (Roche Diagnostics, catalog number: 10708976001 )
      2. Glacial acetic acid (Sigma-Aldrich, catalog number: ARK2183 )
      3. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
    8. 1 kb DNA ladder (New England Biolabs, catalog number: N3232 )
    9. 20x saline sodium citrate (SSC) solution (see Recipes)
      1. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
      2. Sodium citrate (Sigma-Aldrich, catalog number: W302600 )
    10. Denaturing solution (see Recipes)
      1. Sodium chloride (NaCl)
      2. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 71687 )
    11. Neutralising solution (see Recipes)
      1. Tris (pH 7.4)
      2. Sodium chloride (NaCl)
    12. Rep probe primers
      fw: AACTGGACCAATGAGAACTTTCC; rv: AAAAAGTCTTTGACTTCCTGCTT
    13. Amp probe primers
      fw: AATCAGTGAGGCACCTATCTCAGC; rv: AACTCGGTCGCCGCATACACTATT
    14. GoTaq® Colorless Master Mix PCR Kit (Promega, catalog number: M7132 )
    15. QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
    16. dCTPs, [α-32P]- 6,000 Ci/mmol 20 mCi/ml, 250 µCi (PerkinElmer, catalog number: BLU013Z250UC )
    17. Prime-It RmT Random Primer Labeling Kit (Agilent Technologies, catalog number: 300392 )
    18. Nylon Wash solution (see Recipes)
      1. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
      2. Ethylenediaminetetraacetic acid (EDTA)
      3. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )

Equipment

  1. Pipette  
  2. CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraCell 240 )
  3. Hemocytometer
  4. Refrigerated benchtop microcentrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM FrescoTM 17 )
  5. Refrigerated centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM MultifugeTM X3R )
  6. Microbiological safety cabinet (Medical Air Technology, model: BioMAT2 Class II )
  7. NanoDrop spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDrop 1000 or NanoDrop 2000 )
  8. Gel electrophoresis system (Thermo Fisher Scientific, Thermo ScientificTM, model: OwlTM A1 )
  9. Platform shaker (Heidolph Instruments, model: Polymax 1040 )
  10. Glass plate
  11. Glass tray
  12. Ultraviolet crosslinker (UVP, model: CL-1000 )
  13. Hybridisation oven (UVP, model: HB-1000 )
  14. Perspex screen (Thermo Fisher Scientific, Thermo ScientificTM, model: NalgeneTM Acrylic Benchtop Beta Radiation Shield , catalog number: 67002418)
  15. Typhoon Molecular Dynamics phosphor/fluorescence imager (GE Healthcare, model: Trio Variable Mode Imager )
  16. PCR thermal cycler (Eppendorf, model: Mastercycler® )
  17. Heat block (Cole-Parmer, Stuart, catalog number: SBH200DC )
  18. Storage Phosphor screen (GE Healthcare, catalog number: 28-9564-75 )
  19. Exposure cassette (GE Healthcare, catalog number: 63-0035-44 )

Software

  1. ImageQuant analysis software (GE Healthcare)

Procedure

  1. Transfection of 293T cells and AAV production
    1. Seed 4.3 x 106 293T cells in a 10 cm dish in 10 ml of growth medium (DMEM + 10% FBS) for transfection next day. Include an additional plate to determine cell number at the time of infection with adenovirus. Cells are maintained for 24 h at 37 °C and 5% CO2.
    2. Dilute 17.5 µg of pAV2 plasmid in 500 µl of DMEM (no serum added, SF DMEM).
    3. Dilute 94 µl of PEI in 500 µl of SF DMEM and gently mix. The transfection reagents should be at room temperature (RT) before mixing. Incubate 5 min at RT.
    4. Combine the PEI and DNA and gently mix by pipetting. Incubate at RT for 20 min.
    5. Add the mixture to the medium in which the cells are growing. Pipet carefully to avoid detaching the cells.
    6. 4 h after transfection, determine the cell number (for example using a hemocytometer) in the extra plate.
    7. Infect the cells using 5 pfu/cell of adenovirus. Include a condition without adenovirus as a negative control for AAV replication.
    8. 40 to 50 h post transfection a cytopathogenic effect is expected. Cells will round up and eventually detach (Figure 1).
      Note: The amount of adenovirus necessary to fully support AAV replication and to cause a cytopathogenic effect at 40 to 50 h post transfection can vary depending on the health of the cell line used and its passage number. This may need to be optimized before starting this protocol: infect cells with increasing amounts of adenovirus and monitor the appearance of a cytopathogenic effect (Figure 1).


      Figure 1. AAV and adenovirus induced cytopathogenic effect. Left panel: 24 h post-transfection cells are adherent and form a monolayer. Right panel: 55 h post-transfection the cytopathogenic is evident; cells have rounded up and are lifting from the plate. Scale bars = 50 µm.

  2. Extraction of low molecular weight
    1. Collect cells and supernatant in a 15 ml conical tube. Cells should be loosely adherent and can be detached by pipetting. Wash the plate with 2 ml 1x DPBS and add to the 15 ml tube.
    2. Centrifuge for 5 min at 500 x g at RT.
    3. Discard the supernatant, and resuspend the cell pellet in 1 ml DPBS. Transfer 250 µl to a new 1.5 ml tube. The remainder of the cells can be harvested for additional analyses, e.g., gene and protein expression.
    4. Centrifuge for 5 min at 500 x g at RT in a benchtop centrifuge and discard supernatant.
    5. Add 500 µl of Hirt lysis buffer and mix by flicking or inverting the tube.
      Note: The lysate will be very viscous and mixing by pipetting will create bubbles.
    6. Incubate for 10 min at RT.
    7. Add 1.25 µl of proteinase K (50 µg/ml final concentration) and incubate for 1 h at 37 °C.
    8. Add 120 µl of 5 M NaCl (1 M final concentration). Mix gently by flicking/inverting the tube.
    9. Incubate overnight at 4 °C. This step precipitates the high molecular weight (chromosomal) DNA.
    10. Centrifuge for 1 h at maximum speed (17,000 x g) at 4 °C in a refrigerated table-top centrifuge.
    11. Carefully transfer the supernatant (approximately 500 µl) to a new 1.5 ml tube.
      Note: If the supernatant cannot be separated sufficiently from the viscous pellet, a longer centrifugation may be required.
    12. Under a fume hood, add 1 volume (500 µl) of phenol/chloroform/isoamyl alcohol and mix by inverting the tube until homogeneous.
    13. Centrifuge for 10 min at 17,000 x g at 4 °C.
    14. Carefully transfer the upper phase to a new 1.5 ml tube (approximately 450 µl).
    15. Add 50 µl 3 M sodium acetate (0.3 M final concentration) and mix.
    16. Incubate overnight at -20 °C.
    17. Centrifuge for 20 min at 17,000 x g at 4 °C.
    18. Carefully remove and discard the supernatant, then wash the DNA pellet with 100 µl of 70% ethanol.
    19. Centrifuge for 10 min at 17,000 x g at 4 °C.
    20. Remove supernatant and air-dry the DNA pellet.
    21. Resuspend the pellet in 80 µl ddH2O.
    22. Determine the concentration of DNA in the samples using a NanoDrop spectrophotometer. Typical DNA concentration is around 750 ng/µl.

  3. Southern blot assay (gel transfer)
    1. Digest approximately 1 µg of the extracted DNA with DpnI, for 2 h at 37 °C. DpnI specifically digests methylated DNA, e.g., DNA amplified in dam+ bacteria strains. This step ensures that the input plasmid DNA is digested while newly replicated AAV DNA will not be affected.
    2. Separate the digested DNA on a 0.8% agarose gel overnight at 1 V/cm.
    3. Remove the gel from the electrophoresis tank and incubate for 30 min in denaturing solution on a platform shaker at approximately 25 rpm.
    4. Rinse the gel twice in ddH2O.
    5. Incubate the gel for 30 min in neutralizing solution on a platform shaker at approximately 25 rpm.
    6. Replace the neutralizing solution with fresh neutralizing solution and incubate for a further 30 min while shaking.
    7. Rinse in ddH2O, and then proceed with the assembly of the transfer set-up as detailed in Figure 2.


      Figure 2. Scheme of capillary transfer method for Southern blotting. The support plate is a glass plate resting on the sides of the glass tray containing the 20x SSC. The paper towels layer is approximately 15 cm high. The weight used was approximately 600 g.

    8. Incubate overnight to transfer the DNA in the gel to the nylon membrane by the capillary method (Figure 2). To obtain the same sample orientation on the nylon membrane as on the gel, turn the gel upside down.
    9. Disassemble the set-up and carefully remove the membrane.
    10. Rinse the membrane in 2x SSC, and let it air-dry.
    11. To fix the DNA to the membrane, UV cross-link the membrane with 120,000 µJ/cm2.
    12. Pre-hybridize the membrane in 20 ml 0.75x nylon wash solution for at least 1 h at 65 °C in hybridization tubes.

  4. Preparation of the Rep and Amp probes
    1. The template to prepare the Rep and Amp specific probes is obtained by PCR on the pAV2 plasmid using the aforementioned primer pairs and the PCR conditions listed below. The PCR products are gel-purified and quantified using a NanoDrop spectrophotometer. The expected size of the PCR products is 338 base-pairs for the Rep PCR and 587 for the Amp PCR. 20 to 25 ng of purified PCR product DNA is used as DNA template to prepare the probes.
      For both Rep and Amp PCRs, mix in a final volume of 25 µl:
      GoTaq Colorless Master mix (2x)
      12.5 µl
      Fw Rep or Amp primer (10 µM)
      1 µl
      Rv Rep or Amp primer (10 µM)
      1 µl
      Template DNA
      25 ng
      ddH2O
      to 25 µl

      Cycling conditions Rep PCR:


      Cycling conditions Amp PCR:


    2. Reconstitute the reaction mixture (provided as 0.2 ml tubes containing dehydrated random primers, dNTPs, buffer and cofactors) using ddH2O containing 20-25 ng of probe DNA to a final volume of 44 µl, as suggested in the Prime-It RmT Random Primer Labeling Kit. Heat at 95 °C for 5 min.
    3. Add 1.5 µl of 32P-labelled dCTPs, and 3 µl of Magenta polymerase enzyme (tube with red cap in the labelling kit). Incubate for 8-10 min at 37 °C.
    4. Add 2 µl of stop mix (tube with green cap in the labelling kit).
    5. Separate labelled DNA (the probe) from unincorporated labelled nucleotides using the Quick Spin Columns for radiolabeled DNA purification.
    6. Heat the probe at 95 °C for 5 min to denature the DNA.
    7. Place tube on ice for 5 min then quickly spin to collect the probe at the bottom of the tube.

  5. Southern blot assay (membrane hybridization)
    1. Add the probe to the hybridization tube. Depending on the freshness of the radioactivity, one probe prepared as detailed above is sufficient to label 2-4 membranes.
    2. Incubate overnight at 65 °C in a hybridization oven.
    3. Wash membrane in 0.5x nylon wash solution for 20 min at 65 °C.
    4. Wash membrane in 0.1x nylon wash solution for 20 min at 65 °C.
      Note: If additional washing is required, repeat washing step using 0.01x nylon wash solution.
    5. Expose the membrane to the Phosphoimager screen for at least 2 h.
      Note: The length of exposure necessary to obtain the best signal can vary depending on the strength of the signal.
    6. Acquire images using the Typhoon phosphor imager. Image analysis can be performed using the ImageQuant analysis software (GE Healthcare).

Data analysis

The gel images acquired following the procedure described above were analyzed using the ImageQuant analysis software (GE Healthcare). The level of AAV replication under different conditions is proportional to the intensity of the bands corresponding to viral replicative intermediates in DpnI-treated samples hybridized with the viral (Rep) probe (Figure 3). For wt AAV2 virus, monomeric and dimeric molecules of approximately 4.7 kb and 9.4 kb, respectively should be visible. Additional higher molecular weight intermediates can also be present. Membranes hybridized with the Amp probe allow for the assessment of efficiency of transfection and efficacy of DpnI digestion (Figure 3). The ImageQuant software supports band quantification by densitometry if a quantitative band comparison is required.


Figure 3. Analysis of AAV replication intermediates by Southern blot. Left panel: pAAV-GFP (no Rep control), pAV2 (WT AAV plasmid) and pAV2-RepK340H (replication-deficient Rep mutant) were transfected in the presence (+ Ad) or absence of adenovirus. The Amp probe binds to all plasmids containing the ampicillin resistance gene. The Rep probe only binds to DNA containing the AAV Rep gene. Right panel: samples were treated with DpnI digestion to remove all input plasmid; only AAV DNA that is rescued from the plasmid and replicated is detected. Plasmids pAAV-GFP and pAV2 were transfected in the presence (+ Ad) or absence of adenovirus. Hybridisation with the Amp probe confirms that all input plasmid was digested by DpnI treatment. The Rep probe detects replicated AAV DNA. M: monomeric replicative form; D: dimeric replicative form; larger replicative intermediates are visible above. Adapted from Bardelli et al., 2016.

Notes

  1. Steps D3 to E5 should only be performed in areas designated for work with radioactivity. Please follow local directives.
  2. Note on controls: several controls should be used when running this assay.
    1. + AAV, - adenovirus: no replication control. In the absence of adenovirus, AAV replication is absent or very low.
    2. - Rep, + adenovirus: no replication control. Instead of wt AAV, use a replication-deficient AAV mutant or recombinant AAV vector (for example pAAV-GFP). This will also control for the absence of wt AAV contamination in the adenovirus batch used in the experiment.
    3. The Amp probe is used to assure that input DNA is digested.
    4. No DpnI control: allows the comparison of input plasmid between different conditions, and controls for the binding of the Amp probe.

Recipes

  1. Hirt lysis buffer
    6 ml of 10% SDS
    1 ml of 1 M Tris (pH 7.5) solution
    2 ml of 500 mM EDTA (pH 8.0) solution
    Bring the final volume to 100 ml with ddH2O
  2. TAE (Tris-acetate-EDTA) buffer
    Prepare 50x solution:
    242 g Tris
    57.1 ml glacial acetic acid
    100 ml of 500 mM EDTA (pH 8.0) solution
    Bring the final volume to 1 L ddH2O
    Dilute 1:50 in ddH2O to make 1x TAE buffer
  3. 20x saline sodium citrate (SSC) solution
    175.3 g NaCl
    88.2 g sodium citrate
    Bring the final volume to 1 L ddH2O
  4. Denaturing solution
    300 ml of 5 M NaCl solution
    100 ml of 5 M NaOH solution
    Bring to 1 L with ddH2O
  5. Neutralising solution
    500 ml of 1 M Tris (pH 7.4) solution
    300 ml of 5 M NaCl solution
    Bring to 1 L with ddH2O
  6. Nylon wash solution (pH 7.2)
    40.6 g Na2HPO4
    18.65 g EDTA
    500 g SDS
    Bring the final volume to 3.58 L with ddH2O

Acknowledgments

This work was supported by the Pfizer Rare Diseases Consortium Award (to EH), NIH grant RO1-GM092854 (to CRE) and United Kingdom Medical Research Council grant 1001764 (to RML).

References

  1. Bardelli, M., Zarate-Perez, F., Agundez, L., Linden, R. M., Escalante, C. R. and Henckaerts, E. (2016). Identification of a functionally relevant Adeno-associated virus Rep68 oligomeric interface. J Virol 90(15): 6612-6624.
  2. Graham, F. L. and Prevec, L. (1991). Manipulation of adenovirus vectors. Methods Mol Biol 7: 109-128.
  3. Hirt, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 26(2): 365-369.
  4. Im, D. S. and Muzyczka, N. (1990). The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell 61(3): 447-457.
  5. Laughlin, C. A., Tratschin, J. D., Coon, H. and Carter, B. J. (1983). Cloning of infectious adeno-associated virus genomes in bacterial plasmids. Gene 23(1): 65-73.
  6. Rose, J. A., Berns, K. I., Hoggan, M. D. and Koczot, F. J. (1969). Evidence for a single-stranded adenovirus-associated virus genome: formation of a DNA density hybrid on release of viral DNA. Proc Natl Acad Sci U S A 64(3): 863-869.
  7. Samulski, R. J., Berns, K. I., Tan, M. and Muzyczka, N. (1982). Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells. Proc Natl Acad Sci U S A 79(6): 2077-2081.
  8. Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503-17.
  9. Straus, S. E., Sebring, E. D. and Rose, J. A. (1976). Concatemers of alternating plus and minus strands are intermediates in adenovirus-associated virus DNA synthesis. Proc Natl Acad Sci U S A 73(3): 742-746.
  10. Ward, P. (2006). Replication of adeno-associated virus DNA. In: Kerr, J. R., Cotmore, S. F., Bloom, M. E., Linden, R. M. and Parrish, C. R. (Eds.). Parvoviruses. Hodder Arnold pp: 189-211.

简介

腺相关病毒(AAV)是一种小型单链DNA病毒,需要存在辅助病毒,如腺病毒或疱疹病毒,以有效地复制其基因组。 AAV DNA通过滚转发夹机制(Ward,2006)进行复制,并且在复制期间可以检测出几种DNA中间体。该详细方案描述了如何使用改良的Hirt提取物(Hirt,1967)程序和Southern印迹(Southern,1975)分析在AAV复制期间形成的AAV DNA中间体。

背景 AAV DNA复制通过滚动发夹机制在由AAV和辅助病毒如腺病毒或疱疹病毒共感染的细胞中进行(Ward,2006)。 AAV DNA由4.7kb的线性DNA分子和倒置的末端重复(ITR)组成,折叠形成T形发夹结构。 3'末端发夹作为AAV DNA复制的引物。这些发夹结构由AAV Rep蛋白再生,允许进一步复制(Im和Muzyczka,1990)。 AAV DNA的+和 - 链都被包装并且是感染性的(Rose等人,1969)。当分析复制AAV DNA时,可以检测到几种复制中间体(Straus等人,1976)。最丰富的复制中间体是由AAV DNA的一个和一个链形成的线性单体双链体分子,其被认为是将包装在预先形成的衣壳中的后代单链分子的直接前体(Straus ,1976)。二聚体复制中间体也是常见的,AAV复制模型与甚至更大的复制中间体相容。 AAV复制的研究受益于AAV质粒是感染性的发现 - AAV DNA可以从质粒中完全拯救(在辅助病毒存在下),并且其复制模拟天然病毒的复制(Samulski等,1982)。这里详细描述的方法可以研究从AAV质粒引发的DNA复制过程中形成的DNA中间体,并用于比较AAV Rep蛋白的不同突变体的支持AAV复制能力。可以使用相同的方法来研究AAV生命周期中可能影响该病毒DNA复制的其他方面,例如辅助病毒蛋白质或其他限制/增强AAV复制因子的作用。

关键字:腺相关病毒, AAV, DNA复制, 复制中间体, DNA印迹

材料和试剂

  1. 转染293T细胞和野生型(wt)AAV生产
    1. 100毫米的菜肴(康宁,目录号:430293)
    2. 293T细胞(ATCC,目录号:CRL-11268)
    3. pAV2质粒(Laughlin等人,1983,ATCC获得,目录号:37216)
    4. 线性聚乙烯亚胺(PEI),分子量25,000,1mg/ml和pH7.0(Polysciences,目录号:23966-1)
    5. wt的腺病毒血清型5(Graham和Prevec,1991,可得自ATCC,目录号:VR-1516)
    6. Dulbecco改良Eagle's培养基(DMEM)(Thermo Fisher Scientific,目录号:31966021)
    7. 胎牛血清(FBS)(Thermo Fisher Scientific,目录号:10270106)
    8. 1x Dulbecco的磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190094)

  2. 提取低分子量DNA
    1. 15ml锥形管(Corning,目录号:430791)
    2. 1.5 ml管子
    3. Hirt裂解缓冲液(参见食谱)
      1. 十二烷基硫酸钠(SDS)(Thermo Fisher Scientific,Ambion TM,目录号:AM9822)
      2. Tris(pH 7.5)
      3. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
    4. 蛋白酶K溶液(20mg/ml)(Thermo Fisher Scientific,Invitrogen TM,目录号:25530049)
    5. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
    6. 苯酚 - 氯仿 - 异戊醇25:24:1(Sigma-Aldrich,目录号:77617)
    7. 乙酸钠(CH 3 COONa)(Sigma-Aldrich,目录号:71183)
    8. 2-丙醇(EMD Millipore,目录号:109634)
    9. 乙醇(EMD Millipore,目录号:100983)
    10. ddH 2 O(Thermo Fisher Scientific,Ambion TM ,目录号:AM9937)
    11. RNaseA(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:EN0531)

  3. 使用 Rep Amp 探针的Southern印迹分析
    1. Whatman TM 论文(GE Healthcare,目录号:3030-917)
    2. Amersham Hybond-N +膜(GE Healthcare,目录号:RPN203B)
    3. 杂交管(Chemglass Life Sciences,目录号:CG-1140-05)
    4. 快速旋转柱用于放射性标记的DNA纯化(Roche Diagnostics,目录号:11273973001)
    5. I(New England Biolabs,目录号:R0176S)
    6. UltraPure TM琼脂糖(Thermo Fisher Scientific,Invitrogen TM,目录号:16500500)
    7. TAE(Tris-acetate-EDTA)缓冲液(参见食谱)
      1. Tris(Roche Diagnostics,目录号:10708976001)
      2. 冰醋酸(Sigma-Aldrich,目录号:ARK2183)
      3. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
    8. 1 kb DNA ladder(New England Biolabs,目录号:N3232)
    9. 20x盐水柠檬酸钠(SSC)溶液(参见食谱)
      1. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
      2. 柠檬酸钠(Sigma-Aldrich,目录号:W302600)
    10. 变性溶液(见配方)
      1. 氯化钠(NaCl)
      2. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:71687)
    11. 中和解(见配方)
      1. Tris(pH 7.4)
      2. 氯化钠(NaCl)
    12. 探针引物
      fw:AACTGGACCAATGAGAACTTTCC; rv:AAAAAGTCTTTGACTTCCTGCTT
    13. 探针引物
      fw:AATCAGTGAGGCACCTATCTCAGC; rv:AACTCGGTCGCCGCATACACTATT
    14. Go Taq ®无色主混合PCR试剂盒(Promega,目录号:M7132)
    15. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
    16. dCTPs,[α 32 P] -6,000Ci/mmol 20mCi/ml,250μCi(PerkinElmer,目录号:BLU013Z250UC)
    17. Prime-It RmT随机引物标签试剂盒(Agilent Technologies,目录号:300392)
    18. 尼龙洗涤液(参见食谱)
      1. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S3264)
      2. 乙二胺四乙酸(EDTA)
      3. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)

设备

  1. 移液器
  2. CO 2亚型培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:HeraCell 240)
  3. 血细胞计数器
  4. 冷藏台式微量离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Fresco TM 17)
  5. 冷冻离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Multifuge TM X3R)
  6. 微生物安全柜(医用航空技术,型号:BioMAT 2 II级)
  7. NanoDrop分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop 1000或NanoDrop 2000)
  8. 凝胶电泳系统(Thermo Fisher Scientific,Thermo Scientific TM,型号:Owl TM A1)
  9. 平台振动筛(Heidolph Instruments,型号:Polymax 1040)
  10. 玻璃板
  11. 玻璃托盘
  12. 紫外线交联剂(UVP,型号:CL-1000)
  13. 杂交炉(UVP,型号:HB-1000)
  14. Perspex屏幕(Thermo Fisher Scientific,Thermo Scientific TM,型号:Nalgene TM压克力台式Beta辐射防护罩,目录号:67002418)
  15. 台风分子动力学荧光/荧光成像仪(GE Healthcare,型号:Trio Variable Mode Imager)
  16. PCR热循环仪(Eppendorf,型号:Mastercycler )
  17. 热块(Cole-Parmer,Stuart,目录号:SBH200DC)
  18. 存储荧光屏(GE Healthcare,目录号:28-9564-75)
  19. 曝光盒(GE Healthcare,目录号:63-0035-44)

软件

  1. ImageQuant分析软件(GE Healthcare)

程序

  1. 转染293T细胞和AAV生产
    1. 在10ml培养基(DMEM + 10%FBS)的10cm培养皿中种子4.3×10 6个细胞用于转染第二天。包括额外的板以确定腺病毒感染时的细胞数。细胞在37℃和5%CO 2维持24小时。
    2. 在500μl的DMEM(没有添加血清,SF DMEM)中稀释17.5μg的pAV2质粒。
    3. 在500μlSF DMEM中稀释94μlPEI,轻轻混合。转染试剂应在室温(RT)下混合。在室温下孵育5分钟。
    4. 结合PEI和DNA,并通过移液轻轻混合。在室温下孵育20分钟
    5. 将混合物加入细胞生长的培养基中。仔细吸管以避免分离细胞。
    6. 转染后4小时,在额外的板中测定细胞数(例如使用血细胞计数器)
    7. 使用5 pfu /细胞的腺病毒感染细胞。包括没有腺病毒的病毒作为AAV复制的阴性对照。
    8. 转染后40〜50 h预计会产生细胞致病作用。细胞将向上并最终分离(图1) 注意:转染后40至50 h完全支持AAV复制和引起细胞致病作用所需的腺病毒量可能会因使用的细胞系的健康状况及其通过次数而异。这可能需要在开始此协议之前进行优化:用增加量的腺病毒感染细胞并监测细胞致病作用的出现(图1)。


      图1. AAV和腺病毒诱导的细胞致病作用。左图:转染后24小时细胞粘附并形成单层。右图:转染后55 h细胞致病性明显;细胞已经四舍五入,从板上抬起。刻度棒=50μm。

  2. 提取低分子量
    1. 将细胞和上清液收集在15ml锥形管中。细胞应松散粘附,并可通过移液分离。用2ml 1x DPBS洗板,加入15ml管中
    2. 在室温下以500×g离心5分钟。
    3. 弃去上清液,并将细胞沉淀重悬于1ml DPBS中。将250μl转移到新的1.5 ml管中。可以收集剩余的细胞用于额外的分析,例如基因和蛋白质表达。
    4. 在室温下在室温下以500×g离心5分钟,并倒出上清液。
    5. 加入500μlHirt裂解缓冲液,通过轻拂或反转管混合。
      注意:裂解物将非常粘稠,通过移液混合会产生气泡。
    6. 在室温下孵育10分钟。
    7. 加入1.25μl蛋白酶K(50μg/ml终浓度),37℃孵育1 h
    8. 加入120μl5 M NaCl(1M终浓度)。轻轻地/轻轻地混合管。
    9. 在4℃下孵育过夜。该步骤沉淀出高分子量(染色体)DNA
    10. 在4°C的冷藏台式离心机中以最大速度离心1小时(17,000 x g)离心机。
    11. 小心地将上清液(约500μl)转移到新的1.5 ml管中。
      注意:如果上清液不能从粘稠颗粒充分分离,可能需要较长的离心。
    12. 在通风橱下,加入1体积(500μl)的苯酚/氯仿/异戊醇,并通过倒转管混合直到均匀。
    13. 在4℃下以17,000 x g离心10分钟。
    14. 小心地将上层转移到新的1.5 ml管中(约450μl)
    15. 加入50μl3M乙酸钠(0.3M终浓度)并混合
    16. 在-20°C孵育过夜。
    17. 在4℃下以17,000×g离心20分钟。
    18. 仔细取出并弃去上清液,然后用100μl70%乙醇洗涤DNA沉淀
    19. 在4℃下以17,000 x g离心10分钟。
    20. 去除上清液并空气干燥DNA沉淀。
    21. 将沉淀重悬于80μlddH 2 O中。
    22. 使用NanoDrop分光光度计测定样品中DNA的浓度。典型的DNA浓度约为750 ng /μl
  3. Southern印迹测定(凝胶转移)
    1. 将大约1μg的提取的DNA用DpnI消化,在37℃下2小时。 Dnn特异性地消化甲基化的DNA,例如,在dam +细菌菌株中扩增的DNA。该步骤确保输入质粒DNA被消化,而新重复的AAV DNA不会受到影响。
    2. 将消化的DNA在0.8%琼脂糖凝胶上以1V/cm分离过夜
    3. 从电泳槽中取出凝胶,并在平台式搅拌器上以约25 rpm的变性溶液温育30分钟。
    4. 在ddH O中冲洗凝胶两次。
    5. 在平台式搅拌器上以约25 rpm的速度在中和溶液中孵育凝胶30分钟。
    6. 用新鲜的中和溶液代替中和溶液,再振荡30分钟
    7. 在ddH <2> O中冲洗,然后继续安装传输设置,如图2所示。


      图2.用于Southern印迹的毛细管转移方法的方案支撑板是搁置在含有20x SSC的玻璃托盘的侧面上的玻璃板。纸巾层高约15厘米。使用的重量约为600克。

    8. 孵育过夜,通过毛细管法将凝胶中的DNA转移到尼龙膜上(图2)。要在尼龙膜上获得与凝胶上相同的样品取向,请将凝胶倒置。
    9. 拆卸设备,小心取出膜。
    10. 在2x SSC中冲洗膜,让其风干。
    11. 为了将DNA固定在膜上,UV以120,000μJ/cm 2的比例交联膜。
    12. 将膜在20毫升0.75倍尼龙清洗液中预先杂交至65℃的杂交管中至少1小时。

    1. 使用上述引物对和下面列出的PCR条件,通过PCR对pAV2质粒进行PCR获得用于制备 Rep Amp 特异性探针的模板。 PCR产物用NanoDrop分光光度计进行凝胶纯化和定量。 PCR产物的预期大小是PCR反应的338碱基对,而PCR产物的预期大小是587。使用20至25ng纯化的PCR产物DNA作为DNA模板来制备探针 对于 Rep Amp PCR,混合最终体积为25μl:
      Go Taq 无色主混音(2x)
      12.5μl
      Fw Amp 引用(10μM)
      1μl
      Rv 或 Amp 引用(10μM)
      1μl
      模板DNA
      25 ng
      ddH 2 O
      至25μl

      循环条件
      PCR:


      循环条件 PCR:


    2. 使用含有20-25ng探针DNA的ddH 2 O,将反应混合物(提供为含有脱水的随机引物,dNTP,缓冲液和辅因子的0.2ml管)重构成最终体积为44μl,如所建议的在Prime-It RmT随机引物标签试剂盒中。在95℃下加热5分钟
    3. 加入1.5μl的p-d标记的dCTPs和3μlMagenta聚合酶(标记试剂盒中带有红帽的管)。在37°C孵育8-10分钟。
    4. 加入2μl停止混合物(标签试剂盒中带有绿色帽子的管)。
    5. 使用快速旋转柱分离标记的DNA(探针)与未掺入的标记核苷酸进行放射性标记的DNA纯化。
    6. 将探针在95℃加热5分钟使DNA变性
    7. 将管置于冰上5分钟,然后快速旋转以收集管底部的探针。

  4. Southern印迹测定(膜杂交)
    1. 将探针加入杂交管。根据放射性的新鲜度,如上所述制备的一个探针足以标记2-4个膜。
    2. 在杂交炉中在65℃孵育过夜。
    3. 在0.5×尼龙清洗液中,在65℃下将膜洗涤20分钟
    4. 在65℃下将0.1×尼龙洗涤溶液中的膜洗涤20分钟。
      注意:如果需要额外的洗涤,请使用0.01x尼龙清洗液重复洗涤步骤。
    5. 将膜暴露于Phosphoimager屏幕至少2小时。
      注意:获取最佳信号所需的曝光时间长短取决于信号的强度。
    6. 使用台风荧光粉成像仪获取图像。可以使用ImageQuant分析软件(GE Healthcare)进行图像分析。

数据分析

使用ImageQuant分析软件(GE Healthcare)分析按照上述程序获取的凝胶图像。在不同条件下AAV复制的水平与对应于与病毒(Rep)探针杂交的DpnI处理的样品中的病毒复制中间体的条带的强度成比例(图3)。对于wt AAV2病毒,分别为约4.7kb和9.4kb的单体和二聚体分子应该是可见的。还可以存在另外更高分子量的中间体。与Amp 探针杂交的膜片可以评估转染的效率和DpnI消化的功效(图3)。如果需要定量波段比较,ImageQuant软件通过光密度计支持频带量化

图3.通过Southern印迹分析AAV复制中间体。左图:pAAV-GFP(no Rep 对照),pAV2(WT AAV质粒)和pAV2-RepK340H(+ Ad)或不存在腺病毒转染(复制缺陷型突变体)。探针结合所有含氨苄青霉素抗性基因的质粒。 Rep 探针仅与含有AAV Rep 基因的DNA结合。右图:样品用IpI消化处理以除去所有输入质粒;检测到仅从质粒中拯救并复制的AAV DNA。在腺病毒存在(+ Ad)或不存在时转染质粒pAAV-GFP和pAV2。用 Amp 探针杂交确认所有输入质粒通过DpnI处理消化。 Rep 探针检测到复制的AAV DNA。 M:单体复制形式; D:二聚复制形式;较大的复制中间体在上面可见。改编自Bardelli等。,2016。

笔记

  1. 步骤D3至E5应仅在指定用于放射性工作的区域执行。请遵守本地指令。
  2. 关于对照的注意事项:运行该测定时应使用几个对照。
    1. + AAV, - 腺病毒:无复制控制。在没有腺病毒的情况下,AAV复制不存在或非常低
    2. - Rep ,+腺病毒:无复制控制。代替wt AAV,使用复制缺陷型AAV突变体或重组AAV载体(例如pAAV-GFP)。这也将控制在实验中使用的腺病毒批次中不存在wt AAV污染。
    3. 使用 Amp 探针来确保输入DNA被消化。
    4. 否控制:允许不同条件之间输入质粒的比较,以及用于结合Amp 探针的对照。

食谱

  1. Hirt裂解缓冲液
    6 ml 10%SDS
    1毫升1M Tris(pH 7.5)溶液
    2ml 500mM EDTA(pH8.0)溶液
    使用ddH 2 O
    使最终体积达到100ml
  2. TAE(Tris-acetate-EDTA)缓冲液
    准备50x解决方案:
    242克Tris
    57.1毫升冰醋酸
    100ml 500mM EDTA(pH8.0)溶液
    将最终音量设置为1 L ddH 2 O
    在ddH 2 O中稀释1:50,使1x TAE缓冲液
  3. 20x盐水柠檬酸钠(SSC)溶液
    175.3克NaCl
    88.2克柠檬酸钠
    将最终音量设置为1 L ddH 2 O
  4. 变性溶液
    300 ml 5 M NaCl溶液
    100ml 5 M NaOH溶液
    带有ddH 2 O的
  5. 中和解决方案
    500毫升1M Tris(pH 7.4)溶液
    300 ml 5 M NaCl溶液
    带有ddH 2 O的
  6. 尼龙洗液(pH 7.2)
    40.6g Na 2 HPO 4
    18.65g EDTA
    500克SDS
    使最终体积达到3.58L,ddH <2> O

致谢

这项工作得到辉瑞稀有疾病联盟奖(EH),NIH授予RO1-GM092854(CRE)和英国医学研究理事会授予1001764(RML)的支持。

参考

  1. Bardelli,M.,Zarate-Perez,F.,Agundez,L.,Linden,RM,Escalante,CR and Henckaerts,E.(2016)。  确定与功能相关的Adeno相关病毒Rep68寡聚体界面。 J Virol 90(15 ):6612-6624。
  2. Graham,FL和Prevec,L。(1991)。操纵腺病毒载体。方法分子生物学7:109-128。
  3. Hirt,B.(1967)。  选择性提取多瘤来自感染的小鼠细胞培养物的DNA。 J Mol Biol 26(2):365-369。
  4. Im,DS and Muzyczka,N。(1990)。  AAV起始结合蛋白Rep68是具有DNA解旋酶活性的ATP依赖性位点特异性内切核酸酶。细胞 61(3):447-457。
  5. Laughlin,CA,Tratschin,JD,Coon,H.and Carter,BJ(1983)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/6352411"target ="_ blank">细菌质粒中感染性腺相关病毒基因组的克隆基因 23(1):65-73。
  6. Rose,JA,Berns,KI,Hoggan,MD和Koczot,FJ(1969)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/5264145 "target ="_ blank">单链腺病毒相关病毒基因组的证据:病毒DNA释放时形成DNA密度杂合物。美国Proc Natl Acad Sci USA 64(3) ):863-869。
  7. Samulski,RJ,Berns,KI,Tan,M.和Muzyczka,N。(1982)。将腺相关病毒克隆到pBR322中:从人类细胞中的重组质粒中拯救完整病毒。Proc Natl Acad Sci USA 79(6) :2077-2081。
  8. Southern,EM(1975)。  检测特定序列通过凝胶电泳分离的DNA片段。分子生物 98:503-17。
  9. Straus,SE,Sebring,ED和Rose,JA(1976)。 
  • 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: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bardelli, M., Zarate-Perez, F., Agundez, L., Jolinon, N., Linden, R. M., Escalante, C. R. and Henckaerts, E. (2017). Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting. Bio-protocol 7(9): e2271. DOI: 10.21769/BioProtoc.2271.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。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或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。