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Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines
A2和A72 J-Lat细胞系中HIV-1经shRNA沉默后转录活性的流式细胞分析   

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Abstract

The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). To eliminate viral reservoirs, one strategy focuses on reversing HIV-1 latency via ‘shock and kill’ (Deeks, 2012). The basis of this strategy is to overcome the molecular mechanisms of HIV-1 latency by therapeutically inducing viral gene and protein expression under antiretroviral therapy and to cause selective cell death via the lytic properties of the virus, or the immune system now recognizing the infected cells. Recently, a number of studies have described the therapeutic potential of pharmacologically inhibiting members of the bromodomain and extraterminal (BET) family of human bromodomain proteins (Filippakopoulos et al., 2010; Dawson et al., 2011; Delmore et al., 2011) that include BRD2, BRB3, BRD4 and BRDT. Small-molecule BET inhibitors, such as JQ1 (Filippakopoulos et al., 2010; Delmore et al., 2011), I-BET (Nicodeme et al., 2010), I-Bet151 (Dawson et al., 2011), and MS417 (Zhang et al., 2012) successfully activate HIV transcription and reverse viral latency in clonal cell lines and certain primary T-cell models of latency. To identify the mechanism by which BET proteins regulate HIV-1 latency, we utilized small hairpin RNAs (shRNAs) that target BRD2, BRD4 and Cyclin T1, which is a component of the critical HIV-1 cofactor positive transcription elongation factor b (P-TEFb) and interacts with BRD2, and tested them in the CD4+ J-Lat A2 and A72 cell lines. The following protocol describes a flow cytometry-based method to determine the amount of transcriptional activation of the HIV-1 LTR upon shRNA knockdown. This protocol is optimized for studying latently HIV-1-infected Jurkat (J-Lat) cell lines.

Keywords: Human immunodeficiency virus-1(人类免疫缺陷病毒-1), Latency(潜伏期), shRNA knockdown(shRNA沉默), Transcriptional activation(转录激活), Flow cytometry(流式细胞术), HIV-1 LTR(HIV-1 LTR), J-Lat cells lines(J-Lat细胞系), BRD2(BRD2), BRD4(BRD4), Cyclin T1(细胞周期蛋白T1)

Background

A72 J-Lat cells contain a latent HIV minigenome composed of just the HIV promoter in the 5’LTR that drives the expression of the fluorescent marker GFP (LTR-GFP; A72) while in A2 cells transcriptional activity is driven by the viral transactivator Tat (LTR-Tat-IRES-GFP; A2) (Jordan et al., 2001 and 2003). HIV transcription can be induced in both cell lines with TNFα mimicking T cell-receptor engagement. Cells were transduced with lentiviral vectors expressing two different shRNAs targeting each cellular protein or a scrambled control, followed by puromycin treatment to select successfully transduced cells. Cells were then stimulated with a suboptimal or saturating dose of TNFα or were left unstimulated for 24 h, followed by flow cytometry of GFP to assess transcriptional activation of the HIV-1 LTR.

Materials and Reagents

  1. Production of shRNA containing virus particles
    1. 175 cm2 tissue culture flask (Corning, Falcon®, catalog number: 353112 )
    2. Tips
      0.1-10 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-681-440 )
      1-200 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-502 )
      101-1,000 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-509 )
    3. 15 ml conical tube (Fisher Scientific, FisherbrandTM, catalog number: 05-539-5 )
    4. 10 cm dish (Corning, catalog number: 353803 )
    5. 10 ml syringes (BD, catalog number: 309604 )
    6. 0.45 µm syringe filter (EMD Millipore, catalog number: SLHV033RS )
    7. HEK293T cells (ATCC, catalog number: CRL-3216 )
    8. shRNA-expressing lentiviral vectors (Sigma-Aldrich) to knockdown:
      a. BRD2: TRCN0000006308 and TRCN0000006310
      b. BRD4: TRCN0000021424 and TRCN0000021428
      c. Cyclin T1: TRCN0000013673 and TRCN0000013675
      d. Control: pLKO.1 vector containing scramble shRNA
      e. Lentiviral packaging construct pCMVdelta R8.91 (Naldini et al., 1996)
      f. VSV-G glycoprotein-expressing vector (Naldini et al., 1996)
    9. DMEM (Mediatech, catalog number: 10-013-CV )
    10. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
    11. L-glutamine (Mediatech, catalog number: 25-005-Cl )
    12. 100x penicillin/streptomycin (Mediatech, catalog number: 30-002-Cl )
    13. 1x PBS (Mediatech, catalog number: 21-031-CV )
    14. Trypsin-EDTA (Mediatech, catalog number: 25-052-Cl )
    15. Chloroquine diphosphate salt (Sigma-Aldrich, catalog number: C6628 )
    16. HEPES (Sigma-Aldrich, catalog number: H3375 )
    17. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    18. Dextrose (Fisher Scientific, catalog number: BP350-1 )
    19. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
    20. Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, catalog number: BP332-500 )
    21. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
    22. Glycerol (Sigma-Aldrich, catalog number: G5516 )
    23. Nuclease-free H2O (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9937 )
    24. Lenti-XTM p24 Rapid Titer Kit (Takara Bio, Clontech, catalog number: 632200 )
    25. 25 mM chloroquine (see Recipes)
    26. HBSS buffer (see Recipes)
    27. 2 M CaCl2 (see Recipes)
    28. Glycerol shock solution (see Recipes)

  2. Infection of A2 and A72 J-Lat cells
    1. 75 cm2 tissue culture flask (Corning, Falcon®, catalog number: 353110 )
    2. 6 well tissue culture plates (Corning, Falcon®, catalog number: 353224 )
    3. Posi-ClickTM 1.7 ml microcentrifuge tubes (Denville Scientific, catalog number: C2170 )
    4. Tips
      0.1-10 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-681-440 )
      1-200 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-502 )
      101-1,000 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-509 )
    5. A2 and A72 J-Lat cells (Jordan et al., 2003)
    6. RPMI (Mediatech, catalog number: 10-040-CV )
    7. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
    8. L-glutamine (Mediatech, catalog number: 25-005-Cl )
    9. 100x penicillin/streptomycin (Mediatech, catalog number: 30-002-Cl )
    10. Polybrene® (Santa Cruz Biotechnology, catalog number: sc-134220 )
    11. Puromycin (Sigma-Aldrich, catalog number: P7255 )
    12. Polybrene solution (see Recipes)

  3. Analysis of HIV-1 LTR transcriptional activation by flow cytometry
    1. 96-well tissue culture plates and lids (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 249570 and 163320 )
    2. Tips
      0.1-10 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-681-440 )
      1-200 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-502 )
      101-1,000 µl (Fisher Scientific, FisherbrandTM, catalog number: 02-707-509 )
    3. A2 and A72 J-Lat cells (Jordan et al., 2003)
    4. 1x PBS (Mediatech, catalog number: 21-031-CV )
    5. RPMI (Mediatech, catalog number: 10-040-CV )
    6. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
    7. L-glutamine (Mediatech, catalog number: 25-005-Cl )
    8. 100x penicillin/streptomycin (Mediatech, catalog number: 30-002-Cl )
    9. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
    10. TNFα (PeproTech, catalog number: 300-01A ), make stock solution of 100 ng/μl in sterile H2O
    11. 7AAD, Propidium iodide or one of the Zombie viability dyes
    12. JQ1 (Cayman Chemical, catalog number: 11187 ), make stock solution of 10 mM in DMSO
    13. MACSQuant Running buffer (Miltenyi Biotech, catalog number: 130-092-747 )
    14. ApoTox-GloTM Triplex Assay (Promega, catalog number: G6320 )

  4. Confirmation of shRNA knockdown efficiency by SDS-PAGE and Western blot
    1. Posi-ClickTM 1.7 ml microcentrifuge tubes (Denville Scientific, catalog number: C2170 )
    2. Nitrocellulose membrane 0.2 µm (Bio-Rad Laboratories, catalog number: 1620112 )
    3. Whatman paper (GE Healthcare, catalog number: 3030-917 )
    4. High-performance chemiluminescence film (GE Healthcare, catalog number: 28906839 )
    5. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
    6. NP-40 (Igepal CA-630) (Sigma-Aldrich, catalog number: I3021 )
    7. Na-deoxycholate (Deoxycholic acid, sodium salt) (Fisher Scientific, catalog number: BP349-100 )
    8. Sodium dodecyl sulfate (SDS) (Fisher Scientific, catalog number: BP166-500 )
    9. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
    10. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    11. Ethylenediaminetetraacetic acid (EDTA) (Fisher Scientific, catalog number: S311-500 )
    12. Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S6521 )
      Note: This product has been discontinued.
    13. DCTM Protein assay (Reagents A, B and S, Bio-Rad Laboratories, catalog numbers: 5000113 , 5000114 , 5000115 )
    14. Mini-Protean® TGXTM Gels (Bio-Rad Laboratories, catalog number: 4569034 )
    15. PageRulerTM Prestained protein ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26617 )
    16. Antibodies for Western blot
      1. Rabbit polyclonal anti-BRD2 (Cell Signaling, catalog number: 5848 )
      2. Rabbit polyclonal anti-BRD4 (Abcam, catalog number: ab75898 )
      3. Rabbit polyclonal anti-Cyclin T1 (Santa Cruz Biotechnology, catalog number: sc-10750 )
      4. Rabbit polyclonal anti-α-Tubulin (Abcam, catalog number: ab15246 )
      5. Goat-anti-Rabbit IgG, HRP conjugated (Bethyl Laboratories, catalog number: A120-201P )
    17. Tween® 20 (Fisher Scientific, catalog number: BP337-500 )
    18. Glycine (Fisher Scientific, catalog number: BP381-5 )
    19. Methanol (Fisher Scientific, catalog number: A433P-4 )
    20. Blotting-grade blocker (dry milk) (Bio-Rad Laboratories, catalog number: 1706404 )
    21. Lumi-light Western blotting substrate (Roche Diagnostics, catalog number: 12015200001 )
    22. RIPA buffer (see Recipes)
    23. Western blot running buffer (see Recipes)
    24. Western blot transfer buffer (pH 8.3) (see Recipes)
    25. TBS-T buffer (see Recipes)

Equipment

  1. Pipette
  2. Biosafety cabinet ‘Level 2’
  3. Tabletop centrifuge for Eppendorf tubes (Eppendorf, model: 5415 D )
  4. Tabletop centrifuge for 96-well plates, Eppendorf, 15 ml and 50 ml tubes; used for spininfection (Beckman Coulter, model: Allegra X-14R )
  5. CO2 tissue culture incubator, 37 °C (Thermo Electron, model: FormaTM Steri-CultTM CO2 Incubators, catalog number: 3307)
  6. MACSQuant VYB FACS analyzer (Miltenyi Biotech, model: MACSOuant® VYB , catalog number: 130-096-116)
  7. Mini-PROTEAN® Electrophoresis System (Bio-Rad Laboratories, catalog-numbers: 1658006FC and 1703935 )
  8. PowerPacTM HC (Bio-Rad Laboratories, model: Power Pac HC Highe-Current Power Supply , catalog number: 1645052)
  9. Rocker II (Boekel Scientific, catalog number: 260350 )
  10. SpectraMax MiniMaxTM 300 Imaging Cytometer (Molecular Devices, model: SpectraMax MiniMax 300 )

Software

  1. FlowJo 9.9 or never (Tree Star)

Procedure

  1. Production of shRNA containing virus particles in HEK293T cells
    Note: HIV-1 particles are produced after calcium phosphate transfection in HEK293T cells.
    1. HEK293T cell culture
      1. HEK293T cells are cultured in DMEM medium (supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin) in 175 cm2 tissue culture flasks in 35 ml medium.
      2. For maintenance of the HEK293T culture cells are, when approaching confluence (~80%), washed twice with PBS, then trypsinized (0.05% trypsin) and plated after 1/10 dilution in 175 cm2 tissue culture flasks. HEK293T cells are split every 2-3 days.
      3. One day before transfection, plate approximately 2 x 106 of HEK293T cells per 10 cm dish in 10 ml medium.
        Note: After thawing frozen cell vials, HEK293T cells are cultured for at least one week before transfecting them for virus production. In order to maximize viral particle production, HEK293T cells are never kept more than 4 weeks in culture.
    2. Calcium phosphate transfection
      Note: Protocol is described for transfection of one 10 cm dish.
      1. Remove 2 ml medium from dish.
      2. Add 8 µl 25 mM chloroquine for a final concentration of 25 µM, incubate for about 5 min before adding the DNA/CaCl2 mix.
      3. Prepare DNA mix in nuclease-free H2O in a 15 ml conical tube (10 µg shRNA containing vector, 6.5 µg packaging construct pCMVdelta R8.91, and 3.5 µg VSV-G glycoprotein-expressing vector).
      4. Add nuclease-free H2O to the DNA mixture for a final volume of 876 µl.
      5. Add 1 ml of 2x HBSS buffer.
      6. Slowly (dropwise) add 124 μl of 2 M CaCl2 to the diluted DNA.
      7. After addition of CaCl2 gently vortex at the lowest setting for 5 sec (Note: Do not spin down!) and immediately add drop by drop the prepared DNA solution to the cells in the 10 cm dish.
      8. Swirl the plate to distribute the solution evenly.
      9. Culture for 3-4 h at 37 °C.
      10. Remove supernatant.
      11. Add 1 ml of glycerol shock solution, shock for one minute.
        Note: The addition of glycerol results in an osmotic shock and is thought to cause the release of the DNA from the precipitate (Grosjean et al., 2006).
      12. Aspirate shock solution, wash cells in 1 ml DMEM medium (supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin) and aspirate again.
      13. Add 7 ml of DMEM (supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin) medium.
      14. 48 h after transfection, collect supernatant with a 10 ml syringe and filter it through a sterile 0.45 µm PVDF membrane filter.
      15. Titer virus for p24 content using Lenti-XTM p24 Rapid Titer Kit according to manufacturer’s instructions.
      16. Filtered viral supernatant can be used fresh or stored at -80 °C.

  2. Spin infection of A2 and A72 J-Lat cells with shRNA containing virus particles
    Note: A2 and A72 cells are infected by spin infection.
    1. A2 and A72 J-Lat suspension cell culture
      1. A2 and A72 cells are cultured in RPMI medium (supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin) in 75 cm2 tissue culture flasks in 40 ml medium. The flasks are placed horizontally to increase the surface area in which the cells are grown.
      2. Cells are split every 1-2 days and should be kept at a concentration of 2 x 105-1.5 x 106/ml.
      3. One day before transfection split A2 or A72 cells to 5 x 105/ml.
    2. Infection of A2 and A72 J-Lat cells
      1. Count the cells and adjust cell number with new RPMI media (supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin). Add 1 x 106 of A2 or A72 cells to 1.7 ml microcentrifuge tubes.
      2. Centrifuge cell suspension for 3 min at 300 x g (1,800 rpm). Pipette off supernatant completely.
      3. Add 500 µl of shRNA virus (1 ng of p24 per 106 cells), 5 µl 1 M HEPES and 0.5 µl of polybrene (4 µg/ml) in 1.7 ml microcentrifuge tubes.
      4. Centrifuge at 32 °C for 1.5 h at 1,341 x g (2,400 rpm) (Tabletop centrifuge, Beckman).
      5. Pipette off supernatant completely.
      6. Resuspend cells in 3 ml RPMI medium (supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin) in 6-well tissue culture plates.
      7. Culture in CO2 incubator at 37 °C for one day.
      8. If shRNA-virus particles contain a puromycin resistant gene, select cells with 2 µg/ml RPMI medium of puromycin. RPMI media (supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin) with puromycin is changed every two days. Depending on how fast the cells grow and if they are needed for other experiments (e.g., chromatin immunoprecipitation, RNA isolation…) the cells can be grown out by adding media every 2 days and moved to bigger flasks or split to culture them in the 6-well plate.
      9. Culture in CO2 incubator at 37 °C for 4-10 days.

  3. Analysis of HIV-1 LTR transcriptional activation by flow cytometry (please refer to the bio-protocol: Boehm and Ott, 2017)
    1. Count the cells and adjust cell number with new media. About 2 x 105/well of A2 or A72 cells are plated in 96-well tissue culture plates in 195 µl RPMI medium (supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin). For each condition perform three replicates.
    2. Cells are treated with 0.016-10 ng/ml of TNFα (as co-stimulus for maximal activation or to check for synergy), PBS or H2O control (depending on what was used as diluent for TNFα), or left untreated. TNFα and controls are pipetted in 5 µl to reach a total volume of 200 µl/well. Mix cells and TNFα by pipetting up and down at least 3 times.
    3. Culture in CO2 incubator at 37 °C for 18-48 h.
    4. Take the plate from the incubator and centrifuge at room temperature for 2 min at 1341 x g (2,400 rpm) (Tabletop centrifuge, Beckman).
    5. Then proceed directly to flow cytometry analysis. Cells do not have to be washed or fixed.
      Notes:
      1. For flow cytometry, MACSQuant VYB FACS analyzer was used to run 96-well plates. However, other flow cytometers such as Calibur or LSRII are also suitable for these experiments.
      2. If the analysis of cell viability by forward scatter (FSC) vs. side scatter (SSC) is not sufficient for accurate assessment of drug toxicity in drug studies, one of a variety of dyes/stains; for example 7AAD, Propidium iodide or one of the Zombie viability dyes can be used according to manufacturer’s instructions in the flow cytometric analysis in this protocol.
    6. Viability, cytotoxicity and apoptosis was measured with ApoTox-GloTM Triplex Assay (Promega) according to manufacturer’s instructions using a SpectraMax MiniMaxTM 300 Imaging Cytometer.

  4. Confirmation of shRNA knockdown efficiency by SDS-PAGE and Western blot
    SDS-PAGE and Western blot protocols were described elsewhere (Renart et al., 1979; Towbin et al., 1979).
    1. Lyse 1 x 106 cells in 100-500 µl RIPA buffer in 1.7 ml microcentrifuge tubes.
    2. Measure protein concentration by DCTM Protein Assay (Bio-Rad Laboratories) according to manufacturer’s instructions using a SpectraMax MiniMaxTM 300 Imaging Cytometer.
    3. Load PageRulerTM protein ladder in one lane and the other lanes equal amounts of protein from the cell lysates on 6-15% SDS gels (depending on the size of the protein) and electrophorese at 200 V for 1 h to separate the proteins. The expected size in kDa and the appropriate % of acrylamide for each protein are: BRD2, 110 kDa, 8%; BRD4, 171 kDa, 6.5%; Cyclin T1, 87 kDa, 8%; α-Tubulin, 50 kDa, 10%. Since the kDa of the proteins are so different the membranes can be cut to check for equal loading by incubation with α-Tubulin antibody.
    4. Transfer the separated proteins to the nitrocellulose membranes using a Bio-Rad Mini-PROTEAN® Electrophoresis System at 100 mA for 1-3 h or at 35 mA overnight (for proteins larger than 120 kDa).
    5. Block the transferred proteins on the membranes in TBS-T containing 5% nonfat dry milk (blocking buffer) for 1 h at room temperature on a rocker.
    6. Incubate the membranes with primary antibodies diluted (BRD2 1:1,000; BRD4 1 µg/ml; Cyclin T1 1:1,000; α-Tubulin 1:2,000) in TBS-T buffer over night at 4 °C on a rocker.
    7. Wash the membranes three times for approximately 5 min each time with TBS-T buffer and incubate with the HRP-conjugated secondary antibody, diluted 1:10,000 in TBS-T buffer, for 1 h at room temperature on a rocker.
    8. Wash the membranes three times for approximately 5 min each time with TBS-T buffer and incubate for 30 sec in Lumi-light Western blotting substrate.
    9. Develop on chemiluminescence films, expose the films for 10 sec up to 30 min. Depending on if the antibody has been reused and how many times it has been reused the signal will appear weaker, but cleaner (less background bands).

Data analysis

Analysis of HIV-1 LTR transcriptional activation by flow cytometry (Figure 1).


Figure 1. Western blot of BRD2, BRD4, Cyclin T1 and α-Tubulin. A. A72 cells were infected with virus containing shRNA constructs targeting BRD2, BRD4 or a nontargeting scramble control. At 4 days after infection, cells were harvested and lysed in Ripa buffer. Knockdown of BRD2 and BRD4 protein levels were confirmed by immunoblotting with BRD2 and BRD4 antibodies or the control α-Tubulin. B. A72 cells were infected with virus containing shRNA constructs targeting cyclin T1 or a nontargeting control. Knockdown of cyclin T1 protein levels are shown by immunoblotting with cyclin T1 or the control α-Tubulin antibody.

  1. First, set the gate on live J-Lat cells. Cell viability is monitored by forward (FSC-Area) and side scatter (SSC-Area) analysis (Figure 2A).
  2. Gate on singlets (FSC-Height vs. FSC-Area) (Figure 2B).
  3. Set the gate on SSC-Area and GFP/FITC-Area to identify the amount of GFP+ cells (Figure 2C).
  4. Each sample is usually analyzed in triplicate and the experiment is independently repeated three times. Three replicates are averaged by calculating (GFP+ cells Experiment 1 + GFP+ cells Experiment 2 + GFP+ cells Experiment 3)/3. Also calculate standard deviation (STDEV) for error bars.


    Figure 2. Analysis of HIV-1 LTR transcriptional activation by flow cytometry. Gating strategy to analyze A2 or A72 J-Lat cells: A. gating on live J-Lat cells based on size (FSC-Area) and granularity (SSC-Area); B. singlets gate (FSC-Height vs. FSC-Area); C. gating on GFP+ J-Lat cells (SSC-Area vs. GFP-FITC-Area).

Recipes

  1. 25 mM chloroquine
    25 mM chloroquine in PBS
  2. 2x HBSS
    50 mM HEPES
    10 mM KCl
    12 mM dextrose
    280 mM NaCl
    1.5 mM Na2HPO4
    Adjust pH to 7.1
    Note: pH is crucial!
  3. 2 M CaCl2
    2 M CaCl2 in nuclease-free H2O
  4. Glycerol shock solution
    150 nM NaCl
    20 mM HEPES, pH 7.0
    15% glycerol
  5. Polybrene solution
    4 µg/ml in nuclease-free H2O
  6. RIPA buffer
    50 mM Tris buffer, pH 7.4
    1% NP-40
    0.5% Na-deoxycholate
    0.1% SDS
    150 mM NaCl
    2 mM EDTA
    50 mM sodium fluoride
  7. Western blot running buffer
    25 mM Trizma® base
    190 mM glycine
    0.1% SDS
  8. Western blot transfer buffer (pH 8.3)
    25 mM Trizma® base
    190 mM glycine
    20% methanol
  9. TBS-T buffer
    19 mM Trizma® base
    2.7 mM potassium chloride
    137 mM sodium chloride
    1 ml Tween® 20

Acknowledgments

We thank Marielle Cavrois and the Flow cytometry core for the service provided for flow cytometry. This publication was made possible with the help from the University of California, San Francisco – Gladstone Institute of Virology & Immunology Center for AIDS Research (CFAR), a NIH-funded program (P30 AI027763). This research was supported as part of the amfAR Institute for HIV Cure Research, with funding from amfAR grant number 109301. Further, we gratefully acknowledge support from the California HIV/AIDS Research Program (Award number: F13-GI-316) to D.B., an industry-sponsored collaboration with JT Pharma that enabled the shRNA screen to M.O., and grant support from the CARE Collaboratory (U19 AI096113) and the NIH (RO1 AI083139 and RO1 DA043142) to M.O. This protocol was adapted from previous work: ‘BET bromodomain-targeting compounds reactivate HIV from latency via a Tat-independent mechanism’ (Boehm et al., 2013)

References

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  11. Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M. and Trono, D. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272(5259): 263-267.
  12. Nicodeme, E., Jeffrey, K. L., Schaefer, U., Beinke, S., Dewell, S., Chung, C. W., Chandwani, R., Marazzi, I., Wilson, P., Coste, H., White, J., Kirilovsky, J., Rice, C. M., Lora, J. M., Prinjha, R. K., Lee, K. and Tarakhovsky, A. (2010). Suppression of inflammation by a synthetic histone mimic. Nature 468(7327): 1119-1123.
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简介

消除HIV-1患者的主要障碍是后整合延迟(Finzi等人,1999)。抗逆转录病毒治疗仅针对主动复制病毒,而具有低转录活性或无转录活性的潜伏感染仍未得到治疗(Sedaghat等人,2007)。为了消除病毒性水库,一项战略重点是通过“休克和杀死”来逆转HIV-1潜伏期(Deeks,2012)。该策略的基础是通过在抗逆转录病毒治疗下通过治疗性诱导病毒基因和蛋白质表达来克服HIV-1潜伏期的分子机制,并通过病毒的溶解性质或现在识别感染细胞的免疫系统引起选择性细胞死亡。最近,许多研究已经描述了药物抑制人类溴结构域蛋白质的溴结构域和末端(BET)家族的成员的治疗潜力(Filippakopoulos等人,2010; Dawson等人& / em>,2011; Delmore等人,2011),其包括BRD2,BRB3,BRD4和BRDT。小分子BET抑制剂,例如JQ1(Filippakopoulos et al。,2010; Delmore等人,2011),I-BET(Nicodeme等人< / em>。,2010),I-Bet151(Dawson等人,2011)和MS417(Zhang等人,2012)成功地激活了HIV转录和逆转克隆细胞系中的病毒潜伏期和潜伏期的某些初级T细胞模型。为了鉴定BET蛋白质调控HIV-1潜伏期的机制,我们利用靶向BRD2,BRD4和细胞周期蛋白T1的小发夹RNA(shRNA),其是关键HIV-1辅因子阳性转录延伸因子b(P- TEFb)并与BRD2相互作用,并在CD4 + J-Lat A2和A72细胞系中进行测试。以下方案描述了基于流式细胞术的方法,以确定在shRNA敲低时HIV-1LTR的转录激活的量。该协议被优化用于研究潜伏的HIV-1感染的Jurkat(J-Lat)细胞系。

背景 A72 J-Lat细胞含有潜在的HIV微型基因组,其仅由5'LTR中的HIV启动子组成,其驱动荧光标记GFP(LTR-GFP; A72)的表达,而在A2细胞中转录活性由病毒反式激活剂Tat驱动(LTR-Tat-IRES-GFP; A2)(Jordan等人,2001和2003)。可以在模拟T细胞受体结合的TNFα的两种细胞系中诱导HIV转录。用表达两种不同shRNA的慢病毒载体转导细胞,每种shRNA靶向每种细胞蛋白或加扰对照,随后用嘌呤霉素处理以选择成功转导的细胞。然后用次最佳或饱和剂量的TNFα刺激细胞,或将其未刺激24小时,然后用GFP流式细胞术评价HIV-1LTR的转录激活。

关键字:人类免疫缺陷病毒-1, 潜伏期, shRNA沉默, 转录激活, 流式细胞术, HIV-1 LTR, J-Lat细胞系, BRD2, BRD4, 细胞周期蛋白T1

材料和试剂

  1. 含有病毒颗粒的shRNA的产生
    1. 175厘米组织培养瓶(Corning,Falcon ,目录号:353112)
    2. 提示
      0.1-10μl(Fisher Scientific,Fisherbrand TM,目录号:02-681-440)
      1-200μl(Fisher Scientific,Fisherbrand TM ,目录号:02-707-502)
      101-1,000μl(Fisher Scientific,Fisherbrand TM ,目录号:02-707-509)
    3. 15ml锥形管(Fisher Scientific,Fisherbrand TM,目录号:05-539-5)
    4. 10厘米盘(康宁,目录号:353803)
    5. 10ml注射器(BD,目录号:309604)
    6. 0.45μm注射器过滤器(EMD Millipore,目录号:SLHV033RS)
    7. HEK293T细胞(ATCC,目录号:CRL-3216)
    8. 表达shRNA的慢病毒载体(Sigma-Aldrich)击倒:
      一个。 BRD2:TRCN0000006308和TRCN0000006310
      湾BRD4:TRCN0000021424和TRCN0000021428
      C。细胞周期蛋白T1:TRCN0000013673和TRCN0000013675
      天。对照:含有scramble shRNA的pLKO.1载体 即慢病毒包装结构pCMVdelta R8.91(Naldini等人,1996)
      F。 VSV-G糖蛋白表达载体(Naldini等,1996)
    9. DMEM(Mediatech,目录号:10-013-CV)
    10. 胎牛血清(FBS)(Gemini Bio-Products,目录号:100-106)
    11. L-谷氨酰胺(Mediatech,目录号:25-005-Cl)
    12. 100x青霉素/链霉素(Mediatech,目录号:30-002-Cl)
    13. 1x PBS(Mediatech,目录号:21-031-CV)
    14. 胰蛋白酶-EDTA(Mediatech,目录号:25-052-Cl)
    15. 二氯磷酸盐(Sigma-Aldrich,目录号:C6628)
    16. HEPES(Sigma-Aldrich,目录号:H3375)
    17. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
    18. 葡萄糖(Fisher Scientific,目录号:BP350-1)
    19. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
    20. 磷酸氢二钠(Na 2 HPO 4)(Fisher Scientific,目录号:BP332-500)
    21. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C1016)
    22. 甘油(Sigma-Aldrich,目录号:G5516)
    23. 不含Nuclease的H 2 O(Thermo Fisher Scientific,Invitrogen,Sup。,目录号:AM9937)
    24. Lenti-X TM p24快速滴定试剂盒(Takara Bio,Clontech,目录号:632200)
    25. 25毫克氯喹(见食谱)
    26. HBSS缓冲区(见配方)
    27. 2 M CaCl 2 (见配方)
    28. 甘油冲击溶液(见食谱)

  2. 感染A2和A72 J-Lat细胞
    1. 75厘米组织培养瓶(Corning,Falcon ,目录号:353110)
    2. 6孔组织培养板(Corning,Falcon ®,目录号:353224)
    3. Posi-Click TM 1.7ml微量离心管(Denville Scientific,目录号:C2170)
    4. 提示
      0.1-10μl(Fisher Scientific,Fisherbrand TM,目录号:02-681-440)
      1-200μl(Fisher Scientific,Fisherbrand TM ,目录号:02-707-502)
      101-1,000μl(Fisher Scientific,Fisherbrand TM ,目录号:02-707-509)
    5. A2和A72 J-Lat细胞(Jordan等人,2003)
    6. RPMI(Mediatech,目录号:10-040-CV)
    7. 胎牛血清(FBS)(Gemini Bio-Products,目录号:100-106)
    8. L-谷氨酰胺(Mediatech,目录号:25-005-Cl)
    9. 100x青霉素/链霉素(Mediatech,目录号:30-002-Cl)
    10. Polybrene ®(Santa Cruz Biotechnology,目录号:sc-134220)
    11. 嘌呤霉素(Sigma-Aldrich,目录号:P7255)
    12. 聚乙烯溶液(参见食谱)

  3. 通过流式细胞术分析HIV-1 LTR转录激活
    1. 96孔组织培养板和盖子(Thermo Fisher Scientific,Thermo Scientific TM,目录号:249570和163320)
    2. 提示
      0.1-10μl(Fisher Scientific,Fisherbrand TM,目录号:02-681-440)
      1-200μl(Fisher Scientific,Fisherbrand TM ,目录号:02-707-502)
      101-1,000μl(Fisher Scientific,Fisherbrand TM ,目录号:02-707-509)
    3. A2和A72 J-Lat细胞(Jordan等人,2003)
    4. 1x PBS(Mediatech,目录号:21-031-CV)
    5. RPMI(Mediatech,目录号:10-040-CV)
    6. 胎牛血清(FBS)(Gemini Bio-Products,目录号:100-106)
    7. L-谷氨酰胺(Mediatech,目录号:25-005-Cl)
    8. 100x青霉素/链霉素(Mediatech,目录号:30-002-Cl)
    9. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
    10. TNFα(PeproTech,目录号:300-01A),在无菌H 2 O中制备100ng /μl的储备溶液
    11. 7AAD,碘化丙啶或其中一种僵尸活力染料
    12. JQ1(Cayman Chemical,目录号:11187),在DMSO中制备10mM的储备溶液
    13. MACSQuant运行缓冲区(Miltenyi Biotech,目录号:130-092-747)
    14. ApoTox-Glo TM Triplex Assay(Promega,目录号:G6320)

  4. 通过SDS-PAGE和Western blot确定shRNA敲低效率
    1. Posi-Click TM 1.7ml微量离心管(Denville Scientific,目录号:C2170)
    2. 硝酸纤维素膜0.2μm(Bio-Rad Laboratories,目录号:1620112)
    3. Whatman论文(GE Healthcare,目录号:3030-917)
    4. 高性能化学发光膜(GE Healthcare,目录号:28906839)
    5. Trizma ®基质(Sigma-Aldrich,目录号:T1503)
    6. NP-40(Igepal CA-630)(Sigma-Aldrich,目录号:I3021)
    7. 脱氧胆酸钠(脱氧胆酸,钠盐)(Fisher Scientific,目录号:BP349-100)
    8. 十二烷基硫酸钠(SDS)(Fisher Scientific,目录号:BP166-500)
    9. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
    10. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
    11. 乙二胺四乙酸(EDTA)(Fisher Scientific,目录号:S311-500)
    12. 氟化钠(NaF)(Sigma-Aldrich,目录号:S6521)
      注意:本产品已停产。
    13. 蛋白质测定(试剂A,B和S,Bio-Rad Laboratories,目录号:5000113,5000114,5000115)
    14. 凝胶(Bio-Rad Laboratories,目录号:4569034)
      < TG><
    15. PageRuler TM 预染蛋白梯(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:26617)
    16. 免疫印迹的抗体
      1. 兔多克隆抗BRD2(Cell Signaling,目录号:5848)
      2. 兔多克隆抗BRD4(Abcam,目录号:ab75898)
      3. 兔多克隆抗Cyclin T1(Santa Cruz Biotechnology,目录号:sc-10750)
      4. 兔多克隆抗-α-微管蛋白(Abcam,目录号:ab15246)
      5. 山羊抗兔IgG,HRP缀合(Bethyl Laboratories,目录号:A120-201P)
    17. Tween ® 20(Fisher Scientific,目录号:BP337-500)
    18. 甘氨酸(Fisher Scientific,目录号:BP381-5)
    19. 甲醇(Fisher Scientific,目录号:A433P-4)
    20. 印迹级阻滞剂(干牛奶)(Bio-Rad Laboratories,目录号:1706404)
    21. Lumi-light Western印迹底物(Roche Diagnostics,目录号:12015200001)
    22. RIPA缓冲区(请参阅配方)
    23. 蛋白质印迹运行缓冲液(参见食谱)
    24. Western印迹转移缓冲液(pH 8.3)(参见食谱)
    25. TBS-T缓冲(见配方)

设备

  1. 移液器
  2. 生物安全柜“二级”
  3. 用于Eppendorf管的台式离心机(Eppendorf,型号:5415 D)
  4. 用于96孔板,Eppendorf,15ml和50ml管的台式离心机;用于自旋感染(Beckman Coulter,型号:Allegra X-14R)
  5. CO 2组织培养培养箱,37℃(Thermo Electron,型号:Forma< sup> Steri-Cult<> CO 2< / sub >孵化器,目录号:3307)
  6. MACSQuant VYB FACS分析仪(Miltenyi Biotech,型号:MACSOuant ® VYB,目录号:130-096-116)
  7. Mini-PROTEAN ®电泳系统(Bio-Rad Laboratories,catalog-number:1658006FC和1703935)
  8. PowerPac TM HC(Bio-Rad Laboratories,型号:Power Pac HC Highe-Current Power Supply,目录号:1645052)
  9. Rocker II(Boekel Scientific,目录号:260350)
  10. SpectraMax MiniMax TM 300成像细胞仪(Molecular Devices,型号:SpectraMax MiniMax 300)

软件

  1. FlowJo 9.9或永不(Tree Star)

程序

  1. 在HEK293T细胞中产生含有病毒颗粒的shRNA
    注意:在HEK293T细胞中磷酸钙转染后产生HIV-1颗粒
    1. HEK293T细胞培养
      1. 将HEK293T细胞在含有35ml培养基的175cm 2组织培养瓶中的DMEM培养基(补充有10%FBS,1%L-谷氨酰胺和1%青霉素/链霉素)中培养。
      2. 为了维持HEK293T培养细胞,当接近汇合(〜80%)时,用PBS洗涤两次,然后胰蛋白酶消化(0.05%胰蛋白酶),并在175cm 2组织培养物中1/10稀释后铺板瓶。 HEK293T细胞每2-3天分裂一次
      3. 在转染前一天,在10ml培养基中每10cm皿平板约2×10 6个HEK293T细胞。
        注意:解冻冷冻细胞小瓶后,将HEK293T细胞培养至少一周,然后转染它们进行病毒生产。为了使病毒颗粒生产最大化,HEK293T细胞在培养中从未保存超过4周
    2. 磷酸钙转染
      注意:描述了用于转染一个10cm皿的方案。
      1. 从盘中取出2ml培养基。
      2. 加入8μl25mM氯喹,终浓度为25μM,孵育约5分钟,然后加入DNA / CaCl 2 混合物。
      3. 在15ml锥形管(10μg含有载体,6.5μg包装构建体pCMVdelta R8.91和3.5μgVSV-G糖蛋白的载体)中,在无核酸酶的H 2 O 2中制备DNA混合物, 。
      4. 向DNA混合物中加入无核酸酶H 2 O 2,最终体积为876μl。
      5. 加入1 ml 2x HBSS缓冲液。
      6. 慢慢地(滴加)向稀释的DNA中加入124μl2M CaCl 2。
      7. 加入CaCl 2后,在最低设置下轻轻涡旋5秒(注意:不要旋转下来!),并立即将制备的DNA溶液逐滴添加到细胞中在10厘米的盘子里
      8. 旋转平板以平均分配溶液。
      9. 在37°C培养3-4小时。
      10. 去除上清液
      11. 加入1毫升甘油休克溶液,冲击1分钟。
        注意:加入甘油导致渗透性休克,并被认为会导致DNA从沉淀物的释放(Grosjean等,2006)。
      12. 吸出冲击溶液,在1ml DMEM培养基(补充10%FBS,1%L-谷氨酰胺和1%青霉素/链霉素)中洗涤细胞并再次吸出。
      13. 加入7ml DMEM(补充10%FBS,1%L-谷氨酰胺和1%青霉素/链霉素)培养基。
      14. 转染后48 h,用10ml注射器收集上清液,并通过无菌0.45μmPVDF膜过滤器过滤
      15. 根据制造商的说明书,使用Lenti-X TM p24快速滴定试剂盒对p24含量的滴度病毒。
      16. 过滤的病毒上清液可以新鲜使用或储存在-80°C
  2. 具有含有病毒颗粒的shRNA的A2和A72 J-Lat细胞的旋转感染 注意:A2和A72细胞被旋转感染感染。
    1. A2和A72 J-Lat悬浮细胞培养
      1. 将A2和A72细胞在40ml培养基中的75cm 2组织培养瓶中的RPMI培养基(补充有10%FBS,1%L-谷氨酰胺和1%青霉素/链霉素)中培养。将烧瓶水平放置以增加细胞生长的表面积。
      2. 细胞每1-2天分裂一次,并应保持浓度为2×10 5〜15×10 -6 / ml。
      3. 转染前一天,将A2或A72细胞分裂至5×10 5 / ml
    2. 感染A2和A72 J-Lat细胞
      1. 用新的RPMI培养基(补充10%FBS,1%L-谷氨酰胺和1%青霉素/链霉素)计数细胞并调节细胞数。将A2或A72细胞中加入1×10 6个/ ml以上的微量离心管。
      2. 以300×g离心细胞悬液3分钟(1,800rpm)。彻底取出上清液。
      3. 在1.7ml微量离心管中加入500μlshRNA病毒(每10μl6μg细胞1ng p24),5μl1M HEPES和0.5μl聚凝胺(4μg/ ml)。
      4. 在32℃下以1,341 x g(2,400rpm)离心(台式离心机,Beckman)在32℃离心1.5小时。
      5. 彻底清除上清液。
      6. 在6孔组织培养板中将细胞重悬于3ml RPMI培养基(补充有10%FBS,1%L-谷氨酰胺和1%青霉素/链霉素)中。
      7. 在37℃的CO 2培养箱中培养一天。
      8. 如果shRNA病毒颗粒含有嘌呤霉素抗性基因,则选择含2μg/ ml嘌呤霉素RPMI培养基的细胞。使用嘌呤霉素的RPMI培养基(补充有10%FBS,1%L-谷氨酰胺和1%青霉素/链霉素)每两天更换一次。取决于细胞生长的速度以及其它实验(例如,染色质免疫沉淀,RNA分离等)所需要的细胞可以通过每2天加入培养基而生长,并移至较大的培养瓶或分裂培养它们在6孔板。
      9. 在CO 2培养箱中37℃培养4-10天
  3. 通过流式细胞术分析HIV-1 LTR转录激活(请参见生物学协议:Boehm and Ott,2017)
    1. 计数单元格并用新介质调整单元格编号。将约2×10 5个/孔的A2或A72细胞铺在96孔组织培养板中的195μlRPMI培养基(补充有10%FBS,1%L-谷氨酰胺和1%青霉素/链霉素)。每个条件执行三次重复。
    2. 用0.016-10ng / mlTNFα(作为用于最大活化的共同刺激或检查协同作用),PBS或H 2 O对照(取决于用作TNFα的稀释剂)来治疗细胞),或未经处理。将TNFα和对照物以5μl的量移液以达到200μl/孔的总体积。通过上下移动将细胞和TNFα混合至少3次
    3. 在CO 2培养箱中在37℃培养18-48小时。
    4. 从培养箱取板,并在室温下以1341 x g(2,400 rpm)(台式离心机,Beckman)离心2分钟。
    5. 然后直接进行流式细胞分析。细胞不必被洗涤或固定。
      注意:
      1. 对于流式细胞术,使用MACSQuant VYB FACS分析仪来运行96孔板。然而,其他流式细胞仪如Calibur或LSRII也适用于这些实验。
      2. 如果通过前向散射(FSC)与侧向散射(SSC)的细胞活力的分析不足以准确评估药物研究中的药物毒性,则是各种染料/污渍之一;例如7AAD,碘化丙啶或其中一种Zombie活性染料可以按照本协议流程细胞分析中的制造商说明使用。
    6. 使用SpectraMax MiniMax 300成像细胞仪,根据制造商的说明书,使用ApoTox-Glo Triplex Assay(Promega)测量活力,细胞毒性和细胞凋亡。

  4. 通过SDS-PAGE和Western blot确定shRNA敲低效率 SDS-PAGE和Western印迹方案在其他地方描述(Renart等人,1979; Towbin等人,1979)。
    1. 在1.7ml微量离心管中的100-500μlRIPA缓冲液中的Lyse 1×10 6细胞。
    2. 根据制造商的说明书,使用SpectraMax MiniMax 300成像细胞仪测量DC TM蛋白质测定蛋白质浓度(Bio-Rad Laboratories)。
    3. 在一条泳道中加载PageRuler蛋白梯子,另一条泳道在6-15%SDS凝胶上从细胞裂解液中等量的蛋白质(取决于蛋白质的大小),并在200V下电泳1 h分离蛋白质。每个蛋白质的kDa的预期大小和丙烯酰胺的适当百分比是:BRD2,110kDa,8%; BRD4,171kDa,6.5%;细胞周期蛋白T1,87 kDa,8%; α-微管蛋白,50kDa,10%。由于蛋白质的kDa是如此不同,所以可以通过与α-微管蛋白抗体孵育来切割膜以检查相等的载量。
    4. 使用Bio-Rad Mini-PROTEAN电泳系统在100 mA下将分离的蛋白质转移到硝酸纤维素膜上1-3小时或在35 mA下过夜(对于大于120 kDa的蛋白质)。 />
    5. 阻止在含有5%脱脂奶粉(封闭缓冲液)的TBS-T中的转移蛋白质在室温下摇摆。
    6. 在TBS-T缓冲液中,在摇床上4℃下,用一级抗体(BRD2 1:100; BRD41μg/ ml;细胞周期蛋白T1:1,000;α-微管蛋白1:2,000)孵育该膜。 >
    7. 每次用TBS-T缓冲液洗涤膜约3分钟约5分钟,并在室温下在摇床上与在TBS-T缓冲液中以1:10,000稀释的HRP缀合的二抗体孵育1小时。
    8. 每次用TBS-T缓冲液洗涤膜三次约5分钟,并在Lumi-light Western印迹底物中孵育30秒。
    9. 在化学发光膜上进行显影,将膜暴露10秒钟至30分钟。取决于抗体是否已被重复使用,并且多次被重复使用,信号将显得更弱,但更清洁(较少的背景带)。

数据分析

通过流式细胞术分析HIV-1 LTR转录激活(图1)

图1. BRD2,BRD4,细胞周期蛋白T1和α-微管蛋白的蛋白质印迹A.用针对BRD2,BRD4或非靶向加扰对照的含有shRNA构建体的病毒感染A72细胞。在感染后4天,收获细胞并在Ripa缓冲液中裂解。通过用BRD2和BRD4抗体或对照α-微管蛋白的免疫印迹证实BRD2和BRD4蛋白水平的敲低。 B.用靶向细胞周期蛋白T1或非靶向对照的含有shRNA构建体的病毒感染A72细胞。细胞周期蛋白T1蛋白水平的敲低通过用细胞周期蛋白T1或对照α-微管蛋白抗体进行免疫印迹来显示。

  1. 首先,在活的J-Lat细胞上设置门。通过正向(FSC区域)和侧向散射(SSC-Area)分析(图2A)监测细胞活力。
  2. 单刀闸(FSC-Height vs. FSC-Area)(图2B)
  3. 在SSC-Area和GFP / FITC-Area上设置门,以识别GFP + 单元格的数量(图2C)。
  4. 每个样品通常一式三份分析,实验独立重复三次。通过计算(GFP + 细胞实验1 + GFP + 细胞实验2 + GFP + 细胞实验3)/ 3来平均三个重复。还计算误差条的标准偏差(STDEV)。


    图2.通过流式细胞术分析HIV-1 LTR转录激活分析A2或A72 J-Lat细胞的门控策略:A.基于大小(FSC区域)对活J-Lat细胞门控)和粒度(SSC-Area); B.单闸门(FSC-Height vs. FSC-Area); C.Gate on GFP + J-Lat细胞(SSC-Area vs.GFP-FITC-Area)。

食谱

  1. 25毫克氯喹
    25毫克氯喹在PBS中
  2. 2x HBSS
    50 mM HEPES
    10 mM KCl
    12mM葡萄糖
    280 mM NaCl
    1.5mM Na 2 HPO 4
    调节pH到7.1
    注意:pH值至关重要!
  3. 2 M CaCl 2
    不含核酸酶的H 2 O 2中的2M CaCl 2
  4. 甘油冲击液
    150 nM NaCl
    20mM HEPES,pH 7.0
    15%甘油
  5. 聚凝胺溶液
    4毫克/毫升无核酸酶H 2 O O
  6. RIPA缓冲区
    50mM Tris缓冲液,pH 7.4 1%NP-40
    0.5%脱氧胆酸钠
    0.1%SDS
    150 mM NaCl
    2 mM EDTA
    50毫克氟化钠
  7. 蛋白质印迹运行缓冲液
    25 mM Trizma ® base
    190mM甘氨酸
    0.1%SDS
  8. 蛋白质印迹转移缓冲液(pH 8.3)
    25 mM Trizma ® base
    190mM甘氨酸
    20%甲醇
  9. TBS-T缓冲区
    19 mM Trizma ® base
    2.7 mM氯化钾
    137 mM氯化钠
    1 ml吐温® 20

致谢

我们感谢Marielle Cavrois和流式细胞术核心为流式细胞术提供的服务。加利福尼亚大学旧金山Gladstone病毒学研究所的帮助下,这本出版物成为可能。艾滋病研究免疫中心(CFAR),NIH资助计划(P30 AI027763)。这项研究得到了amfAR艾滋病毒治疗研究所的一部分的支持,该研究所获得了来自amfAR拨款109301的资助。此外,我们非常感谢加州艾滋病研究项目(F13-GI-316)获得DB支持,与JT Pharma的行业赞助合作,启用了shRNA筛选MO,并向CARE协作机构(U19 AI096113)和NIH(RO1 AI083139和RO1 DA043142)向MO提供支持该协议从以前的工作改编:“BET溴结构域靶向化合物通过Tat独立机制从延迟重新激活HIV”(Boehm等人,2013)

参考

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Boehm, D. and Ott, M. (2017). Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines. Bio-protocol 7(11): e2314. DOI: 10.21769/BioProtoc.2314.
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