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Cell-based Assays to Monitor AID Activity
基于细胞的监测AID活性的方法   

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Jia Li
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Abstract

The enzyme Activation induced deaminase (AID) underpins antibody affinity maturation and isotype switching through its mutagenic activity of deaminating deoxycytidine to deoxyuridine in DNA. Subsequent processing of the deoxyuridine initiates the processes of somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. Structure-function analysis of AID requires sensitive and biologically relevant methods to measure its various activities. Here we describe simple but effective methods to measure 1) the ability of AID to mutate the Escherichia coli genome, which provides an indication of its catalytic activity; 2) the capacity of AID to perform SHM by complementing a derivative of the DT40 chicken B cell line; 3) the ability of AID to perform CSR by complementing AID-deficient primary mouse B cells. The combination of the three methods, accompanied by the necessary analysis of AID subcellular localization and protein expression levels and stability, as controls, allows detailed structure-function interrogation of AID.

Keywords: Activation induced deaminase(活化诱导脱氨酶), Mutation(突变), Class switch recombination(类别转换重组), Escherichia coli(大肠杆菌), B-lymphocytes(B淋巴细胞)


Part I. Measuring mutagenic activity of AID in E. coli

The following procedure has been adapted from Petersen-Mahrt et al. (2002). It provides a measurement of the capacity of AID to mutate the Escherichia coli genome by selecting for those mutations in the rpoB gene that confer resistance to Rifampicin. It can be used as a proxy for the catalytic activity of the enzyme, although it involves deamination of a transcribed gene, which might influence the results. When comparing different AID variants or mutants, it provides a measurement of their mutagenic activity compared to the wt enzyme, which often but not always correlates with the enzymatic activity of the corresponding recombinant enzymes measured on DNA oligonucleotide substrates.

Materials and Reagents

  1. Sterile multi-channel basin
  2. Competent Δung E coli. We use the BW310 strain (Duncan, 1985) (a gift from Dr. Bernard Weiss Department of Pathology, Emory University School of Medicine, Atlanta)
  3. Plasmids containing AID or AID variants cloned in an inducible prokaryotic expression vector. We use IPTG-inducible pTrcHis vectors (Life Technologies, catalog number: V360-20 )
    Note: Currently, it is “Thermo Fisher Scientific, Invitrogen™, catalog number: V360-20”.
  4. Tryptone (BioShop Canada, catalog number: TRP402 )
  5. Yeast extract (BioShop Canada, catalog number: YEX401 )
  6. Sodium Chloride (NaCl) (BioShop Canada, catalog number: SOD002 )
  7. Agar (bacteriological grade) (BioShop Canada, catalog number: AGR001 )
  8. Na2HPO4.7H2O (BioShop Canada, catalog number: SMP400 )
  9. Potassium Phosphate Monobasic (KH2PO4) (BioShop Canadap, catalog number: PPM666 )
  10. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A0171 )
  11. Ampicillin (stock 100 mg/ml in ddH2O) (BioShop Canada, catalog number: AMP201 )
  12. Carbenicillin (100 mg/ml in ddH2O) (BioShop Canada, catalog number: CAR666 )
  13. Rifampicin (stock 100 mg/ml in DMSO) (Sigma-Aldrich, catalog number: R3501 )
  14. IPTG (stock 1 M in ddH2O) (BioShop Canada, catalog number: IPT001 )
  15. Transparent sterile 96-well plates, flat bottom (Corning, catalog number: 3595 )
  16. LB media (see Recipes)
  17. LB agar (see Recipes)
  18. 2x TY media (see Recipes)
  19. 5x M9 media (see Recipes)

Equipment

  1. Bunsen burner
  2. Sterile glass beads (for bacteria plating) (Genlantis, EZ-spread, catalog number: C400050 )
  3. 37 °C incubator (for bacterial plates)
  4. 37 °C incubator with shaker (for bacterial cultures)
  5. 42 °C water bath
  6. Hand counter
  7. Multichannel pipettor

Procedure

Prepare as many 10 cm LB agar plates supplemented with rifampicin (100 μg/ml) + ampicillin (100 μg/ml) as samples will be tested (selection plates). Prepare also the necessary number of LB agar plates supplemented with ampicillin (100 μg/ml) for the initial transformation and for viability plates in the experiment (see below). For example, to compare 3 constructs prepare 3 plates supplemented with ampicillin for bacterial transformation plus 3 x 5 = 15 plates supplemented with ampicillin for the assay (for a total of 18 ampicillin plates), as well as 15 plates supplemented with rifampicin + ampicillin.

  1. Day 1: Transformations
    1. For each construct, mix 50 ng DNA with 50 μl competent BW310 in a sterile 1.5 ml Eppendorf tube.
    2. Incubate tube on ice for 30 min.
    3. Heat shock for 45 sec at 42 °C in a water bath.
    4. Return tube to ice for 5 min.
    5. Recover bacteria by transferring the whole volume of bacteria to a 1.5 ml Eppendorf tube containing 950 μl 2x TY media and place in shaking incubator at 230 rpm for 45 min at 37 °C.
    6. Plate 40 μl of each bacterial culture from point 5 onto LB-ampicillin plates and incubate at 37 °C overnight.

  2. Day 2: Picking colonies and inducing AID expression
    1. Pick at least 5 isolated colonies from each transformation plate (i.e. construct) into a 15 ml tube containing 1.5 ml LB media supplemented with 100 μg/ml carbenicillin and 1 mM IPTG.
    2. Grow overnight at 37 °C.

  3. Day 3: Plating
    1. Selection plates
      1. Plate 50 μl of each induced culture in 10 cm Petri dishes with LB rifampicin + ampicillin.
      2. Incubate ~48 h at 37 °C until colonies are clearly visible.
        Note: That colony sizes can be heterogeneous, as different mutations will allow different growth rates in rifampicin. In addition, hypo- or hyperactive AID variants may require ad hoc plating (i.e. larger volumes or diluting the sample, respectively) to obtain a number of colonies that can be accurately counted. Do not plate volumes smaller than 50 μl to prevent too rapid absorption and clustering of the colonies.
    2. Viability plates
      1. Make 10-fold serial dilutions of the induced cultures in 1x M9 media using a sterile 96-well plate and a multichannel pipettor for convenience. Dilute up to 10-6.
      2. Plate 50 μl of the 10-6 in 10 cm petri dish LB ampicillin.
        Note: Depending on the conditions of growth, induction and expression, other dilutions (10-4 or 10-5) may be necessary to obtain a number of colonies that can be easily and accurately counted (Figure 1).
      3. Incubate overnight at 37 °C.

  4. Day 4: Count LB ampicillin plates

  5. Day 5: Count LB rifampicin plates
    Note: Count all colonies. In the case of rifampicin plates count colonies of all sizes. If there are too many colonies, (i.e., small and close together) 1/2-1/4 of the plate could be counted to estimate the total number of colonies without affecting the accuracy of the measurement, as long as isolated colonies can be distinguished. If colonies are confluent repeat the experiment, do not replate cultures that were kept overnight since viability diminishes significantly. If there are too few or no colonies the measurement may not be accurate; repeat and adjust the volume or dilution to plate accordingly.


    Figure 1. Example of LB ampicillin + rifampicin plates incubated at 37 °C for two days with bacteria transformed with pTrc-His (Ctrl) and pTrc-His-AID (AID)

  6. Calculating rpoB mutation frequency
    1. For each culture calculate the number (N) of Colony Forming Unit (CFU) per ml using the formula
      N=(n x d x Vo)/Vp
      n: Number of colonies counted in the plate; d: Dilution factor; Vo: Original culture volume (μl); Vp: Plated volume (μl)
      The LB ampicillin plate will provide the viable CFU/ml (NV) and the LB rifampicin plate will provide the mutant CFU/ml (NM)
    2. Calculate the number of Rifampicin resistant (RIFR) mutants per /109 CFU as:

      Calculate the median RifR/109 of all the values obtained for each construct and evaluate significance using the appropriate statistical method depending on the number of samples compared. At least 3 independent experiments with 5 colonies per construct are necessary for accurate comparisons of relative frequencies between AID variants.

Recipes

Note: Media must be autoclaved after preparation and kept sterile.

  1. LB media (for 1 L in ddH2O)        
    10 g tryptone
    5 g yeast extract
    5 g NaCl
  2. LB agar (for 1 L in LB media)        
    LB media
    15 g agar
  3. 2x TY media (for 1 L in ddH2O)    
    16 g tryptone
    10 g yeast extract
    5 g NaCl
  4. 5x M9 media (for 1 L in ddH2O)        
    64 g Na2HPO4.7H2O
    15 g KH2PO4
    2.5 g NaCl
    5 g NH4Cl

Part II. Measuring somatic hypermutation activity of AID in DT40 cells
DT40 cells are a chicken B cell lymphoma line that constitutively expresses IgM at the plasma membrane, as well as AID (Arakawa et al., 2002). Chickens diversify their Ig genes by gene conversion, a homologous recombination-based mechanism. However, the DT40-derived cell line used here (DT40 ΔΨV IgM+) has been engineered to eliminate the V pseudogenes that act as homology donors for gene conversion, causing that AID deaminations accumulate as mutations, mostly at C:G pairs with a bias towards transversion mutations (Arakawa et al., 2004). The protocol below takes advantage of the fact that a fraction of AID-induced mutations at the Ig loci will disrupt IgM expression. This can be quantified by flow cytometry as IgM-loss cells, and is a convenient proxy to monitor SHM frequency. When starting from a DT40 ΔΨV IgM+ population, the proportion of IgM-loss cells is a function of the time the cells spent in culture as well as of the SHM frequency, which depends directly on AID activity. A DT40 ΔΨV IgM+ line in which AID expression has also been eliminated by knock out (KO) can be complemented with expression vectors encoding AID or AID mutants, either tagged with GFP or linked to GFP expression by an internal ribosomal entry site, in order to compare their relative ability for SHM. GFP expression is used to monitor infection efficiency and to evaluate lgM-loss on infected cells only.

Materials and Reagents

  1. 6-well plate, sterile and tissue-treated (Corning, catalog number: 3516 )
  2. 24-well plate, sterile and tissue-treated (Corning, catalog number: 3526 )
  3. 96-well plate, round-bottom, sterile and tissue-treated (Corning, catalog number: 3799 )
  4. 15 ml sterile conical tubes
  5. 1.5 ml Eppendorf tubes
  6. 5 ml round-bottom tubes (facs tube)
  7. HEK 293T cells (ATCC, catalog number: CRL-3216 )
  8. DT40 AID KO ΔΨV IgM+ cells (these are non adherent cells) (Arakawa et al., 2004) (a gift from Dr. J-M Buerstedde, National University of Ireland, Galway)
  9. DMEM culture media (Wisent, catalog number: 319-005-CL )
  10. RPMI 1640 culture media (Wisent, catalog number: 350-000-CL )
  11. FBS (Wisent, catalog number: 080150 )
  12. Chicken serum (Life Technologies, Gibco®, catalog number: 16110-082 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 16110-082 ”.
  13. Penicillin-Streptomycin (Wisent, catalog number: 450-201-EL )
  14. β-Mercaptoethanol (BioShop Canada, catalog number: MER002 )
  15. TransIT-LT1 reagent (Mirus Bio LLC, catalog number MIR 2305 )
  16. Polybrene (8 mg/ml in water) (Sigma-Aldrich, catalog number: H9268 )
  17. HEPES 1 M (Wisent, catalog number: 330-050-EL )
  18. Mouse Anti-Chicken IgM-PE (SouthernBiotech, catalog number: 8310-09 )
  19. PBS (1x) (BioShop Canada, catalog number: PBS404 )
  20. BSA (BioShop Canada, catalog number: ALB001 )
  21. Expression vector encoding the VSV G envelop protein (pVSVG)
  22. Expression vector encoding for MLV polyprotein and reverse transcriptase (pGagPol)
  23. Retroviral expression vector containing AID variants and selection or reporter marker (we use pMXs-ires-GFP)
  24. Direct PCR Lysis reagent (VIAGEN BIOTECH, catalog number: 301-C )
  25. Proteinase K (stock 10 mg/ml in water at -20 °C) (Bio Basic Canada Inc., catalog number: PB0451 )
  26. KOD (Merck Millipore Corporation, Novagen, catalog number: 71086-3 ) or Pfu Turbo (Agilent, catalog number: 600250-52 ) high fidelity DNA polymerase
  27. Any Taq DNA polymerase for A-tailing
  28. QiaexII gel purification Kit (QIAGEN, catalog number: 20021 )
  29. pGEMT easy (Promega Corporation, catalog number: A1360 )
  30. Complete DMEM media (see Recipes)
  31. Complete DT40 media (see Recipes)

Equipment

  1. 40 μm cell strainer
  2. Centrifuge
  3. 37 °C, 5% CO2 cell culture incubator
  4. Flow cytometer
  5. PCR machine
  6. Oven
  7. Vortex (Scientific industries, model: Vortex-genie 2 )

Procedure

  1. Day 1: Plating HEK 293T cells for retrovirus packaging
    Plate 0.5 x 106 cell/well in a 6-well plate in 2 ml complete DMEM media. At the moment of plating, cells should be 75-90% confluent.

  2. Day 2: HEK 293T transfection
    1. Prepare the following for each well of cells to be transfected:
    2. Mix the following in this order in a 1.5 ml Eppendorf tube by gentle tapping at room temperature:
      RPMI 1640 (without additives) 250 μl
      TransIT-LT1 reagent 7.5 μl
      Mix well and then add:
      pGagPol 0.625 μg
      pVSVG 0.625 μg
      Retroviral vector 1.25 μg
      Note: The indicated masses of the plasmids are for achieving a molar DNA ratio of 1 [pVSVG]: 1 [pGagPol]: 2 [Retroviral vector], adjust the masses according to your plasmid sizes.
    3. Let sit 20 min then add to the cells dropwise.
    4. Rock the plate gently and incubate at 37 °C.

  3. Day 3
    1. Carefully remove media from each well of transfected HEK 293T cells.
    2. Add 2 ml complete DT40 media supplemented with 10 mM HEPES to each well.

  4. Day 4: DT40 infection
    1. Transfer the media (viral supernatant) from each transfected HEK 293T cells well to a 15 ml conical tube. Centrifuge at 200 x g, 5 min at room temperature.
    2. In a 24-well plate, plate 1 x 106 DT40 cells/well in 500 μl DT40 media (Cells growing exponentially and harvested at a density of ~0.5-1 x 106 cells/ml should be used).
    3. Add 1.5 ml/well of centrifuged viral supernatant to the plated DT40 cells.
    4. Add Polybrene to 8 μg/ml and HEPES to 10 mM final concentrations to each well.
    5. Centrifuge the plate at 600 x g for 1.5 h at 32 °C.
    6. Place the plate for 4 h in the cell culture incubator (37 °C, 5% CO2).
    7. After 4 h, remove media from each well and add 2 ml DT40 media supplemented with 10 mM HEPES.
    8. GFP infection frequency can be checked by flow cytometry and sorted 24-48 h later. The expected efficiency is ~40-90%, depending on the construct.

  5. Sorting
    Note: Work in sterile conditions.
    1. For each sample, prepare 1 to 4 round-bottom 96-well plates containing 200 μl/well of filtered complete DT40 media.
      Note: The number of plates will depend on the number of cells sorted per well, for single cell deposition prepare 4 plates to account for cloning efficiency, for 5 cells/well two plates are enough, for >5 cells/well one plate will be enough to obtain sufficient populations for the fluctuation analysis.
    2. Transfer 107 infected cells into a sterile FACS tube.
    3. Centrifuge 200 x g, 5 min at 4 °C.
    4. Wash with 4 ml PBS and centrifuge 200 x g, 5 min at 4 °C.
    5. Add anti-chicken IgM-PE (1:200) in 200 μl PBS-1%BSA.
    6. Mix well by vortexing gently and incubate for 30 min on ice and in the dark.
    7. Wash cells with 4 ml PBS and centrifuge 200 x g, 5 min at 4 °C.
    8. Resuspend cells in 300 μl PBS and filter the sample through a 40 μm cell strainer.
    9. Sort 1-3,000 cells/well (GFP+, IgM-).
      Note: The number of cells sorted in each well will depend on the application. The fluctuation analysis for surface IgM-loss will show less variation when the initial populations are large. However, when subsequently analyzing V region sequences, all shared mutations should be removed to prevent biasing by clonal expansion of pre-existing mutations. Using 1 cell per well will yield single cell clones, which have larger variations in the fluctuation analysis but allow a more accurate analysis of SHM upon sequencing. A larger number of clones should be analyzed in this case to obtain accurate median values of IgM-loss.

  6. Identifying clones/populations
    1. Clones/populations will be visible at naked eye 3-5 d after sorting and should be transferred to a 24-well plate containing 1 ml of filtered complete DT40 media. Isolate 12-24 clones/populations for each transduced construct (see Note above).
    2. When cells get confluent (3-4 days after isolating the clones), add 1 ml DT40 media to each well.
    3. Dilute 1:2 by carefully resuspending the cells using a P1000 and swirling around. Remove 1 ml of cells, then add 1 ml of fresh DT40 media to each well, everyday, for 2-4 weeks.
      Note: Monitor cell density by looking at the cells every day. Certain conditions or AID variants may slow down proliferation. Dilution of the cells and the length of expansion should be adjusted accordingly. The frequency of IgM should increase with time; the total time of expansion will depend on the signal to noise ratio of each particular experiment.


  7. Flow cytometry for surface IgM
    1. Transfer 500 μl of cells from each well to a flow cytometry tube.
    2. Centrifuge 200 x g, 5 min at 4 °C.
    3. Wash with 4 ml PBS and centrifuge 200 x g, 5 min at 4 °C.
    4. Add anti-chicken IgM-PE (1:200) in 100 μl PBS-1% BSA.
    5. Mix well by vortexing and let sit for 30 min (on ice and in the dark).
    6. Wash with 4 ml PBS and centrifuge 200 x g, 5 min at 4 °C.
    7. Resuspend in 300 μl PBS.
    8. Analyze samples in a flow cytometer. Acquire 20,000 events. Always have a mixture of IgM+ and IgM- DT40 cells as controls to set the gating (Figure 2).
    9. Analyze IgM-loss % for each clone and calculate median values for each construct or condition. Analyze statistically.


    Figure 2. Example of DT40 AID KO ΔΨV IgM+ cells expressing control vector or pMXs-AID-ires-GFP expanded for 3 weeks and stained for IgM

  8. Variable region sequencing
    The following procedure is for amplifying and sequencing the IgVL region from each DT40 subpopulation. If no phenotypic selections are applied, many sequences will be unmutated. The number of mutations over the total number of bases sequenced will provide an accurate estimation of the mutation frequency. However, obtaining sufficient (>50) independent mutations to evaluate changes in the mutation patterns may require extensive sequencing. Calculating mutation rates will additionally require to measure the doubling time of the cells and record the total number of population doublings during the expansion of the cells. On the other hand, IgM-loss cells can be purified by FACS, which will select cells bearing more mutated sequences on average. The latter is better suited for analyzing mutation patterns but less accurate to calculate mutation frequency.

  9. DNA extraction
    1. Prepare DNA extraction buffer (485 µl Direct PCR + 15 µl 10 mg/ml Proteinase K).
      Note: Direct PCR needs to be warmed up and make sure any precipitates go back in solution before using.
    2. Spin 5 x 104 to 5 x 105 cells for 15 sec at max speed in the tabletop microcentrifuge.
    3. Carefully remove the supernatant.
    4. Resuspend cells gently in 50 μl DNA extraction buffer by pipetting up and down.
    5. Incubate for 5 h at 55 °C. Use an oven rather than a heat block to prevent condensation on the lid (this step can be done overnight).
    6. Incubate for 45 min at 85 °C to inactivate the Proteinase K.
    7. Store DNA at -20 °C or proceed to PCR.

  10. PCR reaction
    1. Use 1-5 μl DNA template from above as template.
    2. Primers:
      5’-gcggggccgtcactgattgccg -3’
      5’-ccccagcctgccgccaagtccaag -3’
    3. PCR has to be made with high fidelity DNA polymerase (such as KOD or Pfu turbo). Use all reagents and concentrations as suggested in the polymerase’s instructions.
    4. Perform PCR in 50 μl final volume.
      Note: Always include a no template control, using some of the left over DNA extraction buffer instead, which controls for possible reagents contamination.
    5. Annealing temperature: perform a touch down PCR of 8 cycles at 68-60 °C followed by 23 cycles at 60 °C for annealing temperatures. Extension time and temperature, as well as denaturing temperature, will depend on the DNA polymerase used. The expected PCR product is 520 base pairs.

  11. Cloning and sequencing
    Note: From here on keep away from the PCR hood to avoid contaminations with final product in future experiments.
    1. Run 50 µl of the PCR product in an agarose gel. A single band of 520 bp should be visible and no band should be visible in the water control.
    2. Gel purify following QIAEX II kit instructions.
    3. Elute the purified fragment in 20 µl of 10 mM Tris pH 8.
    4. Cloning in T-vector (alternatively, restriction sites could be added to the oligonucleotides or use your cloning method of choice).
      Poly-A tailing (high fidelity polymerase do not add A-overhangs):
      Purified PCR product from step K3
      17 µl
      10x DNA pol buffer with Mg2+(2.5 mM Mg2+ final)
      2 µl
      10 mM dATP (0.2 mM final)
      0.4 µl
      Taq DNA Polymerase
      1 µl
      Incubate for 20 min at 72 °C in PCR cycler.
    5. Immediately ligate into pGEM-T easy following the protocol provided by the manufacturer.
    6. Transform the ligation in E. coli DH5α competent bacteria.
    7. Perform minipreps to purify vectors containing cloned PCR products and sequence using Sp6 primer. The total number of sequences to analyze will depend on the average mutation load per sequence and the aim of the experiment.

Recipes

  1. Complete DMEM media
    DMEM
    FBS 10%
    Penicillin-Streptomycin 1%
  2. Complete DT40 media
    RPMI 1640
    FBS 10%
    FCS 1%
    β-Mercaptoethanol 100 μM
    Penicillin-Streptomycin 1%


Part III. Measuring AID class switch recombination activity

This method uses retroviral complementation of primary B cells obtained from the spleen of AID-deficient (Aicda-/-) mice, to assay the capacity of AID variants to produce CSR.

Materials and Reagents

  1. 6-well plate, sterile and tissue-treated (Corning, catalog number: 3516 )
  2. 24-well plate, sterile and tissue-treated (Corning, catalog number: 3526 )
  3. 12-well plate, sterile and tissue-treated (Corning, catalog number: 3513 )
  4. 50 ml sterile conical tubes
  5. 15 ml sterile conical tubes
  6. 1.5 ml eppendorf tubes (Eppendorf or similar)
  7. 10 ml flat syringe plunger
  8. 5 ml pipettes
  9. 5 ml round-bottom tubes (Falcon®, catalog number: 352008 )
  10. 70 μm cell strainer (Corning, catalog number: 352350 )
  11. 40 μm cell strainer
  12. PLAT-E ecotropic retrovirus packaging cells (Morita et al., 2000) (a gift from Dr. T Kitamura, University of Tokyo, Japan)
  13. Aicda-/- mice (Muramatsu et al., 2000) (a gift from Dr. Tasuku Honjo, Kyoto University, Japan)
  14. Retroviral vector encoding AID variants (such as pMX-ires-GFP or pMIG)
  15. RPMI 1640 culture media (Wisent, catalog number: 350-000-CL )
  16. FBS (Wisent, catalog number: 080150 )
  17. Penicillin-Streptomycin (Wisent, catalog number: 450-201-EL )
  18. β -Mercaptoethanol (BioShop Canada, catalog number: MER002 )
  19. TransIT-LT1 transfection reagent (Mirus Bio LLC, catalog number: MIR 2305 )
  20. PBS (1x) (BioShop Canada, catalog number: PBS404 )
  21. BSA (BioShop Canada, catalog number: ALB001 )
  22. EDTA (BioShop Canada, catalog number: EDT001 )
  23. β-Mercaptoethanol (BioShop Canada, catalog number: MER002.100 )
  24. Lympholyte-M (Cedarlane Labs, catalog number: CL5031 )
  25. MACS anti-CD43 microbeads (Miltenyi Biotec, catalog number: 130-049-801 )
  26. Anti-mouse CD180 (BD, Pharmingen, catalog number: 552128 )
  27. HEPES 1M (Wisent, catalog number: 330-050-EL )
  28. Polybrene (stock resuspended at 8 mg/ml in ddH2O) (Sigma-Aldrich, catalog number: H9268 )
  29. LPS (stock resuspended at 1 mg/ml in RPMI without additives stored at -20 °C) (Sigma-Aldrich, catalog number: L-4391 )
  30. Recombinant murine IL-4 (stock resuspended at 5 μg/ml, in PBS + BSA 0.1% stored at -20 °C) (PREPROTECH, catalog number: 214-14 )
  31. Recombinant human TGF-β1 (stock resuspended at 1 μg/ml in 4 mM HCl + 1 mg/ml BSA stored at -20 °C) (R&D Systems, catalog number: 240-B )
  32. FcR Blocking Reagent, mouse (Miltenyi Biotec, catalog number: 130-092-575 )
  33. Biotinylated Rat Anti-Mouse IgG1 (BD, Pharmingen, catalog number: 553441 )
  34. Biotinylated Rat Anti-Mouse IgG3 (BD, Pharmingen, catalog number: 553401 )
  35. Biotinylated Rat Anti-Mouse IgG2b (BD, Pharmingen, catalog number: 553393 )
  36. Anti-biotin–APC (Miltenyi Biotec, catalog number: 130-090-856 )
  37. Propidium Iodide (stock resuspended at 100 mg/ml in ddH2O, stored protected from light at 4 °C) (BioShop Canada, catalog number: PPI777 )
  38. Complete RPMI media (see Recipes)
  39. MACS column buffer (see Recipes)

Equipment

  1. Centrifuge
  2. 37 °C, 5% CO2 tissue culture incubator
  3. AutoMACS Pro Separator (Miltenyi Biotec, catalog number: 130-092-545 )
  4. Flow cytometer

Procedure

Note: All procedures are performed in a tissue culture hood to prevent contaminations.

  1. Day 1: Plating PLAT-E cells

    Transfection
    Plate 0.5 x 106 cell/well in a 6-well plate in 2 ml complete RPMI media.
    Note: At the moment of plating, cells should be 75-90% confluent. The quality of the cells will highly impinge on the transfection frequency and thereby on retroviral production for infection.

    B cell purification
    1. Sacrifice Aicda-/- mice and extract the spleen using sterile utensils and place into a well of a 6-well plate containing sterile PBS.
      Note: From here on work in the cell culture hood. A filmed example of the procedure to remove the spleen and extracting the lymphocytes by the method described in points 2-4 below has been previously published (Zaheen and Martin, 2010).
    2. Put the spleen on a 70 µm cell strainer placed on top of a 50 ml falcon. Add 1 ml complete RPMI media onto the spleen and gently press using the flat end of a 10 ml flat syringe plunger.
    3. Add complete RPMI media 1 ml at a time and continue squishing the spleen until all the cells have been extracted from the organ (should only be left with clear tissue on the strainer and lead to a final volume of 5 ml).
    4. Pipette up and down gently with a 5 ml pipette to produce a homogeneous suspension and transfer cells to a 15 ml conical tube.
    5. Introduce a sterile, long Pasteur pipette in the tube.
    6. Slowly pipette 3 ml of Lympholyte-M into the Pasteur pipette to underlay the cell suspension.
    7. Handle the tube gently to avoid disturbing the phases
    8. Centrifuge 1,250 x g for 20 min at RT-with no break.
      Note: Brake must be turned off in order to maintain the lymphocyte interphase


      Figure 3. Typical aspect of the lymphocyte-rich interphase between medium and lympholyte after centrifugation

    9. Collect the interphase using a 5 ml pipette, avoiding as much Lympholyte-M as possible. Tilt the 15 ml conical tube and place the tip of the pipette slightly on top of the interphase, touching the side of the canonical tube. Aspirate gently avoiding as much of the Lympholyte phase as possible (Figure 3).
    10. Resuspend the cells by pipetting up and down a few times in 15 ml PBS 1% BSA.
    11. Centrifuge 800 x g for 10 min at RT-break is ok now.
    12. Resuspend the cells in 15 ml PBS 1% BSA.
    13. Centrifuge 400 x g for 10 min at RT.
    14. Resuspend in 100 µl PBS 1% BSA.
    15. Add anti-CD43 microbeads. Use 20 µl per spleen.
      Note: Resting B cells are CD43-. Depleting the splenic cell suspension of CD43+ cells will eliminate most T and activated B cells (note that this protocol is very inefficient in extracting dendritic cells or macrophages). A typical purity of >90% naïve B cells is achieved.
    16. Incubate 45 min at 4 °C mixing gently every now and then.
    17. Add 15 ml of cold PBS 1% BSA.
    18. Centrifuge 400 x g for 10 min at 4 °C.
    19. Resuspend in 1 ml MACS column buffer and pass the cells through a 40 μm cell strainer to avoid cell clumps in the AutoMACS.
    20. Proceed to AutoMACS separator and use DEPLETE program with quick washes in between samples.
      Note: Remember this is a depletion of activated cells (i.e., you want the cells that flow through, not the retained ones).
    21. Collect the flow through cells from the AutoMACS and wash with cold PBS 1% BSA, then centrifuge 400 x g for 10 min at 4 °C.
    22. Resuspend in 1 ml complete RPMI media.
    23. Count the cells (do a 1/20 dilution in PBS and then 1/2 with trypan blue).
      Note: The quality of the depletion could be monitored by flow cytometry using anti-CD43 and anti-B220 markers. Typically, this procedure yields >90% pure resting B cells.
    24. Plate 106 B-cells per well in a 24-well plate in 1 ml complete RPMI media. Plate as many wells as constructs you want to test + one well as uninfected control.
    25. Add 1 ml of medium containing 0.5 μg/ml anti-CD180 (final concentrations of 0.5 x 106 B cells/ml and 0.25 μg/ml anti-CD180). Anti-CD180 antibody promotes B cell proliferation without inducing AID expression or CSR.
    26. Put cells in the tissue culture incubator.

  2. Day 3
    Observe the cells, B cells should start to proliferate and clump. Remove media from transfected PLAT-E cells and add 2.5 ml fresh RPMI media supplemented with 10 mM HEPES.

  3. Day 4: B cell infection
    1. Transfer the viral supernatant from transfected PLAT-E cells to a 15 ml conical tube. Centrifuge at 200 x g, 5 min at room temperature.
    2. Transfer viral supernatant to a new tube.
    3. Adjust the concentration of viral supernatant to 20 mM HEPES and 16 μg/ml Polybrene.
    4. Remove 1 ml media from the top of each well containing B cells avoiding disturbing the cells, and add 1 ml of supplemented viral supernatant for a final concentration of 8 μg/ml Polybrene and 10 mM HEPES.
    5. Centrifuge the 24-well plate at 515 x g for 1.5 h at 30 °C.
    6. Place the plate for 4 h in the tissue culture incubator at 37 °C.
    7. After 4 h remove most of the media from each well, and add 2 ml complete RPMI media containing appropriate mitogens and cytokines as follows.
      For IgG1 switching: 5 μg/ml LPS + 25 ng/ml IL-4 (for saturating conditions) or 5 μg/ml LPS + 5 ng/ml (for non-saturating conditions)//
      For IgG3 switching: 25 μg/ml LPS
      For IgG2b switching: 5 μg/ml LPS (+TGF-β1 next day, see below)

  4. Day 5: For IgG2b, add 1 ng/ml TGF-β1 (it is added 24 h after infection because it delays proliferation)

  5. Day 6: For IgG1 or IgG3, transfer cells to a 12-well plate and add 2 ml of fresh RPMI (no cytokines needed). For IgG2b leave cells alone.

  6. Day 7: Measure class switch recombination to IgG1 or IgG3 (Figure 4)


    Figure 4. Example of Aicda-/- B cells transduced with vector control or pMXs-AID-ires-GFP and stimulated as explained above

  7. Day 8: Measure class switch recombination to IgG2b if desired
    Measuring Class Switch Recombination:
    1. Resuspend the cells GENTLY and transfer 500 µl of cells into FACS tubes.
    2. Top up tube with PBS and centrifuge at 400 x g for 5 min at 4 °C.
    3. Block by incubating cells in 100 µl PBS 1% BSA containing 1:10 dilution of FcR block for 10 min at 4 °C.
    4. Add 100 µl PBS 1% BSA containing 1:100 dilution of anti-IgG1/anti-IgG3 or 1:50 dilution of anti-IgG2b.
    5. Incubate at 4 °C for 30 min.
    6. Top up the tube with cold PBS and centrifuge at 400 x g for 10 min at 4 °C.
    7. Resuspend cells in 100 µl of anti-biotin-APC at 1:50 dilution in PBS 1% BSA by gently vortexing.
    8. Incubate 10 min at 4 °C in the dark.
    9. Top up the tube with cold PBS and centrifuge 400 x g for 10 min at 4 °C.
    10. Resuspend the cells in 200 µl PBS.
    11. Bring to flow cytometer.
    12. Add 5 µl of 100 µg/ml propidium iodide per tube to exclude dead cells.
      Note: Infection efficiency can be affected by the AID variant used. With wild type AID in pMX-ires-GFP we typically obtain ~40-60% infection efficiency and ~20% switching to IgG1 among the GFP+ cells.

Recipes

  1. Complete RPMI media
    RPMI 1640
    10% FBS
    100 μM β-Mercaptoethanol
    1% Penicillin-Streptomycin
  2. MACS column buffer
    PBS
    0.5% BSA
    2 mM EDTA

Acknowledgments

The protocols described here were adapted from our publications (Methot et al., 2015; Zahn et al., 2014). Our studies are supported in part by the Canadian Institutes of Health Research, the Cancer Research Society Inc and a Canada Research Chair tier 2 to JMDN. LCL is supported in part by a Cole Foundation Fellowship and SPM is supported by a fellowship from Fonds de recherche Quebec-Santé (FRQ-S).

References

  1. Arakawa, H., Hauschild, J. and Buerstedde, J. M. (2002). Requirement of the activation-induced deaminase (AID) gene for immunoglobulin gene conversion. Science 295(5558): 1301-1306.
  2. Arakawa, H., Saribasak, H. and Buerstedde, J. M. (2004). Activation-induced cytidine deaminase initiates immunoglobulin gene conversion and hypermutation by a common intermediate. PLoS Biol 2(7): E179.
  3. Duncan, B. K. (1985). Isolation of insertion, deletion, and nonsense mutations of the uracil-DNA glycosylase (ung) gene of Escherichia coli K-12. J Bacteriol 164(2): 689-695.
  4. Methot, S. P., Litzler, L. C., Trajtenberg, F., Zahn, A., Robert, F., Pelletier, J., Buschiazzo, A., Magor, B. G. and Di Noia, J. M. (2015). Consecutive interactions with HSP90 and eEF1A underlie a functional maturation and storage pathway of AID in the cytoplasm. J Exp Med 212(4): 581-596.
  5. Morita, S., Kojima, T. and Kitamura, T. (2000). Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7(12): 1063-1066.
  6. Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y. and Honjo, T. (2000). Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102(5): 553-563.
  7. Petersen-Mahrt, S. K., Harris, R. S. and Neuberger, M. S. (2002). AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418(6893): 99-103.
  8. Zaheen, A. and Martin, A. (2010). Induction and assessment of class switch recombination in purified murine B cells. J Vis Exp(42).
  9. Zahn, A., Eranki, A. K., Patenaude, A. M., Methot, S. P., Fifield, H., Cortizas, E. M., Foster, P., Imai, K., Durandy, A., Larijani, M., Verdun, R. E. and Di Noia, J. M. (2014). Activation induced deaminase C-terminal domain links DNA breaks to end protection and repair during class switch recombination. Proc Natl Acad Sci U S A 111(11): E988-997.

简介

酶活化诱导的脱氨酶(AID)通过其将脱氧胞苷脱氨基到DNA中的脱氧尿苷的诱变活性来支持抗体亲和力成熟和同种型转换。脱氧尿苷的后续加工引发B细胞中体细胞超突变(SHM)和类型转换重组(CSR)的过程。 AID的结构功能分析需要灵敏和生物相关的方法来测量其各种活动。在这里我们描述简单但有效的方法来测量1)AID突变大肠杆菌基因组的能力,其提供其催化活性的指示; 2)AID通过补充DT40鸡B细胞系的衍生物来进行SHM的能力; 3)AID通过补充AID缺乏的原代小鼠B细胞来进行CSR的能力。三种方法的组合,伴随着AID亚细胞定位和蛋白质表达水平和稳定性的必要分析作为对照,允许AID的详细结构功能研究。

关键字:活化诱导脱氨酶, 突变, 类别转换重组, 大肠杆菌, B淋巴细胞

第I部分。测量AID在E中的诱变活性。大肠杆菌
以下程序改编自Petersen-Mahrt等人(2002)。它通过选择赋予利福平抗性的rpoB基因中的那些突变提供了AID突变大肠杆菌基因组的能力的测量。它可以用作酶的催化活性的代表,虽然它涉及转录的基因的脱氨作用,其可能影响结果。当比较不同的AID变体或突变体时,其提供与wt酶相比的诱变活性的测量,wt酶经常但不总是与在DNA寡核苷酸底物上测量的相应重组酶的酶活性相关。

材料和试剂

  1. 无菌多通道水槽
  2. 主管大肠杆菌。我们使用BW310菌株(Duncan,1985)(来自亚特兰大Emory大学医学院病理学博士Bernard Weiss博士的礼物)
  3. 含有AID或AID变体的质粒克隆在诱导型原核表达载体中。我们使用IPTG诱导的pTrcHis载体(Life technologies,目录号:V360-20)
    注意:目前,它是"Thermo Fisher Scientific,Invitrogen?,目录号:
  4. 胰蛋白胨(BioShop Canada,目录号:TRP402)
  5. 酵母提取物(BioShop Canada,目录号:YEX401)
  6. 氯化钠(NaCl)(BioShop Canada,目录号:SOD002)
  7. 琼脂(细菌学级)(BioShop Canada,目录号:AGR001)
  8. (BioShop Canada,目录号:SMP400)。
  9. 磷酸二氢钾(KH 2 PO 4)(BioShop Canadap,目录号:PPM666)
  10. 氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:A0171)
  11. 氨苄青霉素(ddH 2 O中储备100mg/ml)(BioShop Canada,目录号:AMP201)
  12. 羧苄青霉素(在ddH 2 O中为100mg/ml)(BioShop Canada,目录号:CAR666
  13. 利福平(DMSO中的100mg/ml储备液)(Sigma-Aldrich,目录号:R3501)
  14. IPTG(ddH 2 O中的储备液1M)(BioShop Canada,目录号:IPT001)
  15. 透明无菌96孔板,平底(Corning,目录号:3595)
  16. LB媒体(见配方)
  17. LB琼脂(见配方)
  18. 2x TY介质(参见配方)
  19. 5x M9介质(参见配方)

设备

  1. 本生灶
  2. 无菌玻璃珠(用于细菌电镀)(Genlantis,EZ-spread,目录号:C400050)
  3. 37℃培养箱(细菌板)
  4. 37°C带振荡器的培养箱(用于细菌培养)
  5. 42℃水浴
  6. 手动计数器
  7. 多通道移液器

程序

制备多个补充有利福平(100μg/ml)+氨苄青霉素(100μg/ml)的10cm LB琼脂平板作为样品进行测试(选择平板)。还准备必需数量的添加氨苄青霉素(100μg/ml)用于初始转化的LB琼脂平板和实验中的活力平板(见下文)。例如,为了比较3个构建体,制备用于细菌转化的补充有氨苄青霉素的3个平板,加上用于测定的氨苄青霉素(总共18个氨苄青霉素平板)的3×5 = 15个平板,以及补充有利福平+氨苄青霉素的15个平板。

  1. 第1天:变革
    1. 对于每个构建体,将50ng DNA与50μl感受态BW310在无菌的1.5ml Eppendorf管中混合
    2. 在冰上孵育30分钟。
    3. 在42℃水浴中热冲击45秒。
    4. 将试管放回冰中5分钟。
    5. 通过将整个体积的细菌转移到含有950μl2×TY培养基的1.5ml Eppendorf管中,并置于振荡培养箱中,在37℃下以230rpm持续45分钟,来回收细菌。
    6. 将40μl来自第5点的每种细菌培养物置于LB-氨苄青霉素板上,并在37℃下孵育过夜

  2. 第2天:挑选菌落并诱导AID表达
    1. 从每个转化平板(即构建体)中挑取至少5个分离的菌落到含有1.5ml补充有100μg/ml羧苄青霉素和1mM IPTG的LB培养基的15ml管中。
    2. 在37℃下生长过夜。

  3. 第3天:电镀
    1. 选择板
      1. 将50μl每个诱导培养物在具有LB利福平+氨苄青霉素的10cm培养皿中平板接种
      2. 在37℃下孵育?48小时,直到菌落清晰可见 注意,集落大小可以是异质的,因为不同的突变将允许利福平的不同生长速率。此外,低活性或活动过度的AID变体可能需要特异性平板(即更大的体积或分别稀释样品),以获得可以精确计数的多个集落。不要将体积小于50μl,以防止菌落的过快吸收和聚集。
    2. 活力板
      1. 为了方便,使用无菌96孔板和多通道移液器,在1×M9培养基中进行10倍连续稀释的诱导培养物。稀释到10 -6
      2. 在50cm培养皿LB氨苄青霉素中加入50μl的10 -6
        注意:取决于生长,诱导和表达的条件,可能需要其它稀释度(10μM-4μM或10μM-50μM)以获得许多菌落可以轻松准确地计数(图1)。
      3. 在37℃下孵育过夜

  4. 第4天:计数LB氨苄青霉素平板

  5. 第5天:计数LB利福平板
    注意:计算所有菌落。在利福平板的情况下,计数所有大小的菌落。如果有太多的菌落(即小的和靠近在一起),可以计数板的1/2-1/4以估计菌落的总数而不影响测量的精确度,只要可以区分分离的菌落。如果菌落是汇合的,重复实验,不复制保持过夜的培养物,因为存活力显着降低。如果菌落太少或没有菌落,测量可能不准确;重复并调整体积或稀释度,以相应地。


    图1.用pTrc-His(Ctrl)和pTrc-His-AID(AID)转化的细菌在37℃下温育两天的LB氨苄青霉素+利福平板的实施例

  6. 计算rpoB突变频率
    1. 对于每个培养物,使用公式计算每ml的菌落形成单位(CFU)的数量(N) N =(n×d×Vo)/Vp
      n:板中计数的菌落数; d:稀释因子; Vo:原始培养体积(μl); Vp:电镀体积(μl)
      LB氨苄青霉素平板将提供活的CFU/ml(N V),LB利福平平板将提供突变体CFU/ml(N )
    2. 计算每10个p CFU的利福平抗性(RIF R )突变体的数目为:

      计算对于每个构建体获得的所有值的中值R f R /10 9,并且使用适当的统计方法根据比较的样品数量评价显着性。为了精确比较AID变体之间的相对频率,至少需要3个独立实验,每个结构具有5个菌落

食谱

注意:培养基必须在制备后进行高压灭菌,并保持无菌。

  1. LB培养基(对于ddH 2 O中的1L)       
    10g胰蛋白胨
    5g酵母提取物
    5克NaCl
  2. LB琼脂(对于LB培养基中的1L)       
    LB媒体
    15克琼脂
  3. 2x TY介质(对于ddH 2 O中的1L)    
    16克胰蛋白酶
    10g酵母提取物
    5克NaCl
  4. 5x M9培养基(对于ddH 2 O中的1L)       
    64g Na 2 HPO 4 sub 。 7H O
    15克KH 2 PO 4 4/
    2.5克NaCl 5g NH 4 Cl

第二部分。测量AID在DT40细胞中的体细胞超突变活性
DT40细胞是在质膜上组成型表达IgM的鸡B细胞淋巴瘤细胞系,以及AID(Arakawa等人,2002)。鸡通过基因转化(基于同源重组的机制)使其Ig基因多样化。然而,这里使用的DT40衍生的细胞系(DT40ΔΨVIgM + )已经被工程化以消除作为基因转化的同源性供体的V假基因,导致AID脱氨作为突变累积,主要在C:G对具有针对颠换突变的偏向(Arakawa等人,2004)。以下方案利用了在Ig基因座处AID诱导的突变的一部分将破坏IgM表达的事实。这可以通过流式细胞术定量为IgM丢失细胞,并且是监测SHM频率的方便代理。当从DT40ΔΨVIgM + 群体开始时,IgM丢失细胞的比例是细胞在培养中花费的时间以及直接取决于AID活性的SHM频率的函数。其中AID表达也已通过敲除(KO)消除的DT40ΔΨVIgM + 品系可以用编码AID或AID突变体的表达载体来补充,所述突变体用GFP标记或通过内部核糖体进入位点,以便比较它们对SHM的相对能力。 GFP表达用于监测感染效率并仅评估感染细胞上的IgM丢失。

材料和试剂

  1. 6孔板,无菌和组织处理(Corning,目录号:3516)
  2. 24孔板,无菌和组织处理(Corning,目录号:3526)
  3. 96孔板,圆底,无菌和组织处理(Corning,目录号:3799)
  4. 15毫升无菌锥形管
  5. 1.5 ml Eppendorf管
  6. 5ml圆底管(facs管)
  7. HEK 293T细胞(ATCC,目录号:CRL-3216)
  8. DT40AIDKOΔΨVIgM +/- 细胞(这些是非贴壁细胞)(Arakawa等人,2004)(来自JM Buerstedde博士,爱尔兰国立大学戈尔韦)
  9. DMEM培养基(Wisent,目录号:319-005-CL)
  10. RPMI 1640培养基(Wisent,目录号:350-000-CL)
  11. FBS(Wisent,目录号:080150)
  12. 鸡血清(Life Technologies,Gibco ,目录号:16110-082)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:
  13. 青霉素 - 链霉素(Wisent,目录号:450-201-EL)
  14. β-巯基乙醇(BioShop Canada,目录号:MER002)
  15. TransIT-LT1试剂(Mirus Bio LLC,目录号MIR 2305)
  16. 聚凝胺(8mg/ml,在水中)(Sigma-Aldrich,目录号:H9268)
  17. HEPES 1M(Wisent,目录号:330-050-EL)
  18. 小鼠抗鸡IgM-PE(SouthernBiotech,目录号:8310-09)
  19. PBS(1x)(BioShop Canada,目录号:PBS404)
  20. BSA(BioShop Canada,目录号:ALB001)
  21. 编码VSV G包膜蛋白(pVSVG)的表达载体
  22. MLV多蛋白和逆转录酶(pGagPol)的表达载体编码
  23. 含有AID变体和选择或报道标记的逆转录病毒表达载体(我们使用pMXs-ires-GFP)
  24. 直接PCR裂解试剂(VIAGEN BIOTECH,目录号:301-C)
  25. 蛋白酶K(-20℃在水中10mg/ml储液)(Bio Basic Canada Inc.,目录号:PB0451??)
  26. KOD(Merck Millipore Corporation,Novagen,目录号:71086-3)或Pfu Turbo(Agilent,目录号:600250-52)高保真DNA聚合酶
  27. 任何用于拖尾的Taq DNA聚合酶
  28. QiaexII凝胶纯化试剂盒(QIAGEN,目录号:20021)
  29. pGEMT easy(Promega Corporation,目录号:A1360)
  30. 完成DMEM介质(参见配方)
  31. 完成DT40媒体(参见配方)

设备

  1. 40μm细胞过滤器
  2. 离心机
  3. 37℃,5%CO 2细胞培养箱中培养
  4. 流式细胞仪
  5. PCR机
  6. 烤箱
  7. Vortex(科学工业,型号:Vortex-genie 2)

程序

  1. 第1天:将HEK 293T细胞铺板用于逆转录病毒包装
    板在2ml完全DMEM培养基中的6孔板中的0.5×10 6个细胞/孔。在电镀的时刻,细胞应该是75-90%汇合
  2. 第2天:HEK 293T转染
    1. 对于要转染的细胞的每个孔准备以下内容:
    2. 通过在室温下轻轻敲打,将以下物质以下述顺序混合在1.5ml Eppendorf管中:
      RPMI 1640(无添加剂)250微升
      TransIT-LT1试剂7.5μl
      混合均匀,然后添加:
      pGagPol0.625μg
      pVSVG0.625μg
      逆转录病毒载体1.25μg
      注意:质粒的指示质量是为了实现1 [pVSVG]:1 [pGagPol]:2 [逆转录病毒载体]的摩尔DNA比,根据质粒大小调整质量。
    3. 让坐20分钟,然后滴加到细胞中。
    4. 轻轻摇动平板并在37℃下孵育。

  3. 第3天
    1. 小心地从转染的HEK 293T细胞的每个孔中除去培养基
    2. 向每个孔中加入2ml补充有10mM HEPES的完全DT40培养基

  4. 第4天:DT40感染
    1. 转移媒体(病毒上清)从每个转染HEK 293T细胞井15毫升锥形管。以200×g离心,室温下5分钟
    2. 在24孔板中,将1×10 6个DT40细胞/孔铺板在500μlDT40培养基中(细胞指数生长并以?0.5-1×10 6个细胞/应使用细胞/ml)。
    3. 将1.5ml /孔的离心的病毒上清液加入铺有镀层的DT40细胞中
    4. 向每个孔中加入聚凝胺至8μg/ml,HEPES至终浓度为10mM
    5. 将板在600×g下在32℃下离心1.5小时
    6. 将板在细胞培养孵育器(37℃,5%CO 2)中放置4小时。
    7. 4小时后,从每个孔中除去培养基并加入2ml补充有10mM HEPES的DT40培养基
    8. GFP感染频率可以通过流式细胞术检查,并在24-48小时后分选。根据结构,预期效率为?40-90%。

  5. 排序
    注意:在无菌条件下工作。
    1. 对于每个样品,制备1至4个圆底96孔板,含有200μl/孔的过滤的完全DT40培养基。
      注意:板数将取决于每孔分选的细胞数目,对于单细胞沉积,制备4个板以考虑克隆效率,对于5个细胞/孔,两个板足以用于> 5个细胞/孔一个板将足以获得足够的群体用于波动分析。
    2. 将10 感染的细胞转移到无菌FACS管中
    3. 在4℃下离心200分钟,5分钟。
    4. 用4ml PBS洗涤并在4℃下离心200×g,5分钟
    5. 在200μlPBS-1%BSA中加入抗鸡IgM-PE(1:200)
    6. 轻轻涡旋混匀,在冰上和黑暗中孵育30分钟
    7. 用4 ml PBS洗涤细胞,并在4°C离心200分钟 5分钟。
    8. 重悬细胞在300微升PBS和过滤样品通过一个40微米的细胞过滤器
    9. 以1-3,000个细胞/孔(GFP + ,IgM - )进行分类。
      注意:每个孔中排序的细胞数量将取决于应用。当初始群体较大时,表面IgM损失的波动分析将显示较小的变化。然而,当随后分析V区序列时,应该去除所有共有的突变以防止由预先存在的突变的克隆扩增引起的偏移。每孔使用1个细胞将产生单个细胞克隆,其在波动分析中具有较大的变化,但允许在测序时更准确地分析SHM。在这种情况下应分析更多的克隆以获得IgM损失的准确中值。

  6. 鉴定克隆/群体
    1. 克隆/群体在分选后3-5天肉眼可见,并应转移到含有1ml经过滤的完全DT40培养基的24孔板中。分离每个转导的构建体的12-24个克隆/群体(参见上面的注释)。
    2. 当细胞汇合时(分离克隆后3-4天),向每个孔中加入1ml DT40培养基。
    3. 稀释1:2仔细重悬细胞使用P1000和旋转。删除1毫升的细胞,然后每天加入1毫升新鲜DT40培养基,每天,2-4周。
      注意:通过每天观察细胞来监测细胞密度。某些条件或AID变体可减缓增殖。细胞的稀释和扩增长度应相应调整。 IgM的频率应随时间增加;扩展的总时间将取决于每个特定实验的信噪比。

  7. 表面IgM的流式细胞术
    1. 转移500微升的细胞从每个孔到流式细胞仪管
    2. 在4℃下离心200分钟,5分钟。
    3. 用4ml PBS洗涤并在4℃下离心200×g,5分钟
    4. 在100μlPBS-1%BSA中加入抗鸡IgM-PE(1:200)
    5. 通过涡旋混合充分,放置30分钟(在冰上和在黑暗中)
    6. 用4ml PBS洗涤并在4℃下离心200×g,5分钟
    7. 重悬于300μlPBS中
    8. 在流式细胞仪中分析样品。获得20,000个事件。始终使用IgM +和IgM-DT40细胞混合物作为对照,以设置门控(图2)。
    9. 分析每个克隆的IgM损失%,并计算每个构建体或条件的中值。统计分析。


    图2.表达对照载体或pMXs-AID-ires-GFP扩增3周并染色IgM 的DT40AIDKOΔΨVIgM + 细胞的实施例。
  8. 可变区序列
    以下程序用于扩增和测序来自每个DT40亚群的IgV L区。如果没有应用表型选择,许多序列将是未突变的。在测序的碱基总数上的突变数目将提供突变频率的精确估计。然而,获得足够的(> 50)独立突变以评价突变模式的变化可能需要大量测序。计算突变率将另外需要测量细胞的倍增时间并记录细胞扩增期间的群体倍增的总数。另一方面,IgM丢失的细胞可以通过FACS纯化,其将选择平均携带更多突变序列的细胞。后者更适合分析突变模式,但不太准确以计算突变频率
  9. DNA提取
    1. 准备DNA提取缓冲液(485微升直接PCR + 15微升10毫克/毫升蛋白酶K) 注意:直接PCR需要预热,并确保任何沉淀物在使用前都溶解在溶液中。
    2. 在台式微量离心机中以最大速度旋转5×10 4至5×10 5个细胞15秒。
    3. 小心地清除上清液。
    4. 通过上下滚动,在50  μ l DNA提取缓冲液中轻轻悬浮细胞。
    5. 在55°C孵育5小时。使用烤箱而不是加热块,以防止盖子上的结露(这一步可以在一夜之间完成)。
    6. 在85℃下孵育45分钟以灭活蛋白酶K.
    7. 将DNA储存在-20°C或进行PCR

  10. PCR反应
    1. 使用1-5μlDNA模板作为模板。
    2. 引文:
      5'-gcggggccgtcactgattgccg -3'
      5'-ccccagcctgccgccaagtccaag -3'
    3. PCR必须用高保真DNA聚合酶(如KOD或Pfu turbo)进行。使用聚合酶说明书中建议的所有试剂和浓度。
    4. 以50μl终体积进行PCR 注意:始终包括一个无模板控制,使用一些剩余的DNA提取缓冲液,控制可能的试剂污染。
    5. 退火温度:在68-60℃下进行8个循环的接触式PCR,接着在60℃下进行23个循环,用于退火温度。延伸时间和温度以及变性温度将取决于所使用的DNA聚合酶。预期的PCR产物为520个碱基对

  11. 克隆和测序
    注意:从这里开始,远离PCR罩,避免在未来的实验中与最终产品混淆。
    1. 在琼脂糖凝胶中运行50μl的PCR产物。一条520 bp的单条带应该是可见的,在水控制中应该看不到条带
    2. 凝胶根据QIAEX II试剂盒说明进行纯化。
    3. 将纯化的片段在20μl10mM Tris pH 8中洗脱
    4. 在T载体中克隆(或者,可以将限制性位点添加到寡核苷酸中或使用所选择的克隆方法) Poly-A尾(高保真聚合酶不添加A突出端):
      来自步骤K3的纯化的PCR产物
      17微升
      含有Mg 2+(2.5mM Mg 2+,最终)的10x DNA聚合缓冲液。 2微升
      10mM dATP(0.2mM终浓度) 0.4μl
      Taq DNA聚合酶 1微升
      在PCR循环仪中于72℃孵育20分钟
    5. 按照制造商提供的方案立即连接到pGEM-T easy中
    6. 在 E中转换连接。大肠杆菌 DH5α感受态细菌
    7. 使用Sp6引物进行微量制备以纯化含有克隆的PCR产物和序列的载体。要分析的序列的总数将取决于每个序列的平均突变负荷和实验的目的

食谱

  1. 完成DMEM媒体
    DMEM
    FBS 10%
    青霉素 - 链霉素1%
  2. 完成DT40媒体
    RPMI 1640
    FBS 10%
    FCS 1%
    β-巯基乙醇100μM
    青霉素 - 链霉素1%


第三部分。测量AID类开关重组活性
该方法使用从AID缺陷( Aicda )小鼠的脾获得的原代B细胞的逆转录病毒互补,的AID变体以产生CSR。

材料和试剂

  1. 6孔板,无菌和组织处理(Corning,目录号:3516)
  2. 24孔板,无菌和组织处理(Corning,目录号:3526)
  3. 12孔板,无菌和组织处理(Corning,目录号:3513)
  4. 50ml无菌锥形管
  5. 15毫升无菌锥形管
  6. 1.5ml eppendorf管(Eppendorf或类似)
  7. 10毫升平针筒柱塞
  8. 5 ml移液器
  9. 5ml圆底管(Falcon ,目录号:352008)
  10. 70μm细胞过滤器(Corning,目录号:352350)
  11. 40μm细胞过滤器
  12. PLAT-E亲性逆转录病毒包装细胞(Morita等人,2000)(来自日本东京大学T Kitamura博士的礼物)
  13. 小鼠(Muramatsu等人,2000)大学,日本)
  14. 逆转录病毒载体编码AID变体(例如pMX-ires-GFP或pMIG)
  15. RPMI 1640培养基(Wisent,目录号:350-000-CL)
  16. FBS(Wisent,目录号:080150)
  17. 青霉素 - 链霉素(Wisent,目录号:450-201-EL)
  18. β-巯基乙醇(BioShop Canada,目录号:MER002)
  19. TransIT-LT1转染试剂(Mirus Bio LLC,目录号MIR 2305)
  20. PBS(1x)(BioShop Canada,目录号:PBS404)
  21. BSA(BioShop Canada,目录号:ALB001)
  22. EDTA(BioShop Canada,目录号:EDT001)
  23. β-巯基乙醇(BioShop Canada,目录号:MER002.100)
  24. 淋巴细胞-M(Cedarlane Labs,目录号:CL5031)
  25. MACS抗CD43微珠(Miltenyi Biotec,目录号:130-049-801)
  26. 抗小鼠CD180(BD,Pharmingen,目录号:552128)
  27. HEPES 1M(Wisent,目录号:330-050-EL)
  28. 聚凝胺(以8mg/ml在ddH 2 O中重悬的原液)(Sigma-Aldrich,目录号:H9268)
  29. LPS(以1mg/ml悬浮于RPMI中,无添加剂储存于-20℃下)(Sigma-Aldrich,目录号:L-4391)
  30. 将重组鼠IL-4(储备液以5μg/ml悬浮,在PBS + BSA 0.1%中储存于-20℃)(PREPROTECH,目录号:214-14)
  31. 将重组人TGF-β1(储存在-20℃下,以1μg/ml悬浮于4mM HCl + 1mg/ml BSA中的储液)(R& D Systems,目录号:240-B)
  32. FcR阻断试剂,小鼠(Miltenyi Biotec,目录号:130-092-575)
  33. 生物素化的大鼠抗小鼠IgG1(BD,Pharmingen,目录号:553441)
  34. 生物素化的大鼠抗小鼠IgG3(BD,Pharmingen,目录号:553401)
  35. 生物素化的大鼠抗小鼠IgG2b(BD,Pharmingen,目录号:553393)
  36. 抗生物素-APC(Miltenyi Biotec,目录号:130-090-856)
  37. 碘化丙啶(在ddH 2 O中以100mg/ml重新悬浮的原液,在4℃下避光保存)(BioShop Canada,目录号:PPI777)
  38. 完成RPMI媒体(参见配方)
  39. MACS列缓冲区(请参阅配方)

设备

  1. 离心机
  2. 37℃,5%CO 2组织培养箱中培养
  3. AutoMACS Pro Separator(Miltenyi Biotec,目录号:130-092-545)
  4. 流式细胞仪

程序

所有程序在组织培养罩中进行以防止污染。

  1. 第1天:电镀PLAT-E电池

    转染
    板在2ml完全RPMI培养基中的6孔板中的0.5×10 6个细胞/孔。
    注意:在铺板的时刻,细胞应该是75-90%汇合。细胞的质量将高度影响转染频率,从而影响感染的逆转录病毒生产。

    B细胞纯化
    1. 牺牲Aicda -/- 小鼠,并使用无菌器具提取脾脏并置于含有无菌PBS的6孔板的孔中。
      注意:从这里在细胞培养罩工作。通过以下2-4点所述的方法除去脾脏和提取淋巴细胞的程序的拍摄实例先前已经公开(Zaheen和Martin,2010)。
    2. 把脾放在70毫米细胞过滤器放置在50毫升猎鹰的顶部。添加1毫升完整的RPMI介质到脾脏,轻轻按压使用10毫升平针筒柱塞的平端。
    3. 每次加入1ml完全RPMI培养基,继续压扁脾脏,直到所有细胞都从器官中提取出来(只有在过滤器上留下清晰的组织,最终体积为5ml)。
    4. 用5ml移液管轻轻地上下移动以产生均匀的悬浮液,并将细胞转移至15ml锥形管。
    5. 在管中引入无菌,长巴氏消毒吸管。
    6. 慢慢吸取3毫升Lympholyte-M到巴斯德吸管中,以覆盖细胞悬液
    7. 轻轻握住管子以避免扰动相位
    8. 在RT下离心1250×g,20分钟,没有断裂。
      注意:必须关闭制动器才能维持淋巴细胞间期。


      图3.离心后介质和淋巴细胞之间富含淋巴细胞的间期的典型方面

    9. 收集中间相使用5毫升移液管,尽可能避免Lympholyte-M尽可能。倾斜15毫升锥形管,并将吸管的尖端略微在相间的顶部,接触规范管的一侧。轻轻吸出尽可能避免Lympholyte相(图3)。
    10. 通过在15ml PBS 1%BSA中上下吹打??几次来重悬细胞
    11. 在RT-break下离心机800 x g 10分钟现在就可以了
    12. 将细胞重悬在15ml PBS 1%BSA中
    13. 在室温下离心400分钟,离心10分钟。
    14. 重悬于100μlPBS 1%BSA中
    15. 加入抗CD43微珠。每脾使用20μl。
      注意:休息的B细胞是CD43 - 。耗尽CD43 + 细胞的脾细胞悬浮液将消除大多数T和活化的B细胞(注意,这种方案在提取树突状细胞或巨噬细胞中非常低效)。实现了> 90%初始B细胞的典型纯度。
    16. 在4°C孵育45分钟,每时每刻轻轻混合
    17. 加入15ml冷PBS 1%BSA
    18. 在4℃下离心400分钟×10分钟。
    19. 重悬于1ml MACS柱缓冲液中,使细胞通过40μm细胞过滤器,以避免AutoMACS中的细胞团。
    20. 进入AutoMACS分离器,使用DEPLETE程序,在样品之间快速清洗 注意:请记住,这是一个耗尽的激活细胞(即你想要的细胞流过,而不是保留的细胞)。
    21. 收集通过来自AutoMACS的细胞的流,并用冷PBS 1%BSA洗涤,然后在4℃下离心400×10分钟。
    22. 重悬于1ml完全RPMI培养基中
    23. 计数细胞(在PBS中1/20稀释,然后用台盼蓝染色1/2) 注意:可以通过使用抗CD43和抗B220标记物的流式细胞术来监测消耗的质量。通常,该方法产生> 90%纯的休眠B细胞。
    24. 平板10 6个B细胞/孔在24孔板中的1ml完全RPMI培养基中。将许多孔作为你想要测试的结构,一个作为未感染的对照
    25. 加入1ml含有0.5μg/ml抗CD180(终浓度为0.5×10 6 B细胞/ml和0.25μg/ml抗CD180)的培养基。抗CD180抗体促进B细胞增殖而不诱导AID表达或CSR
    26. 将细胞放入组织培养箱中。

  2. 第3天
    观察细胞,B细胞应该开始增殖和聚集。从转染的PLAT-E细胞中除去培养基,并加入2.5ml新鲜的RPMI培养基,补充有10mM HEPES
  3. 第4天:B细胞感染
    1. 将转染的PLAT-E细胞的病毒上清转移到15ml锥形管中。以200×g离心,室温下5分钟
    2. 将病毒上清转移到新管中
    3. 将病毒上清液的浓度调节至20mM HEPES和16μg/ml聚凝胺
    4. 从含有B细胞的每个孔的顶部除去1ml培养基,避免扰乱细胞,并加入1ml补充的病毒上清液,终浓度为8μg/ml聚凝胺和10mM HEPES。
    5. 在30℃下,以515×g离心24孔板1.5小时。
    6. 将板在组织培养箱中37℃放置4小时
    7. 4小时后,从每个孔中除去大部分培养基,并如下加入2ml含有适当的有丝分裂原和细胞因子的完全RPMI培养基。
      对于IgG1切换:5μg/ml LPS + 25ng/ml IL-4(对于饱和条件)或5μg/ml LPS + 5ng/ml(对于非饱和条件)//
      对于IgG3切换:25μg/ml LPS
      对于IgG2b切换:5μg/ml LPS(+ TGF-β1第二天,见下文)

  4. 第5天:对于IgG2b,加入1ng/ml TGF-β1(其在感染后24小时加入,因为其延迟增殖)

  5. 第6天:对于IgG1或IgG3,将细胞转移至12孔板并加入2ml新鲜RPMI(不需要细胞因子)。对于IgG2b单独离开细胞
  6. 第7天:测量类别转换重组为IgG1或IgG3(图4


    图4. Aicda -/- B细胞转导的示例与载体对照或pMXs-AID-ires-GFP并如上所述刺激

  7. 第8天:如果需要,测量类别开关重组到IgG2b
    测量类开关重组:
    1. 重悬细胞GENTLY和转移500微升的细胞到FACS管
    2. 用PBS充满管并在4℃下以400×g离心5分钟。
    3. 通过在4℃下将细胞在含有1:10稀释的FcR区段的100μlPBS 1%BSA中孵育10分钟来阻断。
    4. 加入100μl含有1:100稀释的抗IgG1 /抗IgG3或1:50稀释的抗IgG2b的PBS 1%BSA。
    5. 在4℃孵育30分钟。
    6. 用冷PBS补充管并在4℃下以400×g离心10分钟。
    7. 通过轻轻涡旋将细胞以1:50稀释在PBS 1%BSA中的100μl抗生物素-APC重悬。
    8. 在暗处4°C孵育10分钟。
    9. 用冷PBS补充管并在4℃下离心400×10分钟10分钟。
    10. 将细胞重悬于200μlPBS中
    11. 带流式细胞仪。
    12. 每管加入5μl100μg/ml碘化丙啶,以排除死细胞。
      注意:感染效率可能受到所使用的AID变体的影响。使用pMX-ires-GFP中的野生型AID,我们通常在GFP + 细胞中获得?40-60%的感染效率和?20%的转换为IgG1。

食谱

  1. 完成RPMI媒体
    RPMI 1640
    10%FBS
    100μMβ-巯基乙醇 1%青霉素 - 链霉素
  2. MACS列缓冲区
    PBS
    0.5%BSA
    2mM EDTA

致谢

这里描述的方案改编自我们的出版物(Methot等人,2015; Zahn等人,2014)。我们的研究部分由加拿大卫生研究院,癌症研究协会公司和加拿大研究委员会第2级至JMDN支持。 LCL由Cole基金会奖学金部分支持,SPM由魁北克省魁北克省(FQS)的研究金支持。

参考文献

  1. Arakawa,H.,Hauschild,J。和Buerstedde,J.M。(2002)。 免疫球蛋白基因转换的激活诱导脱氨酶(AID)基因的需求。 em> Science 295(5558):1301-1306。
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引用:Litzler, L. C., Methot, S. P., Patenaude, A., Zahn, A. and Di Noia, J. M. (2016). Cell-based Assays to Monitor AID Activity. Bio-protocol 6(3): e1724. DOI: 10.21769/BioProtoc.1724.
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