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Efficient AAV-mediated Gene Targeting Using 2A-based Promoter-trap System
使用基于2A的启动子捕获系统的高效AAV介导的基因打靶

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Abstract

Adeno-associated virus (AAV)-based targeting vectors have 1-4-log higher gene targeting efficiencies compared with plasmid-based targeting vectors. The efficiency of AAV-mediated gene targeting is further increased by introducing a promoter-trap system into targeting vectors. In addition, we found that the use of ribosome-skipping 2A peptide rather than commonly used internal ribosome entry site (IRES) in the promoter-trap system results in significantly higher AAV-mediated gene targeting efficiencies (Karnan et al., 2016). In this protocol, we describe the procedures for AAV-mediated gene targeting exploiting 2A for promoter trapping, including the construction of a targeting vector based on the platform plasmid pAAV-2Aneo or pAAV-2Aneo v2, production of AAV particles, infection of cells with resulting AAV-based targeting vectors, and isolation and verification of gene-targeted cell clones.

Keywords: Adeno-associated virus(腺相关病毒), AAV(AAV), Targeting vector(打靶载体), Gene targeting(基因打靶), Promoter trap(启动子捕获), 2A(2A), Internal ribosome entry site(内部核糖体进入位点), IRES(IRES)

Background

The procedures for AAV-mediated gene targeting in general (corresponding to Sections B-G of this protocol) were previously described in other protocols (Kohli et al., 2004; Rago et al., 2007; Khan et al., 2011; Howes and Schofield, 2015). However, this protocol provides a detailed description of how to perform AAV-mediated gene targeting using a 2A-based promoter–trap system for the first time.

Materials and Reagents

  1. Pipette tips  
  2. 10-cm culture dish
  3. 1.5-ml or 2-ml cryovial
  4. 96-well plate  
  5. 15-ml (or 50-ml) conical tube
  6. 24-well plates
  7. 6-well plates or tissue culture dishes/flasks
  8. 0.22 µm filter
  9. 1.5-ml tubes
  10. Aluminum foil
  11. Disposable pipetting reservoir (AS ONE, catalog number: 2-7844-02 )
  12. E. coli DH5α competent cells (Takara Bio, catalog number: 9057 )
    Note: This product has not been discontinued.
  13. HEK293 or HEK293T cell line
  14. Cell line(s) for gene targeting
  15. pAAV-2Aneo (Addgene, catalog number: 80032 )
  16. pAAV-2Aneo v2 (Addgene, catalog number: 80033 )
  17. Agarose (NIPPON GENE, catalog number: 318-01195 )
  18. Pwo SuperYield DNA polymerase (Roche Diagnostics, catalog number: 04340850001 )
  19. PureLink® Quick Gel Extraction Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: K2100-25 )
  20. PureLink® PCR Purification Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: K3100-02 )
  21. Quick LigationTM Kit (New England BioLabs, catalog number: M2200S )
  22. Dry ice
  23. Restriction enzymes BspEI, MluI, or BsrGI (New England BioLabs, Ipswich, MA)
  24. Restriction enzyme buffers
  25. LB broth (NACALAI TESQUE, catalog number: 20068-75 )
  26. Mini PlusTM Plasmid DNA extraction system (Viogene, catalog number: GF2002 )
  27. BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4337455 )
  28. Alkaline phosphatase, calf intestinal (CIP) (New England BioLabs, catalog number: M0290L )
  29. Custom-made PCR primers (for the amplification of 5’ and 3’ homology arms)
  30. Custom-made sequencing primers (for the sequencing of 5’ and 3’ homology arms)
  31. Custom-made PCR primers (for the screening of gene-targeted clones)
  32. PureLink® HiPure Plasmid Maxiprep Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: K210007 )
  33. Dulbecco’s modified Eagle’s medium (D-MEM) (Wako Pure Chemical Industries, catalog number: 044-29765 )
  34. Fetal bovine serum (FBS) (NICHIREI, catalog number: 172012-500ml )
  35. Opti-MEM® I reduced-serum medium (Thermo Fisher Scientific, GibcoTM, catalog number: 31985-070 )
  36. pRC and pHelper plasmids in AAV Helper-free system (Agilent Technologies, catalog number: 240071 )
  37. TransIT®-293 transfection reagent (Mirus Bio, catalog number: MIR 2705 )
  38. Penicillin-streptomycin solution (x100) (Wako Pure Chemical Industries, catalog number: 168-23191 )
  39. Methanol
  40. Distilled water
  41. RNase-free DNase set (QIAGEN, catalog number: 79254 )
  42. NeoR-Rev #1: 5’-GGCATCAGAGCAGCCGATTG
  43. NeoR-Fwd #1: 5’-CATTCGACCACCAAGCGAAA
  44. NeoR-Rev #2: 5’-CTTGAGCCTGGCGAACAGTT
  45. KOD FX Neo (TOYOBO, catalog number: KFX-201 )
  46. Growth medium appropriate for the cell line(s) undergoing gene targeting
  47. 0.25% (w/v) trypsin solution with phenol red (Wako Pure Chemical Industries, catalog number: 201-18841 )
  48. PCR buffer
  49. dNTPs
  50. SYBR® Green I nucleic acid stain (Lonza, catalog number: 50512 )
  51. PureLink® Genomic DNA Mini Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: K182002 )
  52. Ampicillin sodium (Wako Pure Chemical Industries, catalog number: 012-23303 )
  53. Dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries, catalog number: 048-21985 )
  54. NaCl
  55. KCl
  56. Na2HPO4·12H2O
  57. KH2PO4
  58. Ampicillin working solution (see Recipes)
  59. SYBR Green I working solution (see Recipes)
  60. PBS(-) (see Recipes)

Equipment

  1. Humidified CO2 incubator
  2. VeritiTM thermal cycler (Thermo Fisher Scientific, model: Veriti Thermal Cycler )
  3. Mupid®-2plus submarine-type electrophoresis system (Takara Bio, model: Mupid-2plus System )
  4. NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific)
  5. StepOnePlusTM Real-Time PCR system (Thermo Fisher Scientific, Applied BiosystemTM, model: StepOnePlusTM Real-Time PCR system)
  6. Centrifuge for molecular biology experiments
  7. -20 °C freezer
  8. Tissue culture hood
  9. Phase-contrast microscope
  10. Swing bucket centrifuge for tissue culture
  11. -80 °C deep freezer
  12. Water bath
  13. 12-channel pipettor
  14. Two permanent markers of different colors

Procedure

  1. Construction of an AAV-based targeting vector
    1. Design the PCR primers used to amplify homology arms. To incorporate the amplified homology arms into pAAV-2Aneo (v2) via restriction enzyme digestion, select appropriate restriction enzyme sites from the lineup of the multiple cloning sites MCS-1 (between SacI and SpeI sites) and MCS-2 (between KpnI and XhoI sites) in pAAV-2Aneo (v2). The selected restriction enzyme sites should be absent within the homology arms to be amplified. Then, introduce the selected restriction enzyme sites at the 5’ end of PCR primers. In addition, append 3-6 nucleotides (any sequence; the length depends on the restriction enzyme sites selected) distal to the restriction enzyme sites, which allows the enzymes to cleave PCR products close to the ends. For tips in designing homology arms, refer to the Notes section (Notes 1-3).
    2. Amplify the 5’ and 3’ homology arms by PCR. Use a high-fidelity DNA polymerase; e.g., Pwo SuperYield DNA polymerase.
    3. Analyze the PCR products by agarose gel electrophoresis. Cut out a band of the expected size from the gel, and recover a DNA fragment using the PureLink Quick Gel Extraction Kit. If the PCR product appears as a clean single band and a primer dimer is not detected on the agarose gel, the PCR product may optionally be purified using the PureLink PCR Purification Kit instead of performing gel extraction.
    4. Cleave the PCR products with the selected restriction enzymes, and purify the reaction products using the PureLink PCR Purification Kit.
    5. Since pAAV-2Aneo and pAAV-2Aneo v2 have different ‘frame adjusters’, these vectors are processed in different fashions to adjust reading frames. Follow one of the three options below (steps A5a-c) to process the frame adjuster. In any of the three options, the restriction enzyme-digested DNA solution should be purified using the PureLink PCR Purification Kit or other methods before self-religation. Self-religation is performed using the Quick Ligation Kit.
      1. When pAAV-2Aneo is used and the promoter-trap module is to be incorporated into an intron upon gene targeting (Figure 1): Cleave pAAV-2Aneo with one of the three restriction enzymes (BspEI, MluI, or BsrGI), and religate the vector so that 2Aneo is translated in frame with the 5’ portion of the endogenous target gene (Figure 2).


        Figure 1. Schematic diagram of the AAV-mediated targeting of an intron. Thin dotted lines indicate homology between the endogenous genomic locus and the targeting vector. F1-R1 and F2-R2 indicate pairs of oligonucleotide primers used for screening PCR and confirmatory PCR. The bottom part shows a predicted mRNA expressed from the targeted genomic locus. Gray rectangles: exons; bold black lines: introns; blue hairpin structures: inverted terminal repeats; IVS: synthetic intron; FrAd: frame adjuster; NeoR: the neomycin phosphotransferase gene; pA: polyadenylation site.


        Figure 2. Processing of the frame adjuster in pAAV-2Aneo for targeting introns. An intact frame adjuster (top) and the ones processed by BspEI, MluI, and BsrGI digestion (bottom). Predicted partial polypeptides translated from the region surrounding the frame adjuster are shown under nucleotide sequences. SA: splice acceptor; IVS: synthetic intron; FrAd: frame adjuster.

      2. When the promoter-trap module is to be incorporated into an intron upon gene targeting similar to step A5a (Figure 1), but pAAV-2Aneo v2 is used instead of pAAV-2Aneo: Cleave pAAV-2Aneo v2 with either BspEI or BsrGI and religate the vector, or leave the vector unprocessed, so that 2Aneo is translated in frame with the 5’ portion of the endogenous target gene (Figure 3). Figure 4 schematically depicts the disruption of PIGA intron 5 performed in our previous study as an example of option A5b.


        Figure 3. Processing of the frame adjuster in pAAV-2Aneo v2 for targeting introns. An intact frame adjuster (top) and the ones processed by BspEI and BsrGI digestion (bottom). Predicted partial polypeptides translated from the region surrounding the frame adjuster are shown under nucleotide sequences. SA: splice acceptor; IVS: synthetic intron; FrAd: frame adjuster.


        Figure 4. Schematic description of the disruption of PIGA intron 5. Depicted at the top is the PIGA gene along with the nucleotide sequence of exon 5-intron 5 boundary and a corresponding partial PIGA protein. Shown at the bottom is a partial nucleotide sequence of the targeting vector including a BspE1-truncated frame adjuster. The amino acid sequence under the nucleotide sequence indicates a predicted translation product derived from the targeting vector. IVS: synthetic intron; FrAd: frame adjuster; NeoR: the neomycin phosphotransferase gene; pA: polyadenylation site; SD: splice donor; SA: splice acceptor.

      3. When the promoter-trap module is incorporated into an exon, regardless of the vector platform employed (pAAV-2Aneo or pAAV-2Aneo v2; Figure 5): Cleave pAAV-2Aneo (v2) with BsrGI, and religate the vector so that 2Aneo is translated in frame with the 5’ portion of the endogenous target gene (Figures 6 and 7).


        Figure 5. Schematic diagram of the AAV-mediated targeting of an exon. See the legend for Figure 1 for descriptions and abbreviations.


        Figure 6. The frame adjuster truncated with BsrGI for exon targeting and its adjacent region in pAAV-2Aneo. A predicted partial polypeptide translated from this region is indicated under the nucleotide sequence. The 5’ homology arm should be designed such that the coding exon to be disrupted is translated in frame with the indicated polypeptide. See the legend for Figure 4 for abbreviations.


        Figure 7. The frame adjuster truncated with BsrGI for exon targeting and its adjacent region in pAAV-2Aneo v2. See the legend for Figure 6 for descriptions and abbreviations.

    6. Transform competent cells with the ligation product following standard procedures.
    7. Spread the transformed competent cells onto a LB plate containing 50 µg/ml of ampicillin.
    8. Incubate the cells overnight at 37 °C.
    9. Pick several colonies with pipette tips, and culture the cells in 2.5 ml of LB containing 50 µg/ml of ampicillin, overnight at 37 °C.
    10. Perform mini-prep using the Mini Plus plasmid DNA extraction system as per manufacturer’s instructions.
    11. Confirm that the frame adjuster has been truncated as intended by sequencing the recovered plasmids using the BigDye Terminator v3.1 Cycle Sequencing Kit and a sequencing primer (e.g., NeoR-Rev #1, a primer complementary to the NeoR gene).
    12. To clone one of the PCR-amplified homology arms, cleave the plasmid verified in step A11 with an appropriate restriction enzyme(s). If required, use alkaline phosphatase to prevent self-religation of the plasmid. Purify the linearized plasmid using the PureLink PCR Purification Kit.
    13. Ligate the linearized plasmid with the PCR-amplified homology arm that was trimmed and purified in step A4.
    14. Repeat steps A6-A10 with the ligation product.
    15. Check for the successful insertion of the homology arm by digesting the resultant plasmids (mini-preps) with an appropriate restriction enzyme(s) and resolving the DNA fragments on an agarose gel.
    16. Verify the sequence of the PCR-amplified homology arm using the BigDye Terminator v3.1 Cycle Sequencing Kit and custom-made primers.
      Note: The homology arm is incorporated between an inverted terminal repeat (ITR) and a loxP site, both of which are located close to MCS. Since both ITR and loxP are partially palindromic and thus (particularly ITR) may hinder DNA polymerization, we designed custom-made sequencing primers within the homology arms.
    17. To clone another homology arm, cleave the plasmid from step A16 using an appropriate restriction enzyme(s) with or without alkaline phosphatase, and purify the linearized plasmid using the PureLink PCR purification kit. Then, repeat steps A13-A16.
    18. Perform maxi-prep of the constructed AAV-based targeting plasmid using the PureLink HiPure Plasmid Maxiprep Kit.
    19. Determine the concentration of the maxi-prep plasmid DNA with a NanoDrop spectrophotometer.

  2. AAV production
    1. Plate HEK293 or HEK293T cells onto a 10-cm culture dish at an approximate density of 1 x 106 cells/dish with 10 ml of penicillin/streptomycin-free D-MEM supplemented with 5% FBS. The cells should be 70-80% confluent on the following day.
    2. On the following day, aspirate the medium, and add 5 ml of Opti-MEM I reduced-serum medium.
      Note: Washing the cells prior to the addition of Opti-MEM I reduced-serum medium is optional; however, proceed with caution when washing, as the HEK293(T) cells are loosely adherent to the culture dish.
    3. Pipette the following plasmids into 1.5 ml of Opti-MEM I reduced-serum medium and gently mix:
      pRC                                                               5 µg
      pHelper                                                         5 µg
      AAV-based targeting plasmid                       5 µg
    4. Add 45 μl of TransIT-293 reagent, and gently mix by pipetting.
    5. Let the mixture stand for 15 min at room temperature.
    6. Pipette the DNA-reagent mixture drop-wise onto the HEK293(T) cells in the 10-cm culture dish, and gently mix by rocking the dish.
    7. Incubate the cells in a CO2 incubator at 37 °C for 24 h.
      Notes:
      1. A total of 6.5 ml medium is sufficient for 24 h incubation of cells in a 10-cm culture dish. 
      2. The CO2 incubator is used at 37 °C and 5% CO2 under humidified conditions throughout the study.
    8. After 24 h of transfection, add 5 ml of D-MEM supplemented with 5% FBS and 1% penicillin/streptomycin.

  3. Harvest of AAV particles
    1. After 72 h of transfection, scrape HEK293(T) cells from the 10-cm culture dish by extensive pipetting, and transfer the cells to a 15-ml conical tube with their supernatant.
    2. Centrifuge the 15-ml tube at 400 x g (1,500 rpm) for 5 min at room temperature, and discard the supernatant completely but carefully so as not to remove the cells.
    3. Wash the cells with PBS(-), centrifuge the 15-ml tube at 400 x g for 5 min, and discard the PBS(-) completely but carefully so as not to remove the cells.
    4. Resuspend the cell pellet in 1.0 ml of PBS(-).
      Note: If the cell line that will be infected with the AAV vector in preparation is already known, the cell pellet in step C4 can be resuspended in a medium appropriate for that cell line instead of PBS(-).
    5. Prepare chilled methanol by pouring methanol onto dry ice. Set the temperature of a water bath to 37 °C.
      Note: Wear safety glasses to protect eyes when chilled methanol is being prepared and used.
    6. Dip the 15-ml tube into chilled methanol until the cell suspension is completely frozen.
    7. Transfer the 15-ml tube to the 37 °C water bath, and incubate until the cell suspension is completely thawed.
    8. Repeat steps C6 and C7 thrice (four cycles in total).
    9. Centrifuge the 15-ml tube at 400 x g for 5 min at room temperature, and transfer the supernatant to a 1.5-ml or 2-ml cryovial.
    10. Use the resulting AAV stock immediately for titering or cell infection, or store it at -80 °C until use. Avoid extra freeze-thaw cycles.
      Note: The AAV solution prepared as above can be purified using density gradient centrifugation or affinity columns prior to its use for infection, and these procedures have been described in published protocols (Khan et al., 2011; Howes and Schofield, 2015). Although purification of the AAV solution is critical for in vivo use, however, gene targeting in cell lines in vitro can be achieved by infecting cells with this AAV solution without further purification.

  4. (Optional) AAV titering based on copy number estimation
    Although we titered AAV-based targeting vectors when we assessed the gene targeting efficiencies of individual vectors (Karnan et al., 2012; Konishi et al., 2012; Damdindorj et al., 2014; Karnan et al., 2016), titering is optional when the aim of the experiment is only to obtain gene-targeted cell clones.
    1. Perform a serial 10-fold dilution of the maxi-prep plasmid DNA from which AAV particles were produced, and generate six different concentrations of plasmid solutions (10-1-10-6 ng/μl) for use as standard samples in quantitative PCR (qPCR).
    2. Calculate the copy number of the plasmid in the diluted plasmid solutions. The average molecular weight of deoxyribonucleotides in single-stranded DNA is 303.7. Thus, the approximate copy number of the plasmid N (μl-1) is estimated as:
      N = a x NA x 10-9/(n x 303.7)
      Where,
      a: represents the concentration of plasmid DNA within the solution (ng/μl),
      NA: represents Avogadro’s constant (6.022 x 1023),
      n: represents the size of the plasmid in base pairs.
      Note: In contrast to the single-stranded nature of the AAV vector genome, plasmid DNA is double-stranded under ordinary conditions. However, plasmid DNA is denatured and the copy number is theoretically doubled following the initial denaturation step in qPCR. Therefore, the calculation above was performed accordingly.
    3. In a 1.5-ml tube, mix the following reagents:
      AAV stock (prepared in Procedure C)                                 2.5 μl
      RDD buffer                                                                          5 μl
      DNase I stock solution                                                        1.25 μl
      Distilled water                                                                     41.25 μl
      Note: RDD buffer and DNase I stock solution are components of the RNase-Free DNase Set.
    4. Incubate the reaction mixture above at room temperature (20-25 °C) for 30 min to degrade residual plasmid DNA.
    5. Incubate the reaction mixture at 95 °C for 5 min to inactivate DNase I.
    6. Perform qPCR with triplicate templates, including the AAV solution from step D5, serially diluted standard samples, and distilled water (negative control). Initially, prepare a PCR cocktail by mixing the following solutions:
      2x buffer for KOD FX Neo                                                   7.5 μl x n
      Primer NeoR-Fwd #1 (100 pmol/μl)                                     0.1 μl x n
      Primer NeoR-Rev #2 (100 pmol/μl)                                     0.1 μl x n
      dNTP (10 mM each)                                                            3 μl x n
      KOD FX Neo                                                                       0.1 μl x n
      SYBR Green I working solution                                           0.15 μl x n
      Distilled water                                                                      3.05 μl x n
      n: the number of templates plus some extra
    7. Dispense the cocktail into a 96-well plate (14 μl/well), add 1 μl of templates to individual wells, and mix by pipetting.
    8. Perform qPCR amplification using StepOnePlus with the experimental conditions below. The post-amplification melt curve analysis includes 1 min at 95 °C and 1 min at 55 °C followed by a slow temperature increment to 95 °C.
      Step 1:                        95 °C             2 min
      Step 2 (50 cycles):     95 °C             15 sec
                                         60 °C             15 sec
                                         70 °C             20 sec
    9. Calculate the average value of the triplicate data for each sample. Estimate the copy number of an AAV-based targeting vector by comparing its amplification profile with that of serially diluted standard samples. Note that the AAV solution used for qPCR was diluted 20-fold when it was treated with DNase I in step D3. An example of standard curves generated in qPCRs is shown in Figure 8.


      Figure 8. An example of qPCR standard curves. Red dots indicate threshold cycles in individual samples. Ct: Threshold Cycle.

  5. Cell infection with an AAV-based targeting vector
    1. Propagate the cell line for gene targeting in a 75-cm2 flask with the appropriate medium (growth medium). Cells should be 30-40% confluent on the following day.
    2. (Optional) If G1/S arrest of the cell line for gene targeting is readily achievable with a simple procedure, then arrest the cells at the G1/S boundary prior to AAV infection, as this was reported to enhance the efficiency of AAV-mediated gene targeting (Trobridge et al., 2005). For instance, MCF-10A cells should be incubated with epidermal growth factor (EGF)-free medium for 6-8 h prior to AAV infection, and the medium should be replaced with EGF-containing medium (growth medium) at the time of infection.
    3. The day after cell propagation, thaw the AAV stock stored at -80 °C in a 37 °C water bath.
    4. Aspirate the medium from the 75-cm2 flask, and pour (5-x) ml of growth medium onto the cells, where x (ml) represents a predetermined volume of the AAV stock.
      Note: The volume of the AAV stock used is determined by the multiplicity of infection (MOI) that is used to infect the cells. We generally infect cells with AAV-based targeting vectors at a MOI of 1 x 104 (Karnan et al., 2012; Konishi et al., 2012; Damdindorj et al., 2014; Karnan et al., 2016); however, a range of MOI can be applied depending on the experiment (Russell and Hirata, 1998; Hirata and Russell, 2000; Miller et al., 2003; Porteus et al., 2003). If the aim of the experiment is only to obtain gene-targeted cell clones, use the entire (1.0 ml) AAV stock for the 75-cm2 flask.
    5. Add x ml of the AAV stock to the flask, and mix by gentle rocking. Incubate the cells in the CO2 incubator overnight.
    6. On the next day (16-24 h after infection), add 7 ml of growth medium into the 75-cm2 flask, and incubate the cells in the CO2 incubator for two more days.
    7. AAV infection is considered complete two days later. The cells can now be used for assays or experiments, although G418 selection is subsequently performed in many cases to eliminate uninfected cells. For single cell cloning with the infected cells, proceed to Procedures F and G.

  6. G418 selection in 96-well tissue culture plates
    1. Aspirate the AAV-containing medium from the flask, wash the cells with PBS(-) once, and pipette 2 ml of trypsin into the flask. Incubation time with trypsin in the CO2 incubator varies depending on the cell lines employed.
    2. Swirl the flask and examine it under a microscope to confirm that the cells are dissociated. Then, add the appropriate volume of growth medium to the flask, pipette to further dissociate the cells, and transfer the cell suspension into a 15-ml (or 50-ml) conical tube.
    3. Centrifuge the cells at 400 x g for 5 min, discard the supernatant, and resuspend the cells in 14 ml of growth medium containing the appropriate concentration of G418.
      Note: In this protocol, G418 selection is initiated when cells are seeded into 96-well tissue culture plates as this expedites the selection of cells. However, if the cell line being infected is sensitive to sparse seeding, it is advisable to begin G418 selection 1-3 days after seeding when the cells have attached to the wells. This may increase the number of colonies obtained following G418 selection.
    4. Distribute the cells into seven 96-well plates. First, pour 6 ml of cell suspension to a sterilized pipetting reservoir, and then add 52 ml of growth medium containing G418.
    5. Mix the cell suspension in the reservoir by pipetting, and inoculate the cells in three 96-well plates (200 μl/well) using a 12-channel pipettor.
    6. Pour 4 ml of cell suspension to the reservoir, and add 35 ml of growth medium containing G418.
    7. Mix the cell suspension in the reservoir by pipetting, and inoculate the cells in two 96-well plates (200 μl/well) using a 12-channel pipettor.
    8. Repeat steps F6 and F7 using the remaining (4 ml) cell suspension.
    9. Place the 96-well plates in the CO2 incubator, and maintain the cells until completion of G418 selection and detection of colonies by microscopy (usually 7-14 days after seeding).
      Note: If colonies in the 96-well plates are not clearly visible because of dying cells or dead cells floating in the medium, the visibility can be improved by changing the medium.
    10. Mark wells containing G418-resistant colonies with a permanent marker. Wells that apparently contain multiple colonies should be marked with a different color.
    11. Incubate the 96-well plates in the CO2 incubator for another 7-14 days until cells in the marked wells reach an average of 60-80% confluence.

  7. PCR-based screening of cell clones
    This section describes a PCR-based screening of pooled samples that are generated by manual one-by-one cell pooling. If the number of colonies formed after G418 selection is relatively small, cell pooling can be omitted and the screening of colonies can be performed on a one-by-one basis (Kohli et al., 2004; Rago et al., 2007; Howes and Schofield, 2011). Systematic multi-round cell pooling that we described previously (Konishi et al., 2007b) is usually unnecessary when the constructed targeting vector is based on pAAV-2Aneo (v2).
    1. Decant or aspirate the medium in the 96-well plates, and wash the cells once with PBS(-).
    2. Trypsinize the marked cells with trypsin (20 μl/well).
    3. After cells are dissociated, gently pipette the cell suspension up and down a few times, add growth medium (80 μl/well), and gently pipette the cells again.
    4. Pipette the cell suspension from 8-12 wells (50 μl/well) into a single 1.5-ml tube. Repeat this for all marked wells of the 96-well plates.
      Note: Use a new pipette tip for each cell transfer to prevent cross-contamination of cells.
    5. Add growth medium to the marked wells (150 μl/well), and place the 96-well plates back in the CO2 incubator.
      Note: Use a serum-containing medium to neutralize trypsin in this step. If the use of serum is not optimal for the cell line being engineered, replace the serum-containing medium with the appropriate growth medium after the cells have attached to the wells.
    6. Extract genomic DNA from the cell samples pooled in 1.5-ml tubes using the PureLink Genomic DNA Mini Kit.
    7. Perform screening PCR to identify pooled samples containing gene-targeted cell clones using KOD FX Neo and the genomic DNA extracted in step G6. The PCR-amplified region should encompass one of the homology arms: i.e., one of the PCR primers should correspond to the region unique to the targeting vector, and the other to the region outside of the homology arm (Figures 1 and 5).
    8. Separate the PCR products by agarose gel electrophoresis, and identify pooled samples yielding a PCR product of the expected size.
    9. After the cells in the 96-well plates corresponding to the PCR positive hits grow to at least 50 % confluence, trypsinize and transfer the cells to 24-well plates following standard procedures.
    10. After cells in the 24-well plates grow to at least 50% confluence, aspirate the medium, wash cells once with PBS(-), and treat cells with trypsin (100 μl/well).
    11. After cells are dissociated, gently pipette the cell suspension up and down a few times, add growth medium (900 μl/well), and gently pipette the cells again.
    12. Transfer approximately half of the trypsinized cells from each well into a 1.5-ml tube.
    13. Add growth medium to the 24-well plates up to 1.5 ml/well, and place the plates back in the CO2 incubator.
    14. Extract genomic DNA from the samples in step G12 using the PureLink Genomic DNA Mini Kit, perform a second screening PCR using KOD FX Neo and the same primer pair as in step G7, and identify PCR-positive samples by agarose gel electrophoresis.
    15. Verify the PCR-positive products identified in step G14 by direct sequencing using custom-made primers.
    16. Transfer the PCR-positive cells from the 24-well plates to 6-well plates or tissue culture dishes/flasks. If the positive cells are likely to be of non-single cell origin, perform single cell cloning using a limiting dilution technique, followed by PCR screening of isolated single cell clones using the same primer pair as in steps G7 and G14. Meanwhile, confirm the positivity of PCR encompassing the other homology arm using the genomic DNA obtained in step G14 (primers depicted in Figures 1 and 5). The amplified PCR product of the expected size should be verified by direct sequencing.
      Note: To further confirm the integrity of the isolated gene-targeted cell clones, it would be ideal to sequence the whole genome of the clones. However, as an alternative, we usually perform Southern blotting with the isolated gene-targeted clones to confirm the integrity of the targeted genomic locus and the absence of extra copies of the targeting vector that can be randomly integrated into the genome (Karnan et al., 2012; Konishi et al., 2012; Karnan et al., 2016).

Notes

  1. The promoter-trap module in the targeting vector can be inserted into either an exon or an intron of the endogenous target gene. However, since the neomycin phosphotransferase (NeoR) gene in the targeting vectors is expressed downstream of 2A, the promoter-trap module should be inserted into a coding exon or an intron between the coding exons, and the homology arms should be designed accordingly.
  2. When the promoter-trap module in a targeting vector is inserted into a coding exon, design the PCR reverse primer to amplify the 5’ homology arm so that a reading frame of the target gene coincides with that shown in Figure 6 or 7. Confirm that neither a stop codon nor a polyadenylation signal will be ectopically introduced at the 3’ end of the 5’ homology arm.
  3. The sum of the lengths of 5’ and 3’ homology arms should not exceed 3.0 kb because of the size restriction of the AAV vector.
  4. In amplifying homology arms by PCR to construct a targeting vector, genomic DNA used as a template should ideally be prepared from a cell line used for gene targeting in later steps. The use of a distinct cell line as PCR template potentially results in lower homology between homology arms and the host genome because of polymorphisms within the homologous region. Low homology in the homology arms of a targeting vector results in low gene targeting efficiency. However, to introduce a defined point mutation, deletion, or insertion within a homology arm for targeted knock-in (e.g., Konishi et al., 2007a; Wilsker et al., 2008; Gustin et al., 2009; Konishi et al., 2011), employ a cell line harboring the same genetic alteration with that being knocked-in. Alternatively, perform site-directed mutagenesis after the homology arm is cloned into a vector to introduce the desired alteration.
  5. Although we have listed here the commercially available reagents, kits, and equipment regularly used in our laboratory, most of them can be replaced with equivalent products from other manufacturers.

Recipes

  1. Ampicillin working solution
    1. Dissolve 1 g of ampicillin sodium in 20 ml distilled water (50 mg/ml).
    2. Sterilize the solution using a 0.22 µm filter.
    3. Aliquot the sterilized solution into 1.5-ml tubes (1 ml/tube).
    4. Store the aliquots at -20 °C.
  2. SYBR Green I working solution
    1. Aliquot SYBR Green I to 1.5-ml tubes (1 μl/tube). Immediately wrap the tubes with aluminum foil to protect from light, and label them as ‘80,000x’.
    2. Store the ‘80,000x’ tubes at -20 °C (except for a tube which will be processed as below). Use non-clear boxes for storage.
    3. Label 40 of 1.5-ml tubes as ‘2,000x’.
    4. Dilute 1 μl of SYBR Green I in a ‘80,000x’ tube with 39 μl of DMSO, and aliquot the resultant 40 μl solution to 40 of ‘2,000x’ tubes (again 1μl/tube).
    5. Store the ‘2,000x’ tubes at -20 °C. Use non-clear boxes for storage.
    6. When preparing a qPCR cocktail, thaw a ‘2,000x’ tube and dilute the content with 19 μl of distilled water. This results in the generation of 100x SYBR Green I working solution (20 μl). Discard excess 100x working solution in a thawed tube after qPCR setup.
  3. PBS(-)
    PBS(-) is prepared according to standard procedures.
    1. First, the ingredients below are dissolved in distilled water to produce 10x PBS(-).
      160.2 g NaCl
      4.0 g KCl
      58.0 g Na2HPO4.12H2O
      4.0 g KH2PO4
      Distilled water to 2 L
    2. Then, 50 ml of 10x PBS(-) is diluted with 450 ml of distilled water in a 500-ml bottle and sterilized by autoclave.

Acknowledgments

This protocol was adapted from our previous work, Karnan et al. (2016). This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS; 25460395 to S. K., 15K19561 to A. O., and 25640107 to H. K.), and Takeda Science Foundation (to H. K.).

References

  1. Damdindorj, L., Karnan, S., Ota, A., Hossain, E., Konishi, Y., Hosokawa, Y. and Konishi, H. (2014). A comparative analysis of constitutive promoters located in adeno-associated viral vectors. PLoS One 9(8): e106472.
  2. Gustin, J. P., Karakas, B., Weiss, M. B., Abukhdeir, A. M., Lauring, J., Garay, J. P., Cosgrove, D., Tamaki, A., Konishi, H., Konishi, Y., Mohseni, M., Wang, G., Rosen, D. M., Denmeade, S. R., Higgins, M. J., Vitolo, M. I., Bachman, K. E. and Park, B. H. (2009). Knockin of mutant PIK3CA activates multiple oncogenic pathways. Proc Natl Acad Sci U S A 106(8): 2835-2840.
  3. Hirata, R. K. and Russell, D. W. (2000). Design and packaging of adeno-associated virus gene targeting vectors. J Virol 74(10): 4612-4620.
  4. Howes, R. and Schofield, C. (2015). Genome engineering using Adeno-Associated Virus (AAV). Methods Mol Biol 1239, 75-103.
  5. Karnan, S., Konishi, Y., Ota, A., Takahashi, M., Damdindorj, L., Hosokawa, Y. and Konishi, H. (2012). Simple monitoring of gene targeting efficiency in human somatic cell lines using the PIGA gene. PLoS One 7(10): e47389.
  6. Karnan, S., Ota, A., Konishi, Y., Wahiduzzaman, M., Hosokawa, Y. and Konishi, H. (2016). Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide. Nucleic Acids Res 44(6): e54.
  7. Khan, I. F., Hirata, R. K. and Russell, D. W. (2011). AAV-mediated gene targeting methods for human cells. Nat Protoc 6(4): 482-501.
  8. Kohli, M., Rago, C., Lengauer, C., Kinzler, K. W. and Vogelstein, B. (2004). Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses. Nucleic Acids Res 32(1): e3.
  9. Konishi, H., Karakas, B., Abukhdeir, A. M., Lauring, J., Gustin, J. P., Garay, J. P., Konishi, Y., Gallmeier, E., Bachman, K. E. and Park, B. H. (2007a). Knock-in of mutant K-ras in nontumorigenic human epithelial cells as a new model for studying K-ras mediated transformation. Cancer Res 67(18): 8460-8467.
  10. Konishi, H., Lauring, J., Garay, J. P., Karakas, B., Abukhdeir, A. M., Gustin, J. P., Konishi, Y. and Park, B. H. (2007b). A PCR-based high-throughput screen with multiround sample pooling: application to somatic cell gene targeting. Nat Protoc 2(11): 2865-2874.
  11. Konishi, H., Mohseni, M., Tamaki, A., Garay, J. P., Croessmann, S., Karnan, S., Ota, A., Wong, H. Y., Konishi, Y., Karakas, B., Tahir, K., Abukhdeir, A. M., Gustin, J. P., Cidado, J., Wang, G. M., Cosgrove, D., Cochran, R., Jelovac, D., Higgins, M. J., Arena, S., Hawkins, L., Lauring, J., Gross, A. L., Heaphy, C. M., Hosokawa, Y., Gabrielson, E., Meeker, A. K., Visvanathan, K., Argani, P., Bachman, K. E. and Park, B. H. (2011). Mutation of a single allele of the cancer susceptibility gene BRCA1 leads to genomic instability in human breast epithelial cells. Proc Natl Acad Sci U S A 108(43): 17773-17778.
  12. Konishi, Y., Karnan, S., Takahashi, M., Ota, A., Damdindorj, L., Hosokawa, Y. and Konishi, H. (2012). A system for the measurement of gene targeting efficiency in human cell lines using an antibiotic resistance-GFP fusion gene. Biotechniques 53(3): 141-152.
  13. Miller, D. G., Petek, L. M. and Russell, D. W. (2003). Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks. Mol Cell Biol 23(10): 3550-3557.
  14. Porteus, M. H., Cathomen, T., Weitzman, M. D. and Baltimore, D. (2003). Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks. Mol Cell Biol 23(10): 3558-3565.
  15. Rago, C., Vogelstein, B. and Bunz, F. (2007). Genetic knockouts and knockins in human somatic cells. Nat Protoc 2(11): 2734-2746.
  16. Russell, D. W. and Hirata, R. K. (1998). Human gene targeting by viral vectors. Nat Genet 18(4): 325-330.
  17. Trobridge, G., Hirata, R. K. and Russell, D. W. (2005). Gene targeting by adeno-associated virus vectors is cell-cycle dependent. Hum Gene Ther 16(4): 522-526.
  18. Wilsker, D., Petermann, E., Helleday, T. and Bunz, F. (2008). Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control. Proc Natl Acad Sci U S A 105(52): 20752-20757. 

简介

与基于质粒的靶向载体相比,基于腺相关病毒(AAV)的靶向载体具有1-4对较高的基因靶向效率。 通过将启动子捕获系统引入靶向载体中,AAV介导的基因靶向的效率进一步增加。 此外,我们发现使用核糖体跳跃2A肽而不是通常使用的内部核糖体进入位点(IRES)在启动子捕获系统中导致显着更高的AAV介导的基因靶向效率(Karnan等,2016)。 在该方案中,我们描述了AAV介导的基因靶向开发2A用于启动子捕获的程序,包括基于平台质粒pAAV-2Aneo或pAAV-2Aneo v2的靶向载体的构建,AAV颗粒的产生,细胞感染 基于AAV的靶向载体,以及基因靶向细胞克隆的分离和验证。
【背景】以前在其他方案中描述了AAV介导的基因靶向的程序(对应于本方案的BG部分)(Kohli等人,2004; Rago等人,2007; Khan等人,2011; Howes and Schofield ,2015)。 然而,该方案提供了如何使用基于2A的启动子捕获系统首次进行AAV介导的基因靶向的详细描述。

关键字:腺相关病毒, AAV, 打靶载体, 基因打靶, 启动子捕获, 2A, 内部核糖体进入位点, IRES

材料和试剂

  1. 移液器提示
  2. 10厘米培养皿
  3. 1.5毫升或2毫升冷冻的
  4. 96孔板
  5. 15毫升(或50毫升)锥形管
  6. 24孔板
  7. 6孔板或组织培养皿/烧瓶
  8. 0.22μm过滤器
  9. 1.5 ml管子
  10. 铝箔
  11. 一次性移液池(AS ONE,目录号:2-7844-02)
  12. 电子。大肠杆菌DH5α感受态细胞(Takara Bio,目录号:9057)
    注意:本产品尚未停产。
  13. HEK293或HEK293T细胞系
  14. 用于基因靶向的细胞系
  15. pAAV-2Aneo(Addgene,目录号:80032)
  16. pAAV-2Aneo v2(Addgene,目录号:80033)
  17. 琼脂糖(NIPPON GENE,目录号:318-01195)
  18. Pwo SuperYield DNA聚合酶(Roche Diagnostics,目录号:04340850001)
  19. PureLink ®快速凝胶提取试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:K2100-25)
  20. PureLink ® PCR纯化试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:K3100-02)
  21. 快速连接 TM 试剂盒(New England BioLabs,catalog number:M2200S)
  22. 干冰
  23. 限制性酶Bsp,EI,Mlu I或或Bsr GI(New England BioLabs,Ipswich,MA)
  24. 限制酶缓冲液
  25. LB肉汤(NACALAI TESQUE,目录号:20068-75)
  26. 质粒DNA提取系统(Viogene,目录号:GF2002)
  27. BigDye ®终止者v3.1循环测序试剂盒(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4337455)
  28. 碱性磷酸酶,小肠肠(CIP)(New England BioLabs,目录号:M0290L)
  29. 定制PCR引物(用于扩增5'和3'同源性臂)
  30. 定制测序引物(用于5'和3'同源臂的测序)
  31. 定制PCR引物(用于筛选基因靶向克隆)
  32. PureLink ® HiPure质粒Maxiprep试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:K210007)
  33. Dulbecco改良的Eagle's培养基(D-MEM)(Wako Pure Chemical Industries,目录号:044-29765)
  34. 胎牛血清(FBS)(NICHIREI,目录号:172012-500ml)
  35. Opti-MEM I还原血清培养基(Thermo Fisher Scientific,Gibco TM,目录号:31985-070)
  36. AAV无辅助系统中的pRC和pHelper质粒(Agilent Technologies,目录号:240071)
  37. TransIT ® -293转染试剂(Mirus Bio,目录号:MIR 2705)
  38. 青霉素 - 链霉素溶液(×100)(Wako Pure Chemical Industries,目录号:168-23191)
  39. 甲醇
  40. 蒸馏水
  41. 不含RNA酶的DNA酶(QIAGEN,目录号:79254)
  42. Neo R -Rev#1:5'-GGCATCAGAGCAGCCGATTG
  43. Neo R -Fwd#1:5'-CATTCGACCACCAAGCGAAA
  44. Neo R -Rev#2:5'-CTTGAGCCTGGCGAACAGTT
  45. KOD FX Neo(TOYOBO,目录号:KFX-201)
  46. 生长培养基适合进行基因靶向的细胞系
  47. 和苯酚红0.25%(w / v)胰蛋白酶溶液(Wako Pure Chemical Industries,目录号:201-18841)
  48. PCR缓冲区
  49. dNTPs
  50. 绿色I核酸染色(Lonza,目录号:50512)
  51. PureLink ® Genomic DNA Mini Kit(Thermo Fisher Scientific,Invitrogen TM,目录号:K182002)
  52. 氨苄青霉素钠(和光纯药工业公司,目录号:012-23303)
  53. (DMSO)(和光纯药,目录号:048-21985)
  54. NaCl
  55. KCl
  56. Na 2 HPO 4 / 12H 2 O
  57. KH 2 PO 4
  58. 氨苄青霉素工作液(参见食谱)
  59. SYBR Green I工作解决方案(见配方)
  60. PBS( - )(参见食谱)

设备

  1. 加湿CO 2培养箱
  2. Veriti TM热循环仪(Thermo Fisher Scientific,型号:Veriti Thermal Cycler)
  3. Mupid ® -2plus潜艇式电泳系统(Takara Bio,型号:Mupid-2plus System)
  4. NanoDrop 1000分光光度计(Thermo Fisher Scientific)
  5. StepOnePlus TM实时PCR系统(Thermo Fisher Scientific,Applied Biosystem TM,型号:StepOnePlus TM实时PCR系统) >
  6. 离心机用于分子生物学实验
  7. -20°C冷冻机
  8. 组织文化罩
  9. 相差显微镜
  10. 摇摆式离心机组织培养
  11. -80°C深层冷冻机
  12. 水浴
  13. 12通道移液器
  14. 两种不同颜色的永久标记

程序

  1. 构建基于AAV的靶向载体
    1. 设计用于扩增同源臂的PCR引物。通过限制酶消化将扩增的同源臂结合到pAAV-2Aneo(v2)中,从多克隆位点MCS-1(在SacI和SpeI)之间选择合适的限制酶位点 I网站)和pAAV-2Aneo(v2)中的MCS-2(在 Kpn I和 Xho I网站之间)。选定的限制性内切酶位点在同源臂内不应扩增。然后,在PCR引物的5'末端引入选择的限制酶位点。另外,在限制酶位点的末端附加3-6个核苷酸(任何序列,长度取决于所选择的限制酶位点),这允许酶切割接近末端的PCR产物。有关设计同源性手臂的提示,请参阅注释部分(注1-3)。
    2. 通过PCR扩增5'和3'同源性臂。使用高保真DNA聚合酶;例如,Pwo SuperYield DNA聚合酶。
    3. 通过琼脂糖凝胶电泳分析PCR产物。从凝胶中切出预期大小的条带,并使用PureLink Quick Gel Extraction Kit回收DNA片段。如果PCR产物显示为干净的单一条带,并且在琼脂糖凝胶上未检测到引物二聚体,则PCR产物可以任选地使用PureLink PCR纯化试剂盒进行纯化,而不是进行凝胶提取。
    4. 用选择的限制酶切割PCR产物,并使用PureLink PCR纯化试剂盒纯化反应产物
    5. 由于pAAV-2Aneo和pAAV-2Aneo v2具有不同的“帧调整器”,所以以不同的方式处理这些矢量来调整阅读框。按照以下三个选项之一(步骤A5a-c)处理帧调整器。在三个选项中的任一个中,限制酶消化的DNA溶液应在自我重新连接之前使用PureLink PCR Purification Kit或其他方法进行纯化。使用快速连接套件执行自我重新连接。
      1. l;”>当使用pAAV-2Aneo时启动子 - 陷阱模块在基因靶向后被掺入内含子(图1):用三种限制性内切酶之一(EspI,Mlu)切割pAAV-2Aneo,我或者Bsr GI),并重新连接载体,使得2Aneo与内源性目标基因的5'部分翻译成框架(图2)。


        图1. AAV介导的内含子靶向示意图。 细虚线表示内源基因组座位和靶向载体之间的同源性。 F1-R1和F2-R2表示用于筛选PCR和确认PCR的寡核苷酸引物对。底部显示从目标基因组座位表达的预测mRNA。灰色矩形:外显子;大胆的黑线:内含子;蓝色发夹结构:反向末端重复; IVS:合成内含子FrAd:框架调节器Neo R :新霉素磷酸转移酶基因; pA:多聚腺苷酸化位点。


        图2. pAAV中帧调整器的处理-2Aneo用于定向内含子。完整的框架调整器(顶部)和由 Bsp EI, Mlu I和 Bsr GI消化(底部)。在核苷酸序列下显示了从框架调节子周围区域翻译的预测部分多肽。 SA:接头受体; IVS:合成内含子FrAd:框架调整器。

      2. l;”当启动子陷阱模块以类似于步骤A5a(图1)的基因靶向并入内含子中,但是使用pAAV-2Aneo v2代替pAAV-2Aneo:用bsp EI或em打开pAAV-2Aneo v2 > Bsr GI并重新连接载体,或者将载体未处理,以使2Aneo与内源靶基因的5'部分翻译成框架(图3)。图4示意性地描绘了在之前的研究中作为选项A5b的示例的中断PIGA 内含子5。


        图3. pAAV中帧调整器的处理-2Aneo v2用于靶向内含子。完整的框架调整器(顶部)和由 EI和 Bsr GI消化(底部)处理的框架调整器。在核苷酸序列下显示了从框架调节子周围区域翻译的预测部分多肽。 SA:接头受体; IVS:合成内含子FrAd:框架调整器。


        图4.中断IGA 内含子5。在顶部描述的是PIGA基因以及外显子5-内含子5边界的核苷酸序列和相应的部分PIGA蛋白。显示在底部的是靶向载体的部分核苷酸序列,其包括E1缩短的框架调节器。核苷酸序列下的氨基酸序列表示来自靶向载体的预测翻译产物。 IVS:合成内含子FrAd:框架调节器Neo R :新霉素磷酸转移酶基因; pA:聚腺苷酸化位点; SD:拼接供体; SA:拼接受体。

      3. 当启动子 - 陷阱模块并入外显子时,无论使用的载体平台(pAAV-2Aneo或pAAV-2Aneo v2;图5):用Bsr GI切割pAAV-2Aneo(v2)并连接载体,使2Aneo与内源靶基因的5'部分翻译成框架(图6和7)。


        图5. AAV介导的示意图靶向外显子。 有关说明和缩写,请参见图1的图例。


        图6.框架调整器用 Bsr GI用于外显子靶向及其在pAAV-2Aneo中的相邻区域。 从该区域翻译的预测部分多肽在核苷酸序列下表示。 5'同源臂应设计成使得编码外显子被破坏的内容与指定的多肽一起翻译。有关缩写,请参见图4的图例。


        图7.框架调整器用 Bsr GI用于外显子靶向及其相邻区域的pAAV-2Aneo v2。请参阅图6的图例,了解描述和缩写。

    6. 按照标准程序,用连接产物转化感受态细胞
    7. 将转化的感受态细胞扩散到含有50μg/ ml氨苄青霉素的LB平板上
    8. 在37℃下孵育细胞一夜。
    9. 用移液管吸头挑取几个菌落,并将细胞培养在含有50μg/ ml氨苄青霉素的2.5ml LB中,37℃过夜。
    10. 使用Mini Plus质粒DNA提取系统按照制造商的说明进行微型制剂。
    11. 通过使用BigDye Terminator v3.1循环测序试剂盒和测序引物(例如,Neo R -Rev对序列进行测序,确认帧调节子已被截短#1,与Neo R 基因互补的引物)
    12. 为了克隆PCR扩增的同源臂之一,用合适的限制性酶切割步骤A11中验证的质粒。如果需要,使用碱性磷酸酶来防止质粒自身重新连接。使用PureLink PCR纯化试剂盒纯化线性化质粒。
    13. 用步骤A4修剪和纯化的PCR扩增的同源臂对线性化质粒进行引导。
    14. 用连接产品重复步骤A6-A10。
    15. 通过用适当的限制酶消化所得质粒(小型制品),并在琼脂糖凝胶上拆分DNA片段,检查同源臂的成功插入。
    16. 使用BigDye Terminator v3.1循环测序试剂盒和定制引物验证PCR扩增的同源臂的序列。
      注意:同源臂结合在反向终端重复(ITR)和loxP位点之间,两者都位于靠近MCS的位置。由于ITR和loxP都是部分回文的,因此(特别是ITR)可能阻碍DNA聚合,我们在同源臂内设计定制的测序引物。
    17. 为了克隆另一个同源臂,使用具有或不具有碱性磷酸酶的合适的限制酶切割来自步骤A16的质粒,并使用PureLink PCR纯化试剂盒纯化线性化质粒。然后重复步骤A13-A16。
    18. 使用PureLink HiPure Plasmid Maxiprep Kit完成构建的基于AAV的靶向质粒的最大制备。
    19. 用NanoDrop分光光度计测定maxi-prep质粒DNA的浓度
  2. AAV生产
    1. 将HEK293或HEK293T细胞以约10×10 6细胞/皿的近似密度与10ml含有5%FBS的青霉素/不含链霉素的D-MEM进行培养。细胞在第二天应为70-80%汇合。
    2. 第二天抽取培养基,加入5ml Opti-MEM I还原血清培养基。
      注意:在添加Opti-MEM I还原血清培养基之前洗涤细胞是可选的;然而,洗涤时请小心,因为HEK293(T)细胞松散地粘附在培养皿上。
    3. 将以下质粒移入1.5ml Opti-MEM I还原血清培养基中,轻轻混合:
      pRC 5μg
      pHelper 5μg
      基于AAV的定位质粒 5μg
    4. 加入45μlTransIT-293试剂,轻轻搅拌均匀
    5. 让混合物在室温下静置15分钟。
    6. 将DNA试剂混合物滴入10 cm培养皿中的HEK293(T)细胞上,摇动培养皿轻轻搅拌。
    7. 将细胞孵育在CO 培养箱中37℃24小时 注意:
      1. ”>足以在10厘米培养皿中培养细胞24小时。
      2. ”> CO 2 培养箱在整个研究中在潮湿条件下在37℃和5%CO 2下使用。
    8. 转染24小时后,加入5ml补充有5%FBS和1%青霉素/链霉素的D-MEM。

  3. AAV颗粒的收获
    1. 转染72小时后,通过大量移液从10cm培养皿中刮取HEK293(T)细胞,并将细胞转移到具有上清液的15-ml锥形管中。
    2. 在室温下,以400×g(1500rpm)离心15ml试管5分钟,并小心丢弃上清液,以便不去除细胞。
    3. 用PBS( - )洗涤细胞,以400 xg的速度离心15 ml管5分钟,然后小心丢弃PBS( - ),以免细胞移出。 >
    4. 将细胞沉淀重悬于1.0ml PBS( - )中 注意:如果在制备中将被AAV载体感染的细胞系是已知的,则步骤C4中的细胞沉淀可以重新悬浮在适合于该细胞系而不是PBS( - )的培养基中。 >
    5. 将甲醇倒入干冰中制备冷冻甲醇。将水浴温度设定为37°C 注意:在准备和使用冷冻甲醇时,戴上安全眼镜以保护眼睛。
    6. 将15 ml管浸入冷冻的甲醇中,直到细胞悬液完全冷冻
    7. 将15 ml管转移至37°C水浴,孵育至细胞悬浮液完全解冻。
    8. 重复步骤C6和C7三次(共四个周期)。
    9. 将室温下以400×g离心15ml管5分钟,并将上清液转移至1.5ml或2ml冷冻管中。
    10. 立即使用所得的AAV原药滴定或细胞感染,或将其储存在-80°C直到使用。避免额外的冻融循环。
      注意:如上制备的AAV溶液可以在其用于感染之前使用密度梯度离心或亲和柱纯化,并且这些程序已经在公开的方案中描述(Khan等人,2011; Howes and Schofield,2015 )。尽管AAV溶液的纯化对于体内使用至关重要,但是,通过用这种AAV溶液感染细胞可以在体外实现细胞系中的基因靶向,而无需进一步纯化。

  4. (可选)基于拷贝数估计的AAV滴定
    虽然我们评估了单个载体的基因靶向效率(Karnan等人,2012; Konishi等人,2012年)时,我们滴定了基于AAV的靶向载体,当实验的目的只是为了获得基因靶向的细胞克隆时,滴定是可选的,滴定是可选的。
    1. 对产生AAV颗粒的maxi-prep质粒DNA进行连续10倍稀释,并产生六种不同浓度的质粒溶液(10μg/ ml) > ng /μl)用作定量PCR(qPCR)中的标准样品
    2. 计算稀释质粒溶液中质粒的拷贝数。单链DNA中脱氧核糖核苷酸的平均分子量为303.7。因此,质粒的近似拷贝数(μl -1 )估计为:
      n x 303.7)
      哪里,
      :表示溶液中质粒DNA的浓度(ng /μl),
      :表示Avogadro的常数(6.022 x 10 23 ),
      :表示碱基对质粒的大小。
      注意:与AAV载体基因组的单链性质相反,质粒DNA在常规条件下是双链的。然而,在qPCR中的初始变性步骤之后,质粒DNA被变性并且拷贝数在理论上是双倍的。因此,上面的计算是相应的。
    3. 在1.5 ml管中混合以下试剂:
      AAV原料(以方法C制备) 2.5μl
      RDD缓冲区 5 μl
      DNase I库存解决方案 1.25μl
      蒸馏水 41.25μl
      注意:RDD缓冲液和DNA酶I储备溶液是RNase-Free DNase Set的组件。
    4. 将上述反应混合物在室温(20-25℃)下孵育30分钟以降解残留的质粒DNA
    5. 将反应混合物在95℃孵育5分钟以使DNA酶I失活
    6. 用一式三份的模板进行qPCR,包括步骤D5的AAV溶液,连续稀释的标准样品和蒸馏水(阴性对照)。最初,通过混合以下解决方案制备PCR鸡尾酒:
      KOD FX Neo的2x缓冲区 7.5μlx n
      Primer Neo R -Fwd#1(100 pmol /μl) 0.1μlx
      Primer Neo R -Rev#2(100 pmol /μl) 0.1μlx
      dNTP(每个10 mM) 3μlx
      KOD FX Neo 0.1μlx n
      SYBR Green我的工作解决方案 0.15μlx n
      蒸馏水 3.05微升x
      n :模板数量加上一些额外的
    7. 将鸡尾酒分配到96孔板(14μl/孔)中,向各孔中加入1μl模板,并通过移液混合。
    8. 使用以下实验条件,使用StepOnePlus进行qPCR扩增。后扩增溶解曲线分析包括95℃1分钟和55℃下1分钟,然后缓慢升温至95℃。
      步骤1: 95°C 2分钟
      步骤2(50个周期): 95°C 15秒
      60°C 15秒
      70°C 20秒
    9. 计算每个样本的一式三份数据的平均值。通过将其扩增谱与连续稀释的标准样品进行比较,估算基于AAV的靶向载体的拷贝数。注意,当在步骤D3中用DNase I处理时,用于qPCR的AAV溶液被稀释20倍。在qPCR中生成的标准曲线的一个例子如图8所示

      图8. qPCR标准曲线的示例。红点表示各个样本中的阈值循环。 Ct:阈值循环。

  5. 使用基于AAV的靶向载体进行细胞感染
    1. 在具有合适培养基(生长培养基)的75-cm 2烧瓶中传播用于基因靶向的细胞系。细胞在第二天应为30-40%汇合。
    2. (可选)如果通过简单的程序容易地实现用于基因靶向的细胞系的G1 / S停滞,则在AAV感染之前将细胞停止在G1 / S边界处,因为据报道提高AAV介导的效率基因靶向(Trobridge et al。,2005)。例如,在AAV感染之前,应将MCF-10A细胞与表皮生长因子(EGF) - 培养基孵育6-8h,并且在感染时应将培养基替换为含有EGF的培养基(生长培养基) 。
    3. 在细胞繁殖后的第二天,将37℃水浴中储存在-80℃的AAV储存液解冻
    4. 将培养基从75cm 2烧瓶中吸出,并将(5-x x)ml的生长培养基倒入细胞上,其中 ml)表示AAV原料的预定体积 注意:使用的AAV原料的体积由用于感染细胞的感染复数(MOI)决定。我们通常用1×10 4 MOI的基于AAV的靶向载体感染细胞(Karnan等人,2012; Konishi等人,2012; Damdindorj等人,2014; Karnan等人。,2016);然而,根据实验,可以应用一系列MOI(Russell和Hirata,1998; Hirata和Russell,2000; Miller等,2003; Porteus等,2003)。如果实验的目的只是为了获得基因靶向的细胞克隆,则使用整个(1.0毫升)AAV原料用于75厘米 2的烧瓶。
    5. ml的AAV原料添加到烧瓶中,并通过轻轻摇动进行混合。将CO 培养箱中的细胞孵育过夜。
    6. 在第二天(感染后16-24小时),将7ml生长培养基加入到75-cm 2烧瓶中,并将细胞孵育到CO 2培养箱再两天。
    7. AAV感染被认为是两天后完成的。现在可以将细胞用于测定或实验,尽管随后在许多情况下进行G418选择以消除未感染的细胞。对于用感染细胞进行单细胞克隆,请执行程序F和G.

  6. G418选择96孔组织培养板
    1. 从烧瓶中吸出含有AAV的培养基,用PBS( - )洗涤细胞一次,并将2ml胰蛋白酶吸入烧瓶中。 CO 2培养箱中的胰蛋白酶的孵育时间取决于所用的细胞系。
    2. 旋转烧瓶并在显微镜下检查,以确认细胞解离。然后,将合适体积的生长培养基加入到烧瓶中,移液以进一步离解细胞,并将细胞悬浮液转移到15ml(或50ml)锥形管中。
    3. 以400×g离心细胞5分钟,丢弃上清液,并将细胞重悬于含有适当浓度的G418的14ml生长培养基中。
      注意:在本方案中,当细胞接种到96孔组织培养板中时,开始G418选择,因为这加快了细胞的选择。然而,如果被感染的细胞系对稀疏播种敏感,建议在细胞连接到孔后1-3天播种G418。这可能增加G418选择后获得的菌落数。
    4. 将细胞分配到七个96孔板中。首先,将6ml细胞悬浮液倒入灭菌的移液容器中,然后加入52ml含有G418的生长培养基。
    5. 通过移液将细胞悬浮液混合在储存器中,并使用12通道移液器将细胞接种在三个96孔板(200μl/孔)中。
    6. 将4ml细胞悬浮液倒入储存器中,加入35ml含有G418的生长培养基
    7. 通过移液将细胞悬浮液混合在储存器中,并使用12通道移液器将细胞接种在两个96孔板(200μl/孔)中。
    8. 使用剩余的(4 ml)细胞悬浮液重复步骤F6和F7。
    9. 将96孔板置于CO 2培养箱中,并保持细胞,直到G418选择完成,并通过显微镜检查(通常在播种后7-14天)检测菌落。
      注意:如果96孔板中的菌落由于死亡细胞悬浮在培养基中而不能清晰可见,则可通过更换培养基来改善可见度。
    10. 标记含有G418抗性菌落的孔,并具有永久标记。显然含有多个菌落的孔应该用不同的颜色标记。
    11. 将96孔板在CO 2培养箱中孵育另外7-14天,直到标记孔中的细胞平均达到60-80%汇合。

  7. 基于PCR的细胞克隆筛选
    本节介绍基于PCR的筛选合并样本由手动逐个细胞池生成。如果在G418选择后形成的菌落数量相对较少,可以省略细胞池,并且可以逐个进行筛选菌落(Kohli< et al。 ,2004; Rago et al。 ,2007; Howes and Schofield,2011)。我们之前描述的系统多圆细胞池(Konishi< 等等 )当构建的靶向载体基于pAAV-2Aneo(v2)时通常是不必要的。
    1. 倾倒或吸出96孔板中的培养基,用PBS( - )清洗细胞一次。
    2. 用胰蛋白酶胰蛋白酶消化标记的细胞(20μl/孔)
    3. 细胞解离后,将细胞悬浮液上下移动数次,加入生长培养基(80μl/孔),再次轻轻移液细胞。
    4. 将细胞悬浮液从8-12孔(50μl/孔)中吸取到单个1.5 ml管中。对96孔板的所有标记孔重复此操作。
      注意:为每个细胞转移使用新的移液器吸头,以防止细胞交叉感染。
    5. 向生物标记的孔中加入生长培养基(150μl/孔),并将96孔板放回CO 2培养箱。
      注意:在此步骤中使用含血清培养基中和胰蛋白酶。如果使用血清对于正在工程化的细胞系来说不是最佳的,则在细胞附着于孔后,用合适的生长培养基替换含有血清的培养基。
    6. 使用PureLink Genomic DNA Mini Kit从1.5 ml管中收集细胞样品中提取基因组DNA
    7. 进行筛选PCR以使用KOD FX Neo鉴定含有基因靶向细胞克隆的合并样品,并在步骤G6中提取基因组DNA。 PCR扩增区域应包含同源臂之一:即,引物之一应对应于靶向载体独特的区域,另一个对应于同源臂外的区域(图1和图5)
    8. 通过琼脂糖凝胶电泳分离PCR产物,并鉴定合并的样品,产生预期大小的PCR产物
    9. 在对应于PCR阳性命中的96孔板中的细胞生长至至少50%汇合后,按照标准程序将细胞胰蛋白酶消化并转移至24孔板。
    10. 在24孔板中的细胞生长至少50%汇合后,吸出培养基,用PBS( - )洗涤细胞一次,并用胰蛋白酶(100μl/孔)处理细胞。
    11. 细胞解离后,轻轻地将细胞悬浮液上下移动数次,加入生长培养基(900μl/孔),再次轻轻移液细胞。
    12. 将大约一半的胰蛋白酶化细胞从每个孔转移到1.5-ml管中
    13. 将生长培养基加入到24孔板中至1.5ml /孔,并将板放回CO 2培养箱中。
    14. 使用PureLink Genomic DNA Mini Kit在步骤G12中从样品中提取基因组DNA,使用KOD FX Neo和与步骤G7相同的引物对进行第二次筛选PCR,并通过琼脂糖凝胶电泳鉴定PCR阳性样品。 >
    15. 通过使用定制引物的直接测序验证步骤G14中鉴定的PCR阳性产物。
    16. 将PCR阳性细胞从24孔板转移到6孔板或组织培养皿/烧瓶中。如果阳性细胞可能是非单细胞来源的,则使用有限稀释技术进行单细胞克隆,然后使用与步骤G7和G14相同的引物对,对分离的单细胞克隆进行PCR筛选。同时,使用步骤G14中获得的基因组DNA(图1和图5所示的引物)确认包含其他同源臂的PCR的阳性。应通过直接测序验证预期大小的扩增PCR产物。
      注意:为了进一步确认分离的基因靶向细胞克隆的完整性,将克隆克隆的整个基因组序列是理想的。然而,作为替代方案,我们通常用分离的基因靶向克隆进行Southern印迹,以确认靶向基因组基因座的完整性,并且不存在可以随机整合到基因组中的靶向载体的额外拷贝(Karnan et al。 ,2012; Konishi et al。,2012; Karnan et al。,2016)。

笔记

  1. 靶向载体中的启动子 - 陷阱模块可以插入到内源性靶基因的外显子或内含子中。然而,由于靶向载体中的新霉素磷酸转移酶(Neo R )基因在2A的下游被表达,所以启动子 - 陷阱模块应该插入编码外显子或编码外显子之间的内含子,应该设计同源性武器。
  2. 当将靶向载体中的启动子 - 陷阱模块插入到编码外显子中时,设计PCR反向引物以扩增5'同源臂,使得靶基因的阅读框与图6或7所示的一致。确认在5'同源臂的3'末端不会异位引入终止密码子或多聚腺苷酸化信号。
  3. 由于AAV载体的大小限制,5'和3'同源臂的长度之和不应超过3.0kb。
  4. 在通过PCR扩增同源性臂以构建靶向载体时,理想地从用于后续步骤中的基因靶向的细胞系制备用作模板的基因组DNA。由于同源区域内的多态性,使用不同的细胞系作为PCR模板可能导致同源臂和宿主基因组之间的同源性较低。靶向载体的同源臂的低同源性导致低的基因靶向效率。然而,为了在靶向敲入(例如,,Konishi等人,2007a; Wilsker等人)的同源性臂中引入定义的点突变,缺失或插入, et al。,2008; Gustin等人,2009; Konishi等人,2011),采用与该遗传改变相同的细胞系被敲门或者,在同源臂克隆到载体中以引入所需的改变之后进行定点诱变。
  5. 虽然我们在这里列出了我们实验室定期使用的市售试剂,试剂盒和设备,但大多数可以用其他制造商的等效产品代替。

食谱

  1. 氨苄青霉素工作溶液
    1. 将1μg氨苄青霉素钠溶于20ml蒸馏水(50mg / ml)中。
    2. 使用0.22μm的过滤器灭菌溶液。
    3. 将灭菌的溶液等分成1.5ml管(1ml /管)。
    4. 将等分试样储存在-20°C。
  2. SYBR Green I work solution
    1. 等分SYBR Green I至1.5 ml管(1μl/管)。立即用铝箔包裹管子以防止光照,并将其标记为“80,000x”。
    2. 将“80,000x”管保存在-20°C(将被处理如下的管除外)。使用非清除的盒子进行存储。
    3. 标签为“2000x”的标签为40 ml。 < br />
    4. 在具有39μlDMSO的“80,000”管中稀释1μlSYBR Green I,并将得到的40μl溶液等分至40μl的“ 2,000x'管(再次1μl/管)。
    5. 将“2,000x”管保存在-20°C。使用非清除的盒子进行存储。
    6. 当准备qPCR鸡尾酒时,解冻“2,000x”管,并用19μl蒸馏水稀释内容物。这导致产生100x SYBR Green I工作溶液(20μl)。在qPCR设置后,在解冻的管中丢弃超过100x的工作解决方案。
  3. PBS( - )
    PBS( - )是根据标准程序制备的。
    1. 首先,将以下成分溶于蒸馏水中,生成10倍PBS( - )。
      160.2 g NaCl
      4.0 g KCl
      58.0 g Na 2 HPO 4 .12H 2 O
      4.0 g KH 2 PO 4
      蒸馏水至2升
    2. 然后,将50毫升10倍PBS( - )用450毫升蒸馏水在500毫升瓶中稀释并用高压灭菌灭菌。

致谢

该协议改编自我们以前的工作,Karnan等人。 (2016)。这项工作得到日本科学促进会(JSPS; 25460395至SK,15K19561至AO和25640107至香港)以及武田科学基金会(至香港)的科学研究资助(KAKENHI)的支持。 。

参考

  1. Damdindorj,L.,Karnan,S.,Ota,A.,Hossain,E.,Konishi,Y.,Hosokawa,Y。和Konishi,H。(2014)。 位于腺相关病毒载体中的组成型启动子的比较分析。 PLoS One 9(8):e106472。
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引用:Karnan, S., Ota, A., Konishi, Y., Wahiduzzaman, M., Tsuzuki, S., Hosokawa, Y. and Konishi, H. (2016). Efficient AAV-mediated Gene Targeting Using 2A-based Promoter-trap System. Bio-protocol 6(24): e2058. DOI: 10.21769/BioProtoc.2058.
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