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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

SK Sivasundaram Karnan
Akinobu Ota Akinobu Ota
YK Yuko Konishi
MW Md Wahiduzzaman
ST Shinobu Tsuzuki
YH Yoshitaka Hosokawa
Hiroyuki Konishi Hiroyuki Konishi

发布: 2016年12月20日第6卷第24期 DOI: 10.21769/BioProtoc.2058 浏览次数: 11037

评审: Nicoletta CordaniPinchas TsukermanMartin V Kolev

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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) search AAV (AAV) search Targeting vector (打靶载体) search Gene targeting (基因打靶) search Promoter trap (启动子捕获) search 2A (2A) search Internal ribosome entry site (内部核糖体进入位点) search IRES (IRES) search

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

English
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文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

分子生物学 > DNA > DNA 克隆

分子生物学 > DNA > DNA 重组

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