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
发布: 2016年12月20日第6卷第24期 DOI: 10.21769/BioProtoc.2058 浏览次数: 10185
评审: Nicoletta CordaniPinchas TsukermanMartin V Kolev
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参见作者原研究论文
The authors used this protocol in:
Apr 2016
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
- Pipette tips
- 10-cm culture dish
- 1.5-ml or 2-ml cryovial
- 96-well plate
- 15-ml (or 50-ml) conical tube
- 24-well plates
- 6-well plates or tissue culture dishes/flasks
- 0.22 µm filter
- 1.5-ml tubes
- Aluminum foil
- Disposable pipetting reservoir (AS ONE, catalog number: 2-7844-02 )
- E. coli DH5α competent cells (Takara Bio, catalog number: 9057 )
Note: This product has not been discontinued. - HEK293 or HEK293T cell line
- Cell line(s) for gene targeting
- pAAV-2Aneo (Addgene, catalog number: 80032 )
- pAAV-2Aneo v2 (Addgene, catalog number: 80033 )
- Agarose (NIPPON GENE, catalog number: 318-01195 )
- Pwo SuperYield DNA polymerase (Roche Diagnostics, catalog number: 04340850001 )
- PureLink® Quick Gel Extraction Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: K2100-25 )
- PureLink® PCR Purification Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: K3100-02 )
- Quick LigationTM Kit (New England BioLabs, catalog number: M2200S )
- Dry ice
- Restriction enzymes BspEI, MluI, or BsrGI (New England BioLabs, Ipswich, MA)
- Restriction enzyme buffers
- LB broth (NACALAI TESQUE, catalog number: 20068-75 )
- Mini PlusTM Plasmid DNA extraction system (Viogene, catalog number: GF2002 )
- BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4337455 )
- Alkaline phosphatase, calf intestinal (CIP) (New England BioLabs, catalog number: M0290L )
- Custom-made PCR primers (for the amplification of 5’ and 3’ homology arms)
- Custom-made sequencing primers (for the sequencing of 5’ and 3’ homology arms)
- Custom-made PCR primers (for the screening of gene-targeted clones)
- PureLink® HiPure Plasmid Maxiprep Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: K210007 )
- Dulbecco’s modified Eagle’s medium (D-MEM) (Wako Pure Chemical Industries, catalog number: 044-29765 )
- Fetal bovine serum (FBS) (NICHIREI, catalog number: 172012-500ml )
- Opti-MEM® I reduced-serum medium (Thermo Fisher Scientific, GibcoTM, catalog number: 31985-070 )
- pRC and pHelper plasmids in AAV Helper-free system (Agilent Technologies, catalog number: 240071 )
- TransIT®-293 transfection reagent (Mirus Bio, catalog number: MIR 2705 )
- Penicillin-streptomycin solution (x100) (Wako Pure Chemical Industries, catalog number: 168-23191 )
- Methanol
- Distilled water
- RNase-free DNase set (QIAGEN, catalog number: 79254 )
- NeoR-Rev #1: 5’-GGCATCAGAGCAGCCGATTG
- NeoR-Fwd #1: 5’-CATTCGACCACCAAGCGAAA
- NeoR-Rev #2: 5’-CTTGAGCCTGGCGAACAGTT
- KOD FX Neo (TOYOBO, catalog number: KFX-201 )
- Growth medium appropriate for the cell line(s) undergoing gene targeting
- 0.25% (w/v) trypsin solution with phenol red (Wako Pure Chemical Industries, catalog number: 201-18841 )
- PCR buffer
- dNTPs
- SYBR® Green I nucleic acid stain (Lonza, catalog number: 50512 )
- PureLink® Genomic DNA Mini Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: K182002 )
- Ampicillin sodium (Wako Pure Chemical Industries, catalog number: 012-23303 )
- Dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries, catalog number: 048-21985 )
- NaCl
- KCl
- Na2HPO4•12H2O
- KH2PO4
- Ampicillin working solution (see Recipes)
- SYBR Green I working solution (see Recipes)
- PBS(-) (see Recipes)
Equipment
- Humidified CO2 incubator
- VeritiTM thermal cycler (Thermo Fisher Scientific, model: Veriti Thermal Cycler )
- Mupid®-2plus submarine-type electrophoresis system (Takara Bio, model: Mupid-2plus System )
- NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific)
- StepOnePlusTM Real-Time PCR system (Thermo Fisher Scientific, Applied BiosystemTM, model: StepOnePlusTM Real-Time PCR system )
- Centrifuge for molecular biology experiments
- -20 °C freezer
- Tissue culture hood
- Phase-contrast microscope
- Swing bucket centrifuge for tissue culture
- -80 °C deep freezer
- Water bath
- 12-channel pipettor
- Two permanent markers of different colors
Procedure
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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