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A Simple Method to Generate Gene Knockout Clones in Human Cells Using Transcription Activator-Like Effector Nuclease (TALEN)

采用转录激活因子样效应物核酸酶(TALEN)在人细胞中产生基因敲除克隆的简单方法

CH Christy Hammack

发布: 2015年07月20日第5卷第14期 DOI: 10.21769/BioProtoc.1531 浏览次数: 10416

评审: Pinchas TsukermanAnonymous reviewer(s)

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Abstract

Transcription activator-like effectors (TALEs) are naturally occurring proteins secreted by the plant pathogen, Xanthomonas, and fused to the Fok1 endonuclease to generate TALE nucleases (TALENs). TALEN pairs bind to specific DNA sequences initiating Fok1 dimerization and double-stand cleavage of DNA within the TALEN target site. This cleavage event triggers cellular repair mechanisms that result in insertions and/or deletions (indels), which enable gene knockout. The high specificity and efficiency of TALENs makes them important tools for genome editing. Here, we describe a method for the generation of single-cell clones with targeted gene knockout by TALEN using co-transfection and FACS with a fluorescent reporter. This protocol was designed to knockout cell death-inducing DFFA-like effector b, CIDEB, in Huh7.5 cells; however, this protocol can be applied to a wide range of cell types and genes of interest.

Materials and Reagents

  1. Huh7.5 cells (Charles Rice lab, Rockefeller University)
  2. Dulbecco’s modified Eagle’s medium (DMEM high glucose) (HyClone, catalog number: SH30243.02 )
  3. Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 10437-028 )
  4. Antibiotic Antimycotic solution (10,000 U/ml penicillin G, 10,000 µg/ml streptomycin, 25 µg/ml amphotericin B) (HyClone, catalog number: SV30079.01 )
  5. MEM nonessential amino acids (NEAA) (Corning Incorporated, catalog number: 25025CI )
  6. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: BP39920 )
  7. Trypsin, 0.05% (HyClone, catalog number: SH30236.02 )
  8. TALEN Sure KO plasmids (Cellectis)
    Note: It is custom designed to target the gene of interest and  includes right and left TALEN plasmids to bind both sequences of DNA flanking the target site.
  9. pcDNA3-EGFP expression plasmid (Addgene, plasmid number: 13031 )
  10. Lipofectamine 2000 Transfection Reagent (Life Technologies, InvitrogenTM, catalog number: 11668019 )
  11. Opti-MEM I Reduced-Serum Medium (Life Technologies, Gibco®, catalog number: 31985070 )
  12. QuickExtract DNA Extraction Solution (Epicentre, catalog number: QE09050 )
  13. Platinum PCR SuperMix High Fidelity (Life Technologies, InvitrogenTM, catalog number: 12532016 )
  14. Primers flanking the TALEN target site (custom DNA nucleotides) (Integrated DNA Technologies)
  15. QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
  16. Tris-HCl buffer (pH 6.8, 0.5 M) (Bio-Rad Laboratories, AbD Serotec®, catalog number: 1610799 )
  17. Sodium dodecyl sulfate (20% solution) (Thermo Fisher Scientific, catalog number: BP13111 )
  18. Glycerol (Fisher Scientific, catalog number: BP2291 )
  19. Bromophenol blue (Thermo Fisher Scientific, catalog number: B3925 )
  20. 2-mercaptoethanol (Thermo Fisher Scientific, catalog number: O3446I100 )
  21. 10% DMEM culture media (see Recipes)
  22. 20% DMEM culture media (see Recipes)
  23. FACS buffer (see Recipes)
  24. 2x SDS loading buffer (see Recipes)

Equipment

  1. Cell culture incubator at 37 °C and 5% CO2
  2. Cell culture-treated polystyrene plates (100 mm, 6 well, 12 well, 24 well, 48 well, 96 well) (Corning Incorporated)
  3. Polystyrene Round-Bottom Tube (5 ml FACS tubes) (BD Biosciences, Falcon®, catalog number: 352008 )
  4. Round-Bottom Tube Cap (5 ml) (BD Biosciences, Falcon®, catalog number: 352032 )
  5. 40 micron cell strainer (Falcon®, catalog number: 352340 )
  6. Fluorescence microscope
  7. Table top centrifuge
  8. FACSAria cell sorter (BD Biosciences, catalog number: 656700 )
  9. Multi-channel pipette
  10. Western blotting equipment (Bio-Rad Laboratories, AbD Serotec®)
  11. Heat block (VWR international)
  12. PCR thermocycler (Bio-Rad Laboratories, AbD Serotec®)
  13. Agarose gel electrophoresis system
  14. 3730 DNA analyzer (Life Technologies, Applied Biosystems®, catalog number: 3730S )

Procedure

English
中文翻译

文章信息

版权信息

© 2015 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Hammack, C. (2015). A Simple Method to Generate Gene Knockout Clones in Human Cells Using Transcription Activator-Like Effector Nuclease (TALEN). Bio-protocol 5(14): e1531. DOI: 10.21769/BioProtoc.1531.

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分类

细胞生物学 > 细胞工程 > TALEN技术

分子生物学 > DNA > 诱/突变

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