Published: Vol 5, Iss 14, Jul 20, 2015 DOI: 10.21769/BioProtoc.1531 Views: 9606
Reviewed by: 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
Equipment
Procedure
Representative data
Figure 1. Representative data. A. Representative Western blot for the identification of 4 clones negative for CIDEB protein expression, suggesting successful gene knockout by TALEN. B. Representative DNA sequence analysis of clone 3 confirming that the target region contains TALEN-induced indels leading to loss of gene expression.
Notes
Recipes
Acknowledgments
This work was supported by NIH grant R01 A1079150.
References
Article Information
Copyright
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
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.
Category
Cell Biology > Cell engineering > TALEN
Molecular Biology > DNA > Mutagenesis
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