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Biol Open. 2013 Feb 12;2(4):363-7. doi: 10.1242/bio.20133871. Print 2013 Apr 15.

Quantitative assay for TALEN activity at endogenous genomic loci.

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1
Laboratory for Cardiovascular Molecular Dynamics, Quantitative Biology Center , RIKEN, Furuedai 6-2-3, Suita, Osaka 565-0874 , Japan.

Abstract

Artificially designed nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can induce a targeted DNA double-strand break at the specific target genomic locus, leading to the frameshift-mediated gene disruption. However, the assays for their activity on the endogenous genomic loci remain limited. Herein, we describe a versatile modified lacZ assay to detect frameshifts in the nuclease target site. Short fragments of the genome DNA at the target or putative off-target loci were amplified from the genomic DNA of TALEN-treated or control embryos, and were inserted into the lacZα sequence for the conventional blue-white selection. The frequency of the frameshifts in the fragment can be estimated from the numbers of blue and white colonies. Insertions and/or deletions were easily determined by sequencing the plasmid DNAs recovered from the positive colonies. Our technique should offer broad application to the artificial nucleases for genome editing in various types of model organisms.

KEYWORDS:

Genome editing; LacZ blue–white selection; Quantitative assay; TALEN

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