Quantitative assay for TALEN activity at endogenous genomic loci.
Quantitative assay for TALEN activity at endogenous genomic loci.
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DOI：
10.1242/bio.20133871
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发表时间：
2013-04-15
期刊：
影响因子：
2.4
通讯作者：
Kawahara A
中科院分区：
文献类型：
--
作者：
Hisano Y;Ota S;Arakawa K;Muraki M;Kono N;Oshita K;Sakuma T;Tomita M;Yamamoto T;Okada Y;Kawahara A
人工设计的核酸酶，如锌指核酸酶 (ZFN) 和转录激活子样效应核酸酶 (TALEN)，可以在特定的靶基因组位点诱导靶向 DNA 双链断裂，从而导致移码介导的基因破坏。然而，对其内源基因组基因座活性的测定仍然有限。在此，我们描述了一种通用的改良 lacZ 测定法来检测核酸酶靶位点的移码。从 TALEN 处理或对照胚胎的基因组 DNA 中扩增出目标或推定脱靶位点的基因组 DNA 短片段，并将其插入到 lacZα 序列中以进行传统的蓝白斑选择。片段中移码的频率可以根据蓝色和白色菌落的数量来估计。通过对从阳性菌落回收的质粒 DNA 进行测序，可以轻松确定插入和/或缺失。我们的技术应该为人工核酸酶在各种模型生物体中进行基因组编辑提供广泛的应用。
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.
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影响因子：
46.9
作者：
通讯作者：
--
影响因子：
7
作者：
Kim, Hye Joo;Lee, Hyung Joo;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
影响因子：
14.9
作者：
Doyle EL;Booher NJ;Standage DS;Voytas DF;Brendel VP;Vandyk JK;Bogdanove AJ
通讯作者：
Bogdanove AJ
DOI：
10.1093/bioinformatics/btp698
发表时间：
2010-03-01
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Li H;Durbin R
通讯作者：
Durbin R
影响因子：
64.8
作者：
Kupperman, E;An, SZ;Stainier, DYR
通讯作者：
Stainier, DYR