High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos.
High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos.
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DOI：
10.1242/bio.20133855
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发表时间：
2013-05-15
期刊：
影响因子：
2.4
通讯作者：
Yamamoto T
中科院分区：
文献类型：
--
作者：
Suzuki KT;Isoyama Y;Kashiwagi K;Sakuma T;Ochiai H;Sakamoto N;Furuno N;Kashiwagi A;Yamamoto T
近年来，利用转录激活因子样效应核酸酶（TALENs）进行基因编辑已在生命科学中得到应用。TALENs可以很容易地定制识别特定的DNA序列，并有效地在目标基因组位点引入双链断裂。随后的非同源末端连接修复通过碱基插入、删除或两者同时进行导致靶向基因破坏。为了更容易地评估TALENs在非洲爪蟾胚胎中的效果，我们对酪氨酸酶（tyr）和pax6基因进行了靶向基因破坏，这两个基因分别参与色素沉着和眼睛形成。我们构建了靶向tyr和pax6的TALENs，并将其mrna注射到单细胞期受精卵中。意料之中的是，tyr TALEN mRNA的引入导致了色素沉着的急剧丧失，而且效率很高。同样，对于pax6， TALENs导致注射胚胎的眼睛变形。我们通过限制性内切酶酶切和基因组PCR产物的序列分析证实了目标等位基因的突变。令人惊讶的是，不仅双等位基因，而且在同源基因中也观察到基因破坏。我们的研究结果表明，TALENs的靶向基因破坏提供了一种与反义morpholinos相当的方法来分析非洲爪蟾F0胚胎的基因功能，而且还适用于胚胎发生以外的任何生命阶段。
Recently, gene editing with transcription activator-like effector nucleases (TALENs) has been used in the life sciences. TALENs can be easily customized to recognize a specific DNA sequence and efficiently introduce double-strand breaks at the targeted genomic locus. Subsequent non-homologous end-joining repair leads to targeted gene disruption by base insertion, deletion, or both. Here, to readily evaluate the efficacy of TALENs in Xenopus laevis embryos, we performed the targeted gene disruption of tyrosinase (tyr) and pax6 genes that are involved in pigmentation and eye formation, respectively. We constructed TALENs targeting tyr and pax6 and injected their mRNAs into fertilized eggs at the one-cell stage. Expectedly, introduction of tyr TALEN mRNA resulted in drastic loss of pigmentation with high efficiency. Similarly, for pax6, TALENs led to deformed eyes in the injected embryos. We confirmed mutations of the target alleles by restriction enzyme digestion and sequence analyses of genomic PCR products. Surprisingly, not only biallelic but also paralogous, gene disruption was observed. Our results demonstrate that targeted gene disruption by TALENs provides a method comparable to antisense morpholinos in analyzing gene function in Xenopus F0 embryos, but also applies beyond embryogenesis to any life stage.
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影响因子：
14.9
作者：
Cade L;Reyon D;Hwang WY;Tsai SQ;Patel S;Khayter C;Joung JK;Sander JD;Peterson RT;Yeh JR
通讯作者：
Yeh JR
DOI：
10.1034/j.1600-0749.2003.00064.x
发表时间：
2003-10-01
期刊：
PIGMENT CELL RESEARCH
影响因子：
--
作者：
Kumasaka, M;Sato, S;Yamamoto, H
通讯作者：
Yamamoto, H
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
影响因子：
3
作者：
Rungger-Braendle, Elisabeth;Ripperger, Juergen A.;Rungger, Duri
通讯作者：
Rungger, Duri
影响因子：
46.9
作者：
Miller, Jeffrey C.;Tan, Siyuan;Rebar, Edward J.
通讯作者：
Rebar, Edward J.