Efficient Knockout of Transplanted Green Fluorescent Protein Gene in Medaka Using TALENs
Efficient Knockout of Transplanted Green Fluorescent Protein Gene in Medaka Using TALENs
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DOI：
10.1007/s10126-014-9584-x
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发表时间：
2014-07
影响因子：
3
通讯作者：
Chao Qiu;Bin Cheng;Yunsheng Zhang;Rong Huang;L. Liao;Yongming Li;Daji Luo;Wei Hu;Yaping Wang-Ya
中科院分区：
文献类型：
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作者：
Chao Qiu;Bin Cheng;Yunsheng Zhang;Rong Huang;L. Liao;Yongming Li;Daji Luo;Wei Hu;Yaping Wang-Ya
类转录激活因子效应核酸酶 (TALEN) 用于斑马鱼的基因敲除和基因组编辑研究，这些技术有可能应用于其他鱼类。在这里，我们证明 TALEN 可以通过影响 GFP 的翻译和合成，直接敲除青鳉中的绿色荧光蛋白 (GFP) 转基因。我们构建了携带GFP和红色荧光蛋白（RFP）基因的转基因质粒（pGFP-RFP），并使用改良的TALEN方法为GFP的核心发色团Y66区域组装了一对TALEN。 TALEN信使RNA（mRNA）的胚胎毒性远低于线性化质粒；同时，76.3%的胚胎，TALEN mRNA和线性化质粒共注射后，胚胎的绿色荧光显着降低，但红色荧光没有显着变化。实时定量聚合酶链式反应和测序结果表明，在共注射青鳉胚胎中，近 100% 的 GFP 突变位置在 GFP 的 Y66 区域被破坏，这是由 TALEN 引起的。这导致核苷酸的随机插入-缺失，从而影响 GFP 的翻译并破坏 GFP 的合成。这为在仅已知关键功能域的基因中设计 TALEN 位点提供了新的实验证据。我们的结果表明，改良的 TALEN 方法可以有效且特异性地介导青鳉中的转基因敲除。该报告可能会促进TALEN在斑马鱼以外鱼类基因编辑研究中的应用。
Transcription activator-like effector nucleases (TALENs) are used for gene knockout and genome-editing studies in zebrafish, and these techniques have the potential to be applied to other fish species. Here, we show that TALENs can directly knock out a green fluorescent protein (GFP) transgene in medaka by affecting translation and synthesis of the GFP. We constructed a transgenic plasmid (pGFP-RFP) carrying the GFP and red fluorescent protein (RFP) genes, and used a modified TALEN method to assemble a pair of TALENs for the core chromophore Y66 region of GFP. Embryo toxicity of TALEN messenger RNA (mRNA) was far lower than the linearized plasmid; meanwhile, 76.3 % embryos, green fluorescence of embryos decreased significantly after co-injection of TALEN mRNA and the linearized plasmid, but red fluorescence showed no significant change. Real-time quantitative polymerase chain reaction and sequencing results showed that nearly 100 % mutated GFP position was disrupted at the Y66 region of GFP in the co-injected medaka embryos, caused by TALENs. This led to random insertion-deletion of nucleotides, which affected the translation of GFP and disrupted GFP synthesis. This provides new experimental evidence for designing TALEN sites in genes for which only key functional domains are known. Our results show that a modified TALEN method can efficiently and specifically mediate a transgene knockout in medaka. This report may promote the application of TALENs in gene-editing studies of fish species other than zebrafish.