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首页> 外文期刊>Marine biotechnology >Hybrid Cytomegalovirus-U6 Promoter-based Plasmid Vectors Improve Efficiency of RNA Interference in Zebrafish.
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Hybrid Cytomegalovirus-U6 Promoter-based Plasmid Vectors Improve Efficiency of RNA Interference in Zebrafish.

机译：杂种基于巨细胞病毒-U6启动子的质粒载体可提高斑马鱼中RNA干扰的效率。

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Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.
机译：已显示由RNA聚合酶III（Pol III）或Pol II启动子指导的短发夹RNA（shRNA）能够沉默基因表达，这应允许分析基因功能或作为潜在的治疗工具。但是，shRNA的抑制作用在鱼类中仍然存在问题。我们证明了由由CMV增强子或整个CMV启动子组成的杂种构建体产生的shRNA的沉默效率，该启动子直接位于U6启动子的上游。当测试外源基因时，增强型绿色荧光蛋白（EGFP）靶基因的沉默对于CMVE-U6启动子组是89.18 +/- 5.06％，对于CMV-U6启动子组是88.26 +/- 6.46％。为了测试针对内源基因驱动shRNA效率的杂合启动子，我们使用了针对无尾巴（NTL）基因的shRNA。当在斑马鱼中进行载体化时，杂种构建体会强烈抑制NTL基因表达。对于CMVE-U6启动子和CMV-U6启动子组，NTL表型分别占据52.09 +/- 3.06％和51.56 +/- 3.68％。对于CMVE-U6启动子组，NTL基因表达降低82.17 +/- 2.96％，而对于CMV-U6启动子组，NTL基因表达降低83.06 +/- 2.38％。我们得出的结论是，与单独的CMV启动子或U6启动子相比，位于U6-启动子上游的CMV增强子或整个CMV启动子可以显着提高shRNA诱导的对外源和内源基因的抑制作用。相反，两个杂合的启动子构建体对shRNA的驱动具有相似的作用。

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