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首页> 外文期刊>BMC Biotechnology >Evaluating target silencing by short hairpin RNA mediated by the group I intron in cultured mammalian cells
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Evaluating target silencing by short hairpin RNA mediated by the group I intron in cultured mammalian cells

机译：通过I组内含子介导的短发夹RNA在培养的哺乳动物细胞中评估靶标沉默

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Background The group I intron, a ribozyme that catalyzes its own splicing reactions in the absence of proteins in vitro , is a potential target for rational engineering and attracted our interest due to its potential utility in gene repair using trans-splicing. However, the ribozyme activity of a group I intron appears to be facilitated by RNA chaperones in vivo ; therefore, the efficiency of self-splicing could be dependent on the structure around the insert site or the length of the sequence to be inserted. To better understand how ribozyme activity could be modulated in cultured mammalian cells, a group I intron was inserted into a short hairpin RNA (shRNA), and silencing of a reporter gene by the shRNA was estimated to reflect self-splicing activity in vivo . In addition, we appended a theophylline-binding aptamer to the ribozyme to investigate any potential effects caused by a trans-effector. Results shRNA-expression vectors in which the loop region of the shRNA was interrupted by an intron were constructed to target firefly luciferase mRNA . There was no remarkable toxicity of the shRNA-expression vectors in Cos cells, and the decrease in luciferase activity was measured as an index of the ribozyme splicing activity. In contrast, the expression of the shRNA through intron splicing was completely abolished in 293T cells, although the silencing induced by the shRNA-expressing vector alone was no different from that in the Cos cells. The splicing efficiency of the aptamer-appended intron also had implications for the potential of trans-factors to differentially promote self-splicing among cultured mammalian cells. Conclusions Silencing by shRNAs interrupted by a group I intron could be used to monitor self-splicing activity in cultured mammalian cells, and the efficiency of self-splicing appears to be affected by cell-type specific factors, demonstrating the potential effectiveness of a trans-effector.
机译：背景技术I类内含子是一种核酶，可在体外不存在蛋白质的情况下催化其自身的剪接反应，是合理工程的潜在目标，并且由于其在使用反式剪接进行基因修复中的潜在效用而引起了我们的兴趣。然而，I组内含子的核酶活性似乎在体内由RNA伴侣促进。因此，自我剪接的效率可能取决于插入位点周围的结构或插入序列的长度。为了更好地了解如何在培养的哺乳动物细胞中调节核酶活性，将I组内含子插入短发夹RNA（shRNA）中，并估计shRNA报道基因的沉默可反映体内的自剪活性。此外，我们将茶碱结合适体附加到核酶上，以研究由反式效应子引起的任何潜在作用。结果构建了shRNA表达载体，其中shRNA的环区被内含子打断，靶向萤火虫荧光素酶mRNA。 shRNA表达载体在Cos细胞中没有明显的毒性，并且荧光素酶活性的降低被测量为核酶剪接活性的指标。相反，尽管仅由表达shRNA的载体诱导的沉默与在Cos细胞中的沉默没有区别，但通过内含子剪接的shRNA的表达在293T细胞中被完全消除。适体附加的内含子的剪接效率也对反式因子在培养的哺乳动物细胞中差异性促进自我剪接的潜力具有影响。结论被I组内含子中断的shRNA沉默可用于监测培养的哺乳动物细胞中的自我剪接活性，而自我剪接的效率似乎受到细胞类型特异性因子的影响，证明了反式-内含子的潜在有效性。效应子。

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