RNA Interference Is Responsible for Reduction of Transgene Expression after Sleeping Beauty Transposase Mediated Somatic Integration
RNA Interference Is Responsible for Reduction of Transgene Expression after Sleeping Beauty Transposase Mediated Somatic Integration
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DOI：
10.1371/journal.pone.0035389
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发表时间：
2012-05-03
期刊：
影响因子：
3.7
通讯作者：
Ehrhardt, Anja
中科院分区：
文献类型：
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作者：
Rauschhuber, Christina;Ehrhardt, Anja
工作背景：基于转座因子的整合型非病毒载体在功能基因组学和治疗性基因转移中被广泛用于哺乳动物细胞的基因工程。对于睡美人（SB）转座酶系统，证明了真核细胞中由SB转座酶反向重复序列（IR）驱动的会聚转录发生在体细胞整合之后。这可能导致双链RNA的形成，其潜在地呈现RNA干扰（RNAi）机制的靶标，并随后导致转基因的沉默。因此，我们的目的是调查转基因表达转座下RNA干扰敲低conditions.Principal发现：为了建立RNAi敲低细胞系，我们利用了P19蛋白，这是来自番茄丛矮病毒。P19结合并抑制21个核苷酸长的小干扰RNA，并显示出充分抑制RNAi。我们发现SB介导转座后转基因表达增强，导致转座后集落形成单位（CFU）的量增加3.2倍。相比之下，如果转基因盒通过鸡来源的cHS 4绝缘序列与染色体位置效应的影响绝缘，或者当应用Forg Prince转座子系统时，其仅显示可忽略的转录活性，则获得相似数量的CFU。总的来说，我们首次提供了体细胞整合后转座子衍生的转基因表达受内源性RNAi机制调节的证据。在未来，这一发现将有助于进一步改善SB转座酶载体系统的分子设计。
Background: Integrating non-viral vectors based on transposable elements are widely used for genetically engineering mammalian cells in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB) transposase system it was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs) in eukaryotic cells occurs after somatic integration. This could lead to formation of double-stranded RNAs potentially presenting targets for the RNA interference (RNAi) machinery and subsequently resulting into silencing of the transgene. Therefore, we aimed at investigating transgene expression upon transposition under RNA interference knockdown conditions.Principal Findings: To establish RNAi knockdown cell lines we took advantage of the P19 protein, which is derived from the tomato bushy stunt virus. P19 binds and inhibits 21 nucleotides long, small-interfering RNAs and was shown to sufficiently suppress RNAi. We found that transgene expression upon SB mediated transposition was enhanced, resulting into a 3.2-fold increased amount of colony forming units (CFU) after transposition. In contrast, if the transgene cassette is insulated from the influence of chromosomal position effects by the chicken-derived cHS4 insulating sequences or when applying the Forg Prince transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained.Conclusion: In summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system.