The Nonmonotonic Dose Dependence of Protein Expression in Cells Transfected with Self-Amplifying RNA
The Nonmonotonic Dose Dependence of Protein Expression in Cells Transfected with Self-Amplifying RNA
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自扩增RNA转染细胞中蛋白质表达的非单调剂量依赖性
DOI：
10.1128/jvi.01858-21
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发表时间：
2022
影响因子：
5.4
通讯作者：
Gelbart, W. M.
中科院分区：
文献类型：
--
作者：
Tanimoto, C. R.;Thurm, A. R.;Brandt, D. S.;Knobler, C. M.;Gelbart, W. M.
来自正义RNA病毒基因组的自我扩增（sa） RNA分子——“复制子”——作为基因和疫苗传递载体正受到越来越多的关注。这是因为感兴趣的基因的mRNA形式可以被纳入其中并被强烈扩增，从而增强目标蛋白的表达。在本报告中，我们证明了蛋白质表达对转染的复制子质量的非单调依赖，与通常的非sarna转染的单调情况相反。我们以sa形式的增强黄色荧光蛋白（eYFP）作为报告基因，用脂肪转染了多种细胞系，发现存在一个“甜蜜点”，在这个“甜蜜点”上，蛋白质的表达和细胞活力是最佳的。为了控制给定质量的Lipofectamine转染的复制子RNA的变化质量，将复制子与既不复制也不翻译的“载体”RNA混合；转染RNA的总质量保持不变，同时将复制子的比例从0增加到1。荧光显微镜研究表明，在转染总RNA的1/10的复制子片段中，获得了最佳的蛋白质表达和细胞活力，这些结果通过系统的流式细胞术测量进行了量化。尽管在每个细胞中只有一个基因组拷贝开始病毒生命周期，但由于其RNA复制酶蛋白的强度，阳性RNA病毒通常对其宿主细胞具有细胞毒性作用。它们的非传染性形式——复制子——只包括它们的RNA复制相关基因，也具有很强的自我扩增能力和细胞毒性。因此，当与感兴趣的非病毒基因融合的复制子被转染到细胞中以扩增感兴趣的蛋白质表达时，需要将复制子“剂量”保持在足够低的水平。我们演示了如何控制RNA复制子进入转染细胞的数量，以及复制子剂量的最佳点，以优化蛋白质表达和细胞活力。本文给出了在多种哺乳动物细胞系中带有荧光蛋白报告基因的野田村病毒复制子的例子，这些复制子通过流式细胞术和活/死细胞测定法进行了量化。
Self-amplifying (sa) RNA molecules—“replicons”—derived from the genomes of positive-sense RNA viruses are receiving increasing attention as gene and vaccine delivery vehicles. This is because mRNA forms of genes of interest can be incorporated into them and strongly amplified, thereby enhancing target protein expression. In this report, we demonstrate a nonmonotonic dependence of protein expression on the mass of transfected replicon, in contrast to the usual, monotonic case of non-saRNA transfections. We lipotransfected a variety of cell lines with increasing masses of enhanced yellow fluorescent protein (eYFP) as a reporter gene in sa form and found that there is a “sweet spot” at which protein expression and cell viability are optimum. To control the varying mass of transfected replicon RNA for a given mass of Lipofectamine, the replicons were mixed with a “carrier” RNA that is neither replicated nor translated; the total mass of transfected RNA was kept constant while increasing the fraction of the replicon from zero to one. Fluorescence microscopy studies showed that the optimum protein expression and cell viability are achieved for replicon fractions as small as 1/10 of the total transfected RNA, and these results were quantified by a systematic series of flow cytometry measurements.IMPORTANCEPositive-sense RNA viruses often have a cytotoxic effect on their host cell because of the strength of their RNA replicase proteins, even though only one copy of their genome begins the viral life cycle in each cell. Noninfectious forms of them—replicons—which include just their RNA replication-related genes, are also strongly self-amplifying and cytotoxic. Accordingly, when replicons fused with nonviral genes of interest are transfected into cells to amplify expression of proteins of interest, one needs to keep the replicon “dose” sufficiently low. We demonstrate how to control the number of RNA replicons getting into transfected cells and that there is a sweet spot for the replicon dose that optimizes protein expression and cell viability. Examples are given for the case of Nodamura viral replicons with fluorescent protein reporter genes in a variety of mammalian cell lines, quantified by flow cytometry and live/dead cell assays.
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DOI：
--
发表时间：
1998
期刊：
Cytotechnology (Dordrecht)
影响因子：
--
作者：
E. Schlaeger;K. Lundstrom
通讯作者：
K. Lundstrom
影响因子：
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作者：
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Gelbart, William M.
DOI：
10.1016/j.nano.2013.11.008
发表时间：
2014-05-01
影响因子：
5.4
作者：
Leonhardt, Carolin;Schwake, Gerlinde;Raedler, Joachim O.
通讯作者：
Raedler, Joachim O.
影响因子：
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作者：
Barreau, Carine;Dutertre, Stephanie;Osborne, H. Beverley
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基于复制子的正链 RNA 病毒载体。
DOI：
--
发表时间：
2000
期刊：
Current opinion in molecular therapeutics (Print)
影响因子：
--
作者：
A. Khromykh
通讯作者：
A. Khromykh