Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector.
Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector.
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DOI：
10.1038/s41598-017-02460-2
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发表时间：
2017-05-19
影响因子：
4.6
通讯作者：
Liu J
中科院分区：
文献类型：
--
作者：
Liu Z;Chen O;Wall JBJ;Zheng M;Zhou Y;Wang L;Vaseghi HR;Qian L;Liu J
在单一载体上克隆多个基因，极大地促进了需要多种因子或复杂蛋白的多单位共同表达的基础和转化研究。人们采用了许多策略，其中2A“自切割”肽因其多顺反电子性质、小尺寸和高“切割”效率而受到越来越多的关注。然而，由于缺乏对不同的2As单独或组合的系统比较，限制了2a肽的广泛应用。在这里，我们描述了不同的基因位置和2As对双、三或四顺反子结构中编码的蛋白质表达的影响。以直接心脏重编程为例，我们进一步确定了不同的2As对荧光细胞标记效率和细胞命运转化的影响。我们发现，当荧光团移动到结构的末端时，其表达减少，而在第二个位置的荧光团重编程效率最高。此外，四顺子TPE2A结构比3P2A或PTE2A结构更有效地进行重编程。我们期望本文构建的双、三、四顺反子载体以及不同2A构建体的蛋白表达率的结果可以指导未来2A肽在基础研究和临床应用中的应用。
Cloning of multiple genes in a single vector has greatly facilitated both basic and translational studies that require co-expression of multiple factors or multi-units of complex protein. Many strategies have been adopted, among which 2A “self-cleaving” peptides have garnered increased interest for their polycistronic nature, small size and high “cleavage” efficiency. However, broad application of 2 A peptides is limited by the lack of systematic comparison of different 2As alone or in combination. Here we characterized the effect of varying gene position and 2As on the expression of proteins encoded in bi-, tri-, or quad-cistronic constructs. Using direct cardiac reprogramming as an example, we further determined the effect of varied 2As on the efficiency of fluorescent cell labeling and cell fate conversion. We found that the expression of fluorophores decreased as it was moved towards the end of the construct while reprogramming was most efficient with the fluorophore at the second position. Moreover, quad-cistronic TPE2A constructs resulted in more efficient reprogramming than 3P2A or PTE2A constructs. We expect that the bi-, tri-, and quad-cistronic vectors constructed here and our results on protein expression ratios from different 2A constructs could serve to guide future utilization of 2A peptides in basic research and clinical applications.