Comparison of IRES and F2A-based locus-specific multicistronic expression in stable mouse lines.
Comparison of IRES and F2A-based locus-specific multicistronic expression in stable mouse lines.
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DOI：
10.1371/journal.pone.0028885
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Lufkin T
中科院分区：
文献类型：
--
作者：
Chan HY;V S;Xing X;Kraus P;Yap SP;Ng P;Lim SL;Lufkin T
通过双顺反子或多顺反子载体连接的基因的高效和化学计量表达不仅在基础生物学中已经成为追踪和可视化体内蛋白质的宝贵工具，而且在疫苗开发和基因治疗的临床中也是如此。为了在体内充分比较两种目前流行的共表达策略的有效性-来自小核糖核酸病毒的内部核糖体进入位点（IRES）和来自口蹄疫病毒（FDMV）的2A肽（F2 A），我们分析了两个位点特异性敲入小鼠品系，它们共表达含SRY盒的基因9（Sox 9）和由IRES连接的增强型绿色荧光蛋白（EGFP），（Sox 9 IRES-EGFP）或F2 A（Sox 9 F2 A-EGFP）序列。两种构建体在适当的Sox 9表达结构域中表达Sox 9和EGFP蛋白，与F2 A相比，IRES构建体表达降低水平的EGFP。另一方面，后者产生约42.2%的Sox 9-EGFP融合蛋白，反映了低效的核糖体“跳跃”机制。为了研究“跳跃”过程中的差异是否是基因座依赖性的，我们进一步分析了FLAG 3-Bapx 1F 2A-EGFP小鼠系，发现产生了相似水平的融合蛋白。为了评估EGFP是否阻碍了“跳跃”机制，我们检测了另一种共表达风笛同源框基因1同源物（Bapx 1）、Cre重组酶和EGFP的小鼠品系（Bapx 1F 2A-Cre-F2 A-EGFP）。虽然Bapx 1和Cre之间的“跳跃”是高效的，但Cre和EGFP之间的“跳跃”是非常低效的。因此，我们在我们的比较研究中已经证明，在稳定的小鼠系中可以实现由F2 A连接的基因的有效和接近等同的表达，但是EGFP报告基因可能导致F2 A序列处的“跳跃”的不期望的抑制。因此，当利用F2 A肽时，应探索其他报告基因的使用。
Efficient and stoichiometric expression of genes concatenated by bi- or multi-cistronic vectors has become an invaluable tool not only in basic biology to track and visualize proteins in vivo, but also for vaccine development and in the clinics for gene therapy. To adequately compare, in vivo, the effectiveness of two of the currently popular co-expression strategies - the internal ribosome entry site (IRES) derived from the picornavirus and the 2A peptide from the foot-and-mouth disease virus (FDMV) (F2A), we analyzed two locus-specific knock-in mouse lines co-expressing SRY-box containing gene 9 (Sox9) and enhanced green fluorescent protein (EGFP) linked by the IRES (Sox9IRES-EGFP) or the F2A (Sox9F2A-EGFP) sequence. Both the constructs expressed Sox9 and EGFP proteins in the appropriate Sox9 expression domains, with the IRES construct expressing reduced levels of EGFP compared to that of the F2A. The latter, on the other hand, produced about 42.2% Sox9-EGFP fusion protein, reflecting an inefficient ribosome ‘skipping’ mechanism. To investigate if the discrepancy in the ‘skipping’ process was locus-dependent, we further analyzed the FLAG3-Bapx1F2A-EGFP mouse line and found similar levels of fusion protein being produced. To assess if EGFP was hindering the ‘skipping’ mechanism, we examined another mouse line co-expressing Bagpipe homeobox gene 1 homolog (Bapx1), Cre recombinase and EGFP (Bapx1F2A-Cre-F2A-EGFP). While the ‘skipping’ was highly efficient between Bapx1 and Cre, the ‘skipping’ between Cre and EGFP was highly inefficient. We have thus demonstrated in our comparison study that the efficient and close to equivalent expression of genes linked by F2A is achievable in stable mouse lines, but the EGFP reporter may cause undesirable inhibition of the ‘skipping’ at the F2A sequence. Hence, the use of other reporter genes should be explored when utilizing F2A peptides.
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