Development of a new bicistronic retroviral vector with strong IRES activity.
Development of a new bicistronic retroviral vector with strong IRES activity.
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开发具有强大IRES活性的新型双科逆转录病毒载体。
DOI：
10.1186/1472-6750-6-4
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发表时间：
2006-01-12
影响因子：
3.5
通讯作者：
Pognonec, P
中科院分区：
文献类型：
--
作者：
Martin, P;Albagli, O;Poggi, MC;Boulukos, KE;Pognonec, P
基于内部核糖体进入位点（IRES）的双顺反子载体是当今细胞生物学中的重要工具。在应用中，在独特启动子控制下的两种蛋白质的表达允许通过观察容易检测的示踪剂（例如绿色荧光蛋白（GFP））来监测其生物学功能正在被研究的蛋白质的表达。然而，对利用双顺反子载体的公开结果的分析表明，IRES控制的表达的效率可以从一个载体到另一个载体变化很大，尽管它们的IRES序列明显相同。我们研究了这些差异的分子基础。我们观察到IRES控制的表达与具有相同表观IRES序列的不同双顺反子表达载体的差异高达10倍。我们表明，插入的HindIII位点，在野生型EMCV IRES的起始AUG密码子的地方，是负责从第二顺反子的表达的戏剧性损失，而从第一顺反子的表达保持不受影响。因此，虽然真正的病毒起始AUG由HindIII位点的替代导致HindIII亚克隆cDNA的起始密码子的理论使用，但随后的表达下降显著地减少了双顺反子结构的兴趣。事实上，HindIII位点的插入对IRES功能具有如此负面的影响，以致IRES控制的产物的检测可能是困难的，并且有时甚至低于检测水平。令人惊讶的是，观察到这种有害的修饰广泛存在于可获得的含IRES的载体（包括商业载体）中，尽管文献中的早期报道指出起始密码子的完整性对于最佳IRES功能的重要性。根据这些观察结果，我们设计了一个新的载体家族pPRIG，它尊重EMCV IRES结构，并允许第一顺反子中任何cDNA的容易克隆、标记、测序和表达，同时保持其IRES依赖性第二顺反子（此处编码eGFP）的高水平表达。
Internal Ribosome Entry Site (IRES)-based bicistronic vectors are important tools in today's cell biology. Among applications, the expression of two proteins under the control of a unique promoter permits the monitoring of expression of a protein whose biological function is being investigated through the observation of an easily detectable tracer, such as Green Fluorescent Protein (GFP). However, analysis of published results making use of bicistronic vectors indicates that the efficiency of the IRES-controlled expression can vary widely from one vector to another, despite their apparent identical IRES sequences. We investigated the molecular basis for these discrepancies. We observed up to a 10 fold difference in IRES-controlled expression from distinct bicistronic expression vectors harboring the same apparent IRES sequences. We show that the insertion of a HindIII site, in place of the initiating AUG codon of the wild type EMCV IRES, is responsible for the dramatic loss of expression from the second cistron, whereas expression from the first cistron remains unaffected. Thus, while the replacement of the authentic viral initiating AUG by a HindIII site results in the theoretical usage of the initiation codon of the HindIII-subcloned cDNA, the subsequent drop of expression dramatically diminishes the interest of the bicistronic structure. Indeed, insertion of the HindIII site has such a negative effect on IRES function that detection of the IRES-controlled product can be difficult, and sometimes even below the levels of detection. It is striking to observe that this deleterious modification is widely found in available IRES-containing vectors, including commercial ones, despite early reports in the literature stating the importance of the integrity of the initiation codon for optimal IRES function. From these observations, we engineered a new vector family, pPRIG, which respects the EMCV IRES structure, and permits easy cloning, tagging, sequencing, and expression of any cDNA in the first cistron, while keeping a high level of expression from its IRES-dependent second cistron (here encoding eGFP).
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影响因子：
10.5
作者：
JANG, SK;WIMMER, E
通讯作者：
WIMMER, E
影响因子：
14.9
作者：
Chambard, JC;Pognonec, P
通讯作者：
Pognonec, P
影响因子：
11.4
作者：
KAMINSKI, A;BELSHAM, GJ;JACKSON, RJ
通讯作者：
JACKSON, RJ
影响因子：
4.5
作者：
Ohlmann, T;Jackson, RJ
通讯作者：
Jackson, RJ
影响因子：
5.3
作者：
Okabe, S;Fukuda, T;Tokuhisa, T
通讯作者：
Tokuhisa, T