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首页> 外文期刊>Molecular and Cellular Biology >Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus.
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Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus.

机译：通过使用脑心肌炎病毒的内部核糖体进入位点在小鼠中构建双功能mRNA。

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Picornaviral mRNAs have been shown to possess special structures in their 5' nontranslated regions (5'NTRs) that provide sites for internal binding of ribosomes and thus direct cap-independent translation. The translational cis-acting elements for ribosomal internal entry into the 5'NTR of encephalomyocarditis virus (EMCV), a member of family Picornaviridae, have been named the internal ribosomal entry site (IRES). All of the published experiments regarding the IRES function of the picornavirus 5'NTR, however, were performed with cell extracts in vitro or with tissue culture cells in transient assay systems. In this study, we examined the IRES function of the EMCV 5'NTR in chimeric mouse embryos and demonstrated that this element does in fact work stably in mouse embryos as well as in embryonic stem (ES) cells. By using a dicistronic vector, pWH8, consisting of a promoter-driven neomycin resistance gene (neo) followed by the EMCV 5'NTR-lacZ sequence, we showed that more than half of the ES cells made G418 resistant by the vector stained positive for beta-galactosidase (beta-gal). On Northern (RNA) blots, all of the clones analyzed revealed a transcript of the expected size containing both the beta-gal and the neo cistrons. These results indicate that dicistronic mRNAs are produced from the stably integrated vector in those ES clones and that both of the cistrons are translated to produce functional proteins. The chimeric embryos derived from these ES clones also stained positive for beta-gal, suggesting that the bifunctional mRNAs are active in the embryos. This dicistronic vector system provides a novel tool by which to obtain temporally and spatially coordinated expression of two different genes driven by a single promoter in a single cell in mice.
机译：业已显示，皮科病毒的mRNA在其5'非翻译区（5'NTR）中具有特殊的结构，这些结构为核糖体的内部结合提供了位点，从而直接进行了帽独立的翻译。核糖体内部进入脑心肌炎病毒（EMCV）（Picornaviridae家族的成员）的5'NTR的翻译顺式作用元件被称为内部核糖体进入位点（IRES）。但是，所有有关小核糖核酸病毒5'NTR的IRES功能的已发表实验都是在体外用细胞提取物或在瞬时测定系统中用组织培养细胞进行的。在这项研究中，我们检查了嵌合小鼠胚胎中EMCV 5'NTR的IRES功能，并证明了该元素实际上在小鼠胚胎以及胚胎干（ES）细胞中均能稳定发挥作用。通过使用双顺反子载体pWH8，该载体由启动子驱动的新霉素抗性基因（neo）和随后的EMCV 5'NTR-lacZ序列组成，我们显示，超过一半的ES细胞被该载体染色呈阳性的载体对G418产生了抗性β-半乳糖苷酶（β-gal）。在Northern（RNA）印迹上，所有分析的克隆均显示出预期大小的转录本，其中同时包含β-gal和新的顺反子。这些结果表明，从那些ES克隆中的稳定整合的载体产生了双顺反子mRNA，并且两个顺反子都被翻译以产生功能性蛋白。衍生自这些ES克隆的嵌合胚胎也对β-gal染色呈阳性，表明双功能mRNA在胚胎中具有活性。该双顺反子载体系统提供了一种新颖的工具，通过该工具可以获得在小鼠单细胞中由单个启动子驱动的两个不同基因的时间和空间协调表达。

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