Use of the viral 2A peptide for bicistronic expression in transgenic mice.
Use of the viral 2A peptide for bicistronic expression in transgenic mice.
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DOI：
10.1186/1741-7007-6-40
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发表时间：
2008-09-15
期刊：
影响因子：
5.4
通讯作者：
Srinivas, Shankar
中科院分区：
文献类型：
--
作者：
Trichas, Georgios;Begbie, Jo;Srinivas, Shankar
转基因动物在生物医学研究和生物技术中有着广泛的应用。多顺反子结构，其中几种蛋白质由单个信使RNA编码，通常用于基因工程动物。目前，这是通过使用内部核糖体进入位点来分离不同编码区来完成的。2多肽导致蛋白质的共翻译‘裂解’，是内部核糖体进入位点的一种有吸引力的替代。它们比内部核糖体进入部位更可靠，并导致多个顺反子在等摩尔水平上的表达。它们在各种各样的真核细胞中发挥作用，但到目前为止还没有被证明在转基因小鼠中以可遗传的方式发挥作用。为了测试2A在转基因小鼠中的功能并揭示其广泛表达的任何可能的毒性，我们构建了一个双顺反子报告结构，其中包含膜定位红色荧光蛋白(MyR-TdTomato)和核定位绿色荧光蛋白(H2B-GFP)的编码序列，由2A序列分隔。当本报告被转染到HeLa细胞中时，两个荧光蛋白正确地定位于相互排斥的细胞室，表明双顺反子结构是2A功能的可靠读数。当报告被电穿孔到鸡神经管细胞中时，这两种荧光蛋白也正确定位。我们建立了两个独立的转基因小鼠系，它们普遍表达双顺反子报告。对于这两个品系，转基因小鼠以孟德尔频率出生，并被发现是健康和可生育的。MYR-TdTomato和H2B-GFP在从胚胎到成人的广泛发育阶段被检查的所有组织中分离到相互排斥的细胞室。1个转基因品系在雌性植株中表现出X连锁遗传，在雌性植株中表现为花叶表达，而在雄性植株中表达一致，表明该转基因植株已整合到X染色体上。在通过生殖系遗传的转基因小鼠中，2A肽有效地介导了共翻译切割。小鼠在整个发育过程中和成年后都普遍表达它，这似乎是正常的。因此，它是用于基因工程小鼠的一种可行的工具，是广泛使用的内部核糖体进入位点的一种更好的替代方案。
Transgenic animals are widely used in biomedical research and biotechnology. Multicistronic constructs, in which several proteins are encoded by a single messenger RNA, are commonly used in genetically engineered animals. This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the co-translational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site. They are more reliable than the internal ribosomal entry site and lead to expression of multiple cistrons at equimolar levels. They work in a wide variety of eukaryotic cells, but to date have not been demonstrated to function in transgenic mice in an inheritable manner. To test 2A function in transgenic mice and uncover any possible toxicity of widespread expression of the 2A peptide, we made a bicistronic reporter construct containing the coding sequence for a membrane localised red fluorescent protein (Myr-TdTomato) and a nuclear localised green fluorescent protein (H2B-GFP), separated by a 2A sequence. When this reporter is transfected into HeLa cells, the two fluorescent proteins correctly localise to mutually exclusive cellular compartments, demonstrating that the bicistronic construct is a reliable readout of 2A function. The two fluorescent proteins also correctly localise when the reporter is electroporated into chick neural tube cells. We made two independent transgenic mouse lines that express the bicistronic reporter ubiquitously. For both lines, transgenic mice are born in Mendelian frequencies and are found to be healthy and fertile. Myr-TdTomato and H2B-GFP segregate to mutually exclusive cellular compartments in all tissues examined from a broad range of developmental stages, ranging from embryo to adult. One transgenic line shows X-linked inheritance of the transgene and mosaic expression in females but uniform expression in males, indicating that the transgene has integrated into the X chromosome in this line. The 2A peptide efficiently mediates co-translational cleavage in transgenic mice in which it has been inherited through the germ-line. Mice expressing it ubiquitously throughout development and into adulthood appear normal. It is therefore a viable tool for use in genetically engineered mice and represents a superior alternative to the widely used internal ribosomal entry site.
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