The production of viral vectors designed to express large and difficult to express transgenes within neurons.
The production of viral vectors designed to express large and difficult to express transgenes within neurons.
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DOI：
10.1186/s13041-015-0100-7
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发表时间：
2015-02-24
期刊：
影响因子：
3.6
通讯作者：
Ploski JE
中科院分区：
文献类型：
--
作者：
Holehonnur R;Lella SK;Ho A;Luong JA;Ploski JE
病毒载体经常被用来在许多模式生物的神经系统内以空间和时间受限的方式传递和指导转基因的表达。尽管通常使用病毒载体来引导转基因在神经系统内的异位表达，但创造能够表达非常大的转基因或难以表达的转基因的高滴度病毒载体带来了独特的挑战。在这里，我们描述了腺相关病毒(AAV)和慢病毒的发展，这些病毒旨在表达N-甲基-D-天冬氨酸受体(NMDA)受体的大而难以表达的GluN2A或GluN2B亚单位，特别是在神经元中。我们利用8个广泛使用的启动子(RSV、EFS、TRE3G、0.4αCaMKII、1.3αCaMKII、0.5Synapsin、1.1Synapsin和CMV)的短启动子序列以及非常短的(~75bps)3‘非翻译序列，创建了一些针对大型转基因而优化的定制AAV和慢病毒载体。不足为奇的是，这些启动子表达GluN2亚单位的能力不同，然而令人惊讶的是，我们发现神经元特异性突触素和αCaMKII启动子不能实现全长GluN2亚单位的可检测表达，并且只有当转基因包括内含子或如果GluN2亚单位转基因被截断为仅包括GluN2跨膜结构域的编码区时，这些启动子才能实现可检测的表达。我们确定了病毒包装限制、转基因启动子和转基因内含子的存在都是成功开发出能够以神经元特异性方式运送和表达GluN2转基因的病毒载体的重要因素。由于这些载体已经被优化以适应大的开放阅读框，并且在某些情况下包含内含子以促进难以表达的转基因的表达，因此这些病毒载体可能有助于以神经元特异性的方式传递和表达许多大的或难以表达的转基因。本文的在线版本(doi：10.1186/s13041-0150100-7)包含补充材料，授权用户可以使用。
Viral vectors are frequently used to deliver and direct expression of transgenes in a spatially and temporally restricted manner within the nervous system of numerous model organisms. Despite the common use of viral vectors to direct ectopic expression of transgenes within the nervous system, creating high titer viral vectors that are capable of expressing very large transgenes or difficult to express transgenes imposes unique challenges. Here we describe the development of adeno-associated viruses (AAV) and lentiviruses designed to express the large and difficult to express GluN2A or GluN2B subunits of the N-methyl-D-aspartate receptor (NMDA) receptor, specifically within neurons. We created a number of custom designed AAV and lentiviral vectors that were optimized for large transgenes, by minimizing DNA sequences that were not essential, utilizing short promoter sequences of 8 widely used promoters (RSV, EFS, TRE3G, 0.4αCaMKII, 1.3αCaMKII, 0.5Synapsin, 1.1Synapsin and CMV) and utilizing a very short (~75 bps) 3′ untranslated sequence. Not surprisingly these promoters differed in their ability to express the GluN2 subunits, however surprisingly we found that the neuron specific synapsin and αCaMKII, promoters were incapable of conferring detectable expression of full length GluN2 subunits and detectable expression could only be achieved from these promoters if the transgene included an intron or if the GluN2 subunit transgenes were truncated to only include the coding regions of the GluN2 transmembrane domains. We determined that viral packaging limit, transgene promoter and the presence of an intron within the transgene were all important factors that contributed to being able to successfully develop viral vectors designed to deliver and express GluN2 transgenes in a neuron specific manner. Because these vectors have been optimized to accommodate large open reading frames and in some cases contain an intron to facilitate expression of difficult to express transgenes, these viral vectors likely could be useful for delivering and expressing many large or difficult to express transgenes in a neuron specific manner. The online version of this article (doi:10.1186/s13041-015-0100-7) contains supplementary material, which is available to authorized users.
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影响因子：
3.6
作者：
Choi JH;Yu NK;Baek GC;Bakes J;Seo D;Nam HJ;Baek SH;Lim CS;Lee YS;Kaang BK
通讯作者：
Kaang BK
影响因子：
82.9
作者：
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通讯作者：
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影响因子：
64.8
作者：
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通讯作者：
DAVIS, M
影响因子：
2.4
作者：
Holehonnur, Roopashri;Luong, Jonathan A.;Ploski, Jonathan E.
通讯作者：
Ploski, Jonathan E.
影响因子：
5.3
作者：
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通讯作者：
Roche, KW