Improved antibiotic-free plasmid vector design by incorporation of transient expression enhancers.
Improved antibiotic-free plasmid vector design by incorporation of transient expression enhancers.
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通过掺入瞬态表达增强子，改善了无抗生素质粒载体设计。
DOI：
10.1038/gt.2010.149
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发表时间：
2011-04
期刊：
影响因子：
5.1
通讯作者：
Williams JA
中科院分区：
文献类型：
--
作者：
Luke JM;Vincent JM;Du SX;Gerdemann U;Leen AM;Whalen RG;Hodgson CP;Williams JA
基因医学和基因疫苗接种应用需要改善质粒介导的转基因表达的方法。为了保持插入诱变介导的基因激活的低风险，表达增强序列将理想地起作用以改善瞬时转染的完整质粒的转基因表达，但不改善假基因组整合载体的转基因表达。我们在此报告了有效的最小的，无寄生虫的，高生产产量的哺乳动物表达载体的发展纳入合理设计的添加剂组合的表达增强子。将SV 40 72 bp增强子掺入巨细胞病毒（CMV）增强子的上游，选择性地提高了染色体外转基因表达。瞬时转染后，CMV启动子下游的人嗜T淋巴细胞病毒I型（HTLV-I）R区显著增加了mRNA翻译效率，但未增加总体mRNA水平。类似的mRNA翻译效率的增加，观察到质粒载体纳入和表达蛋白激酶R抑制腺病毒相关（VA）1 RNA。引人注目的是，HTLV-IR和VA 1在基因组整合后不增加转基因表达或质粒DNA的mRNA翻译效率。当与电穿孔递送组合时，载体平台进一步增加了转基因表达，并提高了兔中HIV-1 gp 120 DNA疫苗诱导的中和抗体滴度。这些包含瞬时表达增强子的无寄生虫载体是更安全、更有效的替代物，以改善用于DNA治疗或疫苗接种的转基因表达。本文的在线版本（doi：10.1038/gt.2010.149）包含补充材料，可供授权用户使用。
Methods to improve plasmid-mediated transgene expression are needed for gene medicine and gene vaccination applications. To maintain a low risk of insertional mutagenesis-mediated gene activation, expression-augmenting sequences would ideally function to improve transgene expression from transiently transfected intact plasmid, but not from spurious genomically integrated vectors. We report herein the development of potent minimal, antibiotic-free, high-manufacturing-yield mammalian expression vectors incorporating rationally designed additive combinations of expression enhancers. The SV40 72 bp enhancer incorporated upstream of the cytomegalovirus (CMV) enhancer selectively improved extrachromosomal transgene expression. The human T-lymphotropic virus type I (HTLV-I) R region, incorporated downstream of the CMV promoter, dramatically increased mRNA translation efficiency, but not overall mRNA levels, after transient transfection. A similar mRNA translation efficiency increase was observed with plasmid vectors incorporating and expressing the protein kinase R-inhibiting adenoviral viral associated (VA)1 RNA. Strikingly, HTLV-I R and VA1 did not increase transgene expression or mRNA translation efficiency from plasmid DNA after genomic integration. The vector platform, when combined with electroporation delivery, further increased transgene expression and improved HIV-1 gp120 DNA vaccine-induced neutralizing antibody titers in rabbits. These antibiotic-free vectors incorporating transient expression enhancers are safer, more potent alternatives to improve transgene expression for DNA therapy or vaccination. The online version of this article (doi:10.1038/gt.2010.149) contains supplementary material, which is available to authorized users.
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影响因子：
3.5
作者：
Carnes, Aaron E.;Luke, Jeremy M.;Vincent, Justin M.;Anderson, Sheryl;Schukar, Angela;Hodgson, Clague P.;Williams, James A.
通讯作者：
Williams, James A.
影响因子：
5.4
作者：
Angulo, A;Kerry, D;Ghazal, P
通讯作者：
Ghazal, P
DOI：
10.1016/s0006-291x(02)02486-5
发表时间：
2002-11-08
影响因子：
3.1
作者：
Blomberg, P;Eskandarpour, M;Islam, KB
通讯作者：
Islam, KB
影响因子：
3.7
作者：
Lei, M;Liu, Y;Samuel, CE
通讯作者：
Samuel, CE
影响因子：
1.6
作者：
Mariati;Ho, Steven C. L.;Yang, Yuansheng
通讯作者：
Yang, Yuansheng