Evaluating post-transcriptional regulatory elements for enhancing transient gene expression levels in CHO K1 and HEK293 cells
Evaluating post-transcriptional regulatory elements for enhancing transient gene expression levels in CHO K1 and HEK293 cells
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DOI：
10.1016/j.pep.2009.08.010
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发表时间：
2010-01-01
影响因子：
1.6
通讯作者：
Yang, Yuansheng
中科院分区：
文献类型：
--
作者：
Mariati;Ho, Steven C. L.;Yang, Yuansheng
5个转录后调节元件，（i）人热休克蛋白70 mRNA（Hsp 70）的5'非翻译区（UTR），（ii）衍生自血管内皮生长因子（SP 163）的5' UTR的163-bp长的剪接变体，和（iii）与主要晚期启动子增强子（TM）连接的人腺病毒mRNA的三联前导序列，使用萤火虫荧光素酶（Fluc）、干扰素γ（IFN）和曲妥珠单抗单克隆抗体，评价（iv）人巨细胞病毒立即早期基因的第一内含子（内含子A）和（v）来源于土拨鼠肝炎病毒（WPRE）的转录后调节元件在两种工业细胞系HEK 293和CHO K1中增强瞬时基因表达水平。除了Hsp 70没有作用外，所有其他元件都增强表达，但表现出细胞特异性和基因特异性作用。TM提供了最普遍和最高的基因表达水平的增强。它使HEK 293细胞中所有三种蛋白质的表达和CHO K1细胞中两种蛋白质（Fluc和IFN）的表达增强3.6至7.6倍。其余元件使至少一种细胞系中的一种或多种蛋白质的表达增强1.7至3.2倍。将WPRE与内含子A、SPI 63或TM组合对基因表达具有累积效应。这些组合可以使HEK 293细胞中的Fluc表达增加高达10.5倍。这些结果为改进HEK 293和CHO K1细胞中高水平瞬时基因表达的载体提供了有价值的信息。(C)2009 Elsevier Inc. All rights reserved.
Five post-transcriptional regulatory elements, (i) the 5' untranslated region (UTR) of human heat shock protein 70 mRNA (Hsp70), (ii) the 163-bp long splice variant derived from the 5' UTR of vascular endothelial growth factor (SP163), and (iii) the tripartite leader sequence of human adenovirus mRNA linked with a major late promoter enhancer (TM), (iv) the first intron of human cytomegalovirus immediate early gene (Intron A), and (v) the post-transcriptional regulatory element derived from woodchuck hepatitis virus (WPRE), are evaluated for enhancing transient gene expression levels in two industrial cell lines, HEK293 and CHO K1 using firefly luciferase (Fluc), interferon gamma (IFN), and Trastuzamab monoclonal antibody. Except for the Hsp70 which has no effects, all other elements enhance expression but exhibit cell-specific and gene-specific effects. TM provides the Most universal and highest enhancement of gene expression levels. It enhances the expression of all three proteins in HEK293 cells and two proteins, Fluc and IFN in CHO K1 cells by 3.6- to 7.6-fold. The remaining elements enhance expression of one or more proteins in at least one cell line by 1.7- to 3.2-fold. Combining WPRE with either Intron A, SPI 63, or TM has cumulative effects on gene expression. The combinations can increase Fluc expression by up to 10.5-fold in HEK293 cells. These results provide valuable information to improve vectors for high level transient gene expressions in HEK293 and CHO K1 cells. (C) 2009 Elsevier Inc. All rights reserved.