The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from retroviral vectors
The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from retroviral vectors
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DOI：
10.1038/sj.gt.3302979
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发表时间：
2007-09-01
期刊：
影响因子：
5.1
通讯作者：
Malik, P.
中科院分区：
文献类型：
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作者：
Higashimoto, T.;Urbinati, F.;Malik, P.
土拨鼠肝炎病毒转录后调节元件（WPRE）增加转基因表达从各种病毒载体，虽然确切的机制是未知的。WPRE在位于转基因下游，靠近多聚腺苷酸化信号时最有效。我们假设WPRE可能通过改善转录终止来减少病毒mRNA通读转录，这反过来会增加病毒滴度和表达。使用Cre-lox介导的基于质粒的测定，我们发现来自g-逆转录病毒载体（RV）长末端重复序列（wt RV-LTR）和具有自失活缺失的RV LTR（SIN RV-LTR）的显著通读转录。WPRE，当被放置在RV LTR的上游时，显著减少通读转录。SIN HIV-1 LV-LTR的通读水平低得多，WPRE也降低了通读水平。当置于RV载体中时，WPRE增加了总RV基因组mRNA;并使瞬时转染的293 T细胞和稳定的PG 13生产细胞的病毒滴度增加了7至15倍。滴度和表达增加的机制不是由于核mRNA输出增加、病毒转录速率增加或病毒mRNA半衰期显著增加。我们的结果表明，WPRE改善了载体基因组转录终止，以增加RV的滴度和表达。
The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression from a variety of viral vectors, although the precise mechanism is not known. WPRE is most effective when placed downstream of the transgene, proximal to the polyadenylation signal. We hypothesized that WPRE likely reduces viral mRNA readthrough transcription by improving transcript termination, which in turn would increase viral titers and expression. Using a Cre-lox-mediated plasmid-based assay, we found significant readthrough transcription from g- retroviral vector ( RV) long terminal repeat (wt RV-LTR) and RV LTR with a self-inactivating deletion ( SIN RV-LTR). WPRE, when placed upstream of the RV LTRs, significantly reduced readthrough transcription. Readthrough, present at much lower levels with the SIN HIV-1 LV-LTR, was also reduced with WPRE. When placed in RV vectors, WPRE increased total RV genomic mRNA; and increased viral titers from transiently transfected 293T cells and stable PG13 producer cells by 7- to 15-fold. The mechanism of increased titers and expression was not due to increased nuclear mRNA export, increased rate of viral transcription or a significant increase in viral mRNA half-life. Our results showed that WPRE improved vector genomic transcript termination to increase titers and expression from RVs.