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首页> 外文期刊>Applied Microbiology and Biotechnology >A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr
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A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr

机译：使用短发夹RNA靶向可扩增dhfr的3'-UTR的单质粒载体，用于转基因扩增

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摘要

Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80 % in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q (EPO)) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q (EPO) increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.
机译：使用二氢叶酸还原酶基因（dhfr）和甲氨蝶呤（MTX）进行的基因扩增已广泛用于哺乳动物细胞中的重组蛋白生产，并且通常在DHFR缺陷型中国仓鼠卵巢（CHO）细胞系中进行。 DHFR缺陷型细胞的产生可通过掺入靶向内源dhfr的3'非翻译区（UTR）的短发夹RNA（shRNA）的表达载体来实现。因此，将shRNA设计为靶向内源性dhfr的3'-UTR，并且shRNA-2有效地下调了CHO-K1细胞中的dhfr表达。在Flp-In CHO细胞中，shRNA-2的单基因拷贝也使DHFR的翻译水平降低了80％。然后将shRNA-2掺入表达人促红细胞生成素（EPO）和外源性DHFR的质粒载体中，以在Flp-In系统中发育产生EPO的细胞。通过逐步增加MTX浓度可以提高EPO的比生产率（q（EPO）），并且在表达shRNA-2的Flp-In CHO细胞中观察到扩增速率的差异。此外，在500 nM MTX存在下，q（EPO）增加了2.5倍以上。 dhfr的mRNA表达水平和基因拷贝数与细胞生产力的提高相关，这受内源性dhfr的抑制影响。这项研究表明，包含靶向内源性dhfr 3'-UTR的shRNA的表达载体可通过产生DHFR缺陷型细胞来提高转基因扩增速率和生产率。该方法可用于扩增野生型细胞系中的外源基因，作为通用的单质粒载体。

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