caa a22 4500 605504903 CHVBK 20210128100618.0 cr unu---uuuuu 210128e20151201xx s 000 0 eng 10.1007/s00253-015-6856-y doi (NATIONALLICENCE)springer-10.1007/s00253-015-6856-y A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3′-UTR of amplifiable dhfr [Elektronische Daten] [Shin-Young Kang, Yeon-Gu Kim, Hong Lee, Eun Lee] 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 500nM 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. Springer-Verlag Berlin Heidelberg, 2015 Gene amplification nationallicence Short hairpin RNA (shRNA) nationallicence Untranslated region (UTR) nationallicence DHFR nationallicence Chinese hamster ovary (CHO) nationallicence Kang Shin-Young Department of Bioprocess Engineering, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, 305-350, Daejeon, Republic of Korea aut Kim Yeon-Gu Department of Bioprocess Engineering, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, 305-350, Daejeon, Republic of Korea aut Lee Hong Department of Bioprocess Engineering, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, 305-350, Daejeon, Republic of Korea aut Lee Eun Department of Bioprocess Engineering, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, 305-350, Daejeon, Republic of Korea aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/23(2015-12-01), 10117-10126 0175-7598 99:23<10117 2015 99 253 https://doi.org/10.1007/s00253-015-6856-y text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6856-y text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kang Shin-Young Department of Bioprocess Engineering, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, 305-350, Daejeon, Republic of Korea aut NATIONALLICENCE 700 1- Kim Yeon-Gu Department of Bioprocess Engineering, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, 305-350, Daejeon, Republic of Korea aut NATIONALLICENCE 700 1- Lee Hong Department of Bioprocess Engineering, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, 305-350, Daejeon, Republic of Korea aut NATIONALLICENCE 700 1- Lee Eun Department of Bioprocess Engineering, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, 305-350, Daejeon, Republic of Korea aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/23(2015-12-01), 10117-10126 0175-7598 99:23<10117 2015 99 253