title: A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr.
abstract: 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.
authors.affiliation: Department of Bioprocess Engineering, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 305-350, Republic of Korea.
authors.forename: Shin-Young
authors.initials: SY
authors.lastname: Kang
authors.affiliation: Department of Bioprocess Engineering, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 305-350, Republic of Korea.|Biotechnology Process Engineering Center, KRIBB, 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gu, Cheongju-si, Chungcheongbuk-do, 363-883, Republic of Korea.
authors.forename: Yeon-Gu
authors.initials: YG
authors.lastname: Kim
authors.affiliation: Department of Bioprocess Engineering, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 305-350, Republic of Korea.|Biotechnology Process Engineering Center, KRIBB, 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gu, Cheongju-si, Chungcheongbuk-do, 363-883, Republic of Korea.
authors.forename: Hong Weon
authors.initials: HW
authors.lastname: Lee
authors.affiliation: Department of Bioprocess Engineering, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 305-350, Republic of Korea. eglee@kribb.re.kr.|Biotechnology Process Engineering Center, KRIBB, 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gu, Cheongju-si, Chungcheongbuk-do, 363-883, Republic of Korea. eglee@kribb.re.kr.
authors.forename: Eun Gyo
authors.initials: EG
authors.lastname: Lee
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics; D055785:Gene Knockdown Techniques / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008727:Methotrexate / Q000378:metabolism; D010957:Plasmids; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D019076:Transgenes; D015854:Up-Regulation
chemical_list: D020413:3' Untranslated Regions; D034741:RNA, Small Interfering; D004921:Erythropoietin; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate
keywords: Chinese hamster ovary (CHO); DHFR; Gene amplification; Short hairpin RNA (shRNA); Untranslated region (UTR)