中國倉鼠卵巢細胞(Chinese hamster ovary, CHO)與二氫葉酸還原脢(dihydrofolate reductase, dhfr)/Methotrexate (MTX)基因增幅選擇法所建立之穩定細胞株，已廣泛應用於高品質重組藥用蛋白質之產製，例如醫療性抗體。大部份的單株抗體藉由嵌合型或者是擬人化抗體降低免疫反應。本實驗中利用中國倉鼠卵巢細胞表現系統表現抗日本腦炎中和性嵌合型抗體IgG1-2H2和 IgG1-E3.3。干擾載體psd2，被證實可以在dhfr/MTX基因增幅選擇法的製程中，提高CHO/dhfr-（dhfr基因表現缺失的細胞）株穩定純系細胞株表現外源抗體蛋白。
Mir-30 RNA為基礎的sd2干擾載體與一個非專一性對於二氫葉酸還原脢基因干擾載體應用於改善提升穩定增幅細胞株生產的嵌合性抗體IgG1-2H2。結果顯示，在含有MTX的情況下穩定增幅細胞株具有干擾載體psd2和pMir-30 sd2產量約高於未具有干擾載體和非專一性對於二氫葉酸還原脢基因(pscramble) 干擾載體100%。此一方法成功地篩選出高穩定度與高產量的CHO/dhfr-細胞株。利用本研究所提出之RNA干擾載體於dhfr/MTX基因增幅選擇法，可篩選出高穩定性與高產量的穩定增幅細胞株，用以生產抗體或其他生技藥品，對生技製藥產業而言，極富應用性。

Chinese hamster ovary cells (CHO) and dihydrofolate reductase (dhfr)/methotrexate (MTX) gene amplification system are commonly used to generate stable high-producer CHO cell clones in biopharmaceutical industries, such as antibodies, at the present time. Most biopharmaceutical antibody-based products display favorable safety profiles such as chimeric or humanized antibodies. Simulation of bio-pharmaceutical industrial process for antibody production, the expression system using CHO/dhfr- cell as a host cell to express anti-Japanese encephalitis virus (JEV) neutralizing chimeric antibody IgG1-2H2 and IgG1-E3.3. The silencing shRNA vector psd2, which was previously demonstrated the most effective in silencing dhfr RNA transcripts, improved antibody production in dhfr-deficient CHO (CHO/dhfr-¬) cells through dhfr/MTX gene amplification.
Initially, a chimeric mouse-human antibody expression vector was used to express JEV neutralizing chimeric antibodies, IgG1-2H2 and IgG1-E3.3. Then, in addition to psd2, other silencing vectors that were mir-30 based sd2 silencing vector (pmir-30 sd2) and nonspecific of dhfr gene silencing vector (pscramble) were adopted to improve IgG1-2H2 expression in CHO/dhfr- cells. Compare to the average expression level of IgG1-2H2 of CHO/dhfr stable clones, the clones with the silencing vectors pMir-30 sd2 and psd2 were about 100% higher than that with pscramble silencing vector and without silencing vector. Higher level of IgG expression and more stable productivity in MTX-free medium was achieved in amplified stable clones containing psd2 or pMir30-sd2. The method proposed here can be applied to screen high producer cell clones for recombinant antibody or other biologics expression in CHO/dhfr- cells with efficient stable transfection. It can be beneficial for the production of recombinant protein therapeutics in bio-industry.

Assaraf, Y. G. & Schimke, R. T. (1987). Identification of methotrexate transport deficiency in mammalian cells using fluoresceinated methotrexate and flow cytometry. Proc Natl Acad Sci U S A 84, 7154-7158.
Baik, J. Y., Joo, E. J., Kim, Y. H. & Lee, G. M. (2008). Limitations to the comparative proteomic analysis of thrombopoietin producing Chinese hamster ovary cells treated with sodium butyrate. Journal of biotechnology 133, 461-468.
Baldi, L., Muller, N., Picasso, S., Jacquet, R., Girard, P., Thanh, H. P., Derow, E. & Wurm, F. M. (2005). Transient gene expression in suspension HEK-293 cells: application to large-scale protein production. Biotechnol Prog 21, 148-153.
Beasley, D. W., Li, L., Suderman, M. T., Guirakhoo, F., Trent, D. W., Monath, T. P., Shope, R. E. & Barrett, A. D. (2004). Protection against Japanese encephalitis virus strains representing four genotypes by passive transfer of sera raised against ChimeriVax-JE experimental vaccine. Vaccine 22, 3722-3726.
Bell, A. C. & Felsenfeld, G. (1999). Stopped at the border: boundaries and insulators. Curr Opin Genet Dev 9, 191-198.
Benton, T., Chen, T., McEntee, M., Fox, B., King, D., Crombie, R., Thomas, T. C. & Bebbington, C. (2002). The use of UCOE vectors in combination with a preadapted serum free, suspension cell line allows for rapid production of large quantities of protein. Cytotechnology 38, 43-46.
Birch, J. R. & Racher, A. J. (2006). Antibody production. Advanced drug delivery reviews 58, 671-685.
Brummelkamp, T. R., Bernards, R. & Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550-553.
Butler, M. (2005). Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals. Applied microbiology and biotechnology 68, 283-291.
Cullen, B. R. (2004). Transcription and processing of human microRNA precursors. Molecular cell 16, 861-865.
Cullen, B. R. (2006). Induction of stable RNA interference in mammalian cells. Gene therapy 13, 503-508.
Dinnis, D. M. & James, D. C. (2005). Engineering mammalian cell factories for improved recombinant monoclonal antibody production: lessons from nature? Biotechnology and bioengineering 91, 180-189.
Du, G., Yonekubo, J., Zeng, Y., Osisami, M. & Frohman, M. A. (2006). Design of expression vectors for RNA interference based on miRNAs and RNA splicing. The FEBS journal 273, 5421-5427.
Dykxhoorn, D. M., Novina, C. D. & Sharp, P. A. (2003). Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4, 457-467.
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. & Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.
Gandor, C., Leist, C., Fiechter, A. & Asselbergs, F. A. (1995). Amplification and expression of recombinant genes in serum-independent Chinese hamster ovary cells. FEBS letters 377, 290-294.
Girod, P. A., Nguyen, D. Q., Calabrese, D., Puttini, S., Grandjean, M., Martinet, D., Regamey, A., Saugy, D., Beckmann, J. S., Bucher, P. & Mermod, N. (2007). Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells. Nature methods 4, 747-753.
Girod, P. A., Zahn-Zabal, M. & Mermod, N. (2005). Use of the chicken lysozyme 5' matrix attachment region to generate high producer CHO cell lines. Biotechnology and bioengineering 91, 1-11.
Goncalvez, A. P., Men, R., Wernly, C., Purcell, R. H. & Lai, C. J. (2004). Chimpanzee Fab fragments and a derived humanized immunoglobulin G1 antibody that efficiently cross-neutralize dengue type 1 and type 2 viruses. J Virol 78, 12910-12918.
Gwizdek, C., Ossareh-Nazari, B., Brownawell, A. M., Doglio, A., Bertrand, E., Macara, I. G. & Dargemont, C. (2003). Exportin-5 mediates nuclear export of minihelix-containing RNAs. J Biol Chem 278, 5505-5508.
Haber, D. A., Beverley, S. M., Kiely, M. L. & Schimke, R. T. (1981). Properties of an altered dihydrofolate reductase encoded by amplified genes in cultured mouse fibroblasts. J Biol Chem 256, 9501-9510.
Henikoff, S. (1992). Position effect and related phenomena. Curr Opin Genet Dev 2, 907-912.
Hong, W. W. & Wu, S. C. (2007). A novel RNA silencing vector to improve antigen expression and stability in Chinese hamster ovary cells. Vaccine 25, 4103-4111.
Hong, W. W., Yen, Y. H. & Wu, S. C. (2007). Enhanced antibody affinity to Japanese encephalitis virus E protein by phage display. Biochem Biophys Res Commun.
Huang, Y., Li, Y., Wang, Y. G., Gu, X., Wang, Y. & Shen, B. F. (2007). An efficient and targeted gene integration system for high-level antibody expression. Journal of immunological methods 322, 28-39.
Janeway, C., Travers, P. & Walport, M. S., M. (2005). Immunobiology: the immune system in health and disease.
Kabat, E. A. & Wu, T. T. (1991). Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites. J Immunol 147, 1709-1719.
Kanda, Y., Yamada, T., Mori, K., Okazaki, A., Inoue, M., Kitajima-Miyama, K., Kuni-Kamochi, R., Nakano, R., Yano, K., Kakita, S., Shitara, K. & Satoh, M. (2007). Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types. Glycobiology 17, 104-118.
Kaufman, R. J. (1990). Selection and coamplification of heterologous genes in mammalian cells. Methods in enzymology 185, 537-566.
Kaufman, R. J. & Schimke, R. T. (1981). Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line. Mol Cell Biol 1, 1069-1076.
Kellems, R. E. (1991). Gene amplification in mammalian cells: strategies for protein production. Current opinion in biotechnology 2, 723-729.
Kim, J. M., Kim, J. S., Park, D. H., Kang, H. S., Yoon, J., Baek, K. & Yoon, Y. (2004). Improved recombinant gene expression in CHO cells using matrix attachment regions. J Biotechnol 107, 95-105.
Kim, S. H. & Lee, G. M. (2007). Down-regulation of lactate dehydrogenase-A by siRNAs for reduced lactic acid formation of Chinese hamster ovary cells producing thrombopoietin. Applied microbiology and biotechnology 74, 152-159.
Kim, V. N. (2005). MicroRNA biogenesis: coordinated cropping and dicing. Nature reviews 6, 376-385.
Kito, M., Itami, S., Fukano, Y., Yamana, K. & Shibui, T. (2002). Construction of engineered CHO strains for high-level production of recombinant proteins. Applied microbiology and biotechnology 60, 442-448.
Kwaks, T. H., Barnett, P., Hemrika, W., Siersma, T., Sewalt, R. G., Satijn, D. P., Brons, J. F., van Blokland, R., Kwakman, P., Kruckeberg, A. L., Kelder, A. & Otte, A. P. (2003). Identification of anti-repressor elements that confer high and stable protein production in mammalian cells. Nature biotechnology 21, 553-558.
Lim, S. F., Chuan, K. H., Liu, S., Loh, S. O., Chung, B. Y., Ong, C. C. & Song, Z. (2006). RNAi suppression of Bax and Bak enhances viability in fed-batch cultures of CHO cells. Metabolic engineering 8, 509-522.
Lin, C. W. & Wu, S. C. (2003). A functional epitope determinant on domain III of the Japanese encephalitis virus envelope protein interacted with neutralizing-antibody combining sites. J Virol 77, 2600-2606.
Lin, C. W. & Wu, S. C. (2004). Identification of mimotopes of the Japanese encephalitis virus envelope protein using phage-displayed combinatorial peptide library. Journal of molecular microbiology and biotechnology 8, 34-42.
McManus, M. T. & Sharp, P. A. (2002). Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 3, 737-747.
Moremen, K. W. & Molinari, M. (2006). N-linked glycan recognition and processing: the molecular basis of endoplasmic reticulum quality control. Curr Opin Struct Biol 16, 592-599.
Mori, K., Kuni-Kamochi, R., Yamane-Ohnuki, N., Wakitani, M., Yamano, K., Imai, H., Kanda, Y., Niwa, R., Iida, S., Uchida, K., Shitara, K. & Satoh, M. (2004). Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA. Biotechnology and bioengineering 88, 901-908.
Ngantung, F. A., Miller, P. G., Brushett, F. R., Tang, G. L. & Wang, D. I. (2006). RNA interference of sialidase improves glycoprotein sialic acid content consistency. Biotechnology and bioengineering 95, 106-119.
Nissom, P. M., Sanny, A., Kok, Y. J., Hiang, Y. T., Chuah, S. H., Shing, T. K., Lee, Y. Y., Wong, K. T., Hu, W. S., Sim, M. Y. & Philp, R. (2006). Transcriptome and proteome profiling to understanding the biology of high productivity CHO cells. Molecular biotechnology 34, 125-140.
Novina, C. D. & Sharp, P. A. (2004). The RNAi revolution. Nature 430, 161-164.
Ober, R. J., Martinez, C., Lai, X., Zhou, J. & Ward, E. S. (2004a). Exocytosis of IgG as mediated by the receptor, FcRn: an analysis at the single-molecule level. Proc Natl Acad Sci U S A 101, 11076-11081.
Ober, R. J., Martinez, C., Vaccaro, C., Zhou, J. & Ward, E. S. (2004b). Visualizing the site and dynamics of IgG salvage by the MHC class I-related receptor, FcRn. J Immunol 172, 2021-2029.
Omasa, T. (2002). Gene amplification and its application in cell and tissue engineering. J Biosci Bioeng 94, 600-605.
Pallavicini, M. G., DeTeresa, P. S., Rosette, C., Gray, J. W. & Wurm, F. M. (1990). Effects of methotrexate on transfected DNA stability in mammalian cells. Molecular and cellular biology 10, 401-404.
Pascoe, D. E., Arnott, D., Papoutsakis, E. T., Miller, W. M. & Andersen, D. C. (2007). Proteome analysis of antibody-producing CHO cell lines with different metabolic profiles. Biotechnology and bioengineering 98, 391-410.
Pavlou, A. K. & Reichert, J. M. (2004). Recombinant protein therapeutics--success rates, market trends and values to 2010. Nat Biotechnol 22, 1513-1519.
Rauthan, M., Kaur, R., Appaiahgari, M. B. & Vrati, S. (2004). Oral immunization of mice with Japanese encephalitis virus envelope protein synthesized in Escherichia coli induces anti-viral antibodies. Microbes and infection / Institut Pasteur 6, 1305-1311.
Running Deer, J. & Allison, D. S. (2004). High-level expression of proteins in mammalian cells using transcription regulatory sequences from the Chinese hamster EF-1alpha gene. Biotechnology progress 20, 880-889.
Schimke, R. T., Alt, F. W., Kellems, R. E., Kaufman, R. J. & Bertino, J. R. (1978). Amplification of dihydrofolate reductase genes in methotrexate-resistant cultured mouse cells. Cold Spring Harb Symp Quant Biol 42 Pt 2, 649-657.
Seth, G., Charaniya, S., Wlaschin, K. F. & Hu, W. S. (2007). In pursuit of a super producer-alternative paths to high producing recombinant mammalian cells. Current opinion in biotechnology 18, 557-564.
Siminovitch, L. (1976). On the nature of hereditable variation in cultured somatic cells. Cell 7, 1-11.
Sung, Y. H., Hwang, S. J. & Lee, G. M. (2005). Influence of down-regulation of caspase-3 by siRNAs on sodium-butyrate-induced apoptotic cell death of Chinese hamster ovary cells producing thrombopoietin. Metabolic engineering 7, 457-466.
Trill, J. J., Shatzman, A. R. & Ganguly, S. (1995). Production of monoclonal antibodies in COS and CHO cells. Curr Opin Biotechnol 6, 553-560.
Trummer, E., Ernst, W., Hesse, F., Schriebl, K., Lattenmayer, C., Kunert, R., Vorauer-Uhl, K., Katinger, H. & Muller, D. (2008). Transcriptional profiling of phenotypically different Epo-Fc expressing CHO clones by cross-species microarray analysis. Biotechnology journal 3, 924-937.
Urlaub, G. & Chasin, L. A. (1980). Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci U S A 77, 4216-4220.
Walsh, G. (2005). Biopharmaceuticals: recent approvals and likely directions. Trends Biotechnol 23, 553-558.
Walsh, G. (2006). Biopharmaceutical benchmarks 2006. Nat Biotechnol 24, 769-776.
Walsh, G. & Jefferis, R. (2006). Post-translational modifications in the context of therapeutic proteins. Nature biotechnology 24, 1241-1252.
Wong, D. C., Wong, K. T., Lee, Y. Y., Morin, P. N., Heng, C. K. & Yap, M. G. (2006). Transcriptional profiling of apoptotic pathways in batch and fed-batch CHO cell cultures. Biotechnology and bioengineering 94, 373-382.
Wu, S. C. (2009). RNA interference technology to improve recombinant protein production in Chinese hamster ovary cells. Biotechnology advances 27, 417-422.
Wu, S. C., Hong, W. W. & Liu, J. H. (2008). Short hairpin RNA targeted to dihydrofolate reductase enhances the immunoglobulin G expression in gene-amplified stable Chinese hamster ovary cells. Vaccine 26, 4969-4974.
Wu, S. C., Lian, W. C., Hsu, L. C. & Liau, M. Y. (1997). Japanese encephalitis virus antigenic variants with characteristic differences in neutralization resistance and mouse virulence. Virus Res 51, 173-181.
Wurm, F. & Bernard, A. (1999). Large-scale transient expression in mammalian cells for recombinant protein production. Curr Opin Biotechnol 10, 156-159.
Wurm, F. M. (2004). Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22, 1393-1398.
Wurm, F. M., Gwinn, K. A. & Kingston, R. E. (1986). Inducible overproduction of the mouse c-myc protein in mammalian cells. Proc Natl Acad Sci U S A 83, 5414-5418.
Yee, J. C., de Leon Gatti, M., Philp, R. J., Yap, M. & Hu, W. S. (2008). Genomic and proteomic exploration of CHO and hybridoma cells under sodium butyrate treatment. Biotechnology and bioengineering 99, 1186-1204.
Yi, R., Qin, Y., Macara, I. G. & Cullen, B. R. (2003). Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17, 3011-3016.
Yoshikawa, T., Nakanishi, F., Ogura, Y., Oi, D., Omasa, T., Katakura, Y., Kishimoto, M. & Suga, K. (2000). Amplified gene location in chromosomal DNA affected recombinant protein production and stability of amplified genes. Biotechnol Prog 16, 710-715.
Zahn-Zabal, M., Kobr, M., Girod, P. A., Imhof, M., Chatellard, P., de Jesus, M., Wurm, F. & Mermod, N. (2001). Development of stable cell lines for production or regulated expression using matrix attachment regions. Journal of biotechnology 87, 29-42.