Optimization of genetic constructs for high-level expression of the darbepoetin gene in mammalian cells

详细信息    查看全文

• 作者：R. R. Shukurov (1)
  K. Yu. Kazachenko (1)
  D. G. Kozlov (2)
  A. A. Nurbakov (1)
  E. N. Sautkina (2)
  R. A. Khamitov (2)
  Yu. A. Seryogin (1)
  
• 关键词：CHO cells ; codon optimization ; darbepoetin ; erythropoietin ; expression vectors ; promoter
• 刊名：Applied Biochemistry and Microbiology
• 出版年：2014
• 出版时间：December 2014
• 年：2014
• 卷：50
• 期：9
• 页码：802-811
• 全文大小：1,111 KB
• 参考文献：1. Powell, J.S., Berkner, K.L., Lebo, R.V., and Adamson, J.W., Human erythropoietin gene: high level expression in stably transfected mammalian cells and chromosome localization, / Proc. Natl. Acad. Sci. USA, 1986, vol. 83, pp. 6465-469. CrossRef
  2. Wasley, L.C., Timony, G., Murtha, P., Stoudemire, J., Dorner, A.J., Caro, J., Krieger, M., and Kaufman, R.J., The importance of N- and O-linked oligosaccharides for the biosynthesis and / in vitro and / in vivo biologic activities of erythropoietin, / Blood, 1991, vol. 77, pp. 2524-632.
  3. Cases, A., Darbepoetin alfa: a novel erythropoiesisstimulating protein, / Drugs Today (Barc.), 2003, vol. 39, pp. 477-95. CrossRef
  4. Ibbotson, T. and Goa, K.L., Darbepoetin alfa, / Drugs, 2001, vol. 61, pp. 2097-104. CrossRef
  5. Smith, R., Applications of darbepoietin-alpha, a novel erythropoiesis-stimulating protein, in oncology, / Curr. Opin. Hematol., 2002, vol. 9, pp. 228-33. CrossRef
  6. Macdougall, I.C., Darbepoetin alfa: a new therapeutic agent for renal anemia, / Kidney Int. Suppl., 2002, vol. 80, pp. 55-1. CrossRef
  7. Qin, J.Y., Zhang, L., Clift, K.L., Hulur, I., Xiang, A.P., Ren, B.Z., and Lahn, B.T., Systematic comparison of constitutive promoters and the doxycycline-inducible promoter, / PLoS One, 2010, vol. 5, p. e10611. CrossRef
  8. Magnusson, T., Haase, R., Schleef, M., Wagner, E., and Ogris, M., Sustained, high transgene expression in liver with plasmid vectors using optimized promoterenhancer combinations, / J. Gene Med., 2011, vol. 13, pp. 382-91. CrossRef
  9. Tokushige, K., Moradpour, D., Wakita, T., Geissler, M., Hayashi, N., and Wnads, J.R., Comparison between cytomegalovirus promoter and elongation factor-1 alpha promoter-driven constructs in the establishment of cell lines expressing hepatitis C virus core protein, / J. Virol. Meth., 1997, vol. 64, pp. 73-0. CrossRef
  10. Teschendorf, C., Warrington jr K.H., Siemann, D.W., and Muzyczka, N., Comparison of the EF-1 alpha and the CMV promoter for engineering stable tumor cell lines using recombinant adeno-associated virus, / Anticancer Res., 2002, vol. 22, pp. 3325-330.
  11. Bode, J., Beham, C., Knopp, A., and Mielke, C., Transcriptional augmentation: modulation of gene expression by scaffold/matrix-attached regions (S/MAR elements), / Crit. Rev. Eukaryotic Gene Expr., 2000, vol. 10, pp. 73-0.
  12. Bode, J., Schlake, T., Rios-Ramirez, M., Mielke, C., Stengert, M., Kay, V., and Klehr-Wirth, D., Scaffold/matrix-attached regions: structural properties creating transcriptionally active loci, / Int. Rev. Cytol., 1995, vol. 162A, pp. 389-54.
  13. Zahn-Zabal, M., Kobr, M., Girod, P.A., Imhof, M., Chatellard, P., de Jesus, M., Wurm, F., and Mermod, N., Development of stable cell lines for production or regulated expression using matrix attachment regions, / J. Biotechnol., 2001, vol. 87, pp. 29-2. CrossRef
  14. Schubeler, D., Mielke, C., Maass, K., and Bode, J., Scaffold/matrix-attached regions act upon transcription in a context-dependent manner, / Biochemistry, 1996, vol. 35, pp. 11160-1169. CrossRef
  15. Grote, A., Hiller, K., Scheer, M., Munch, R., Nortemann, B., Hempel, D.C., and Hahn, D., JCat: a novel tool to adapt codon usage of a target gene to its potential expression host, / Nucleic Acids Res., 2005, vol. 33, pp. 526-31. CrossRef
  16. Comeron, J.M. and Aguade, M., An evaluation of measures of synonymous codon usage bias, / J. Mol. Evol., 1998, vol. 47, pp. 268-74. CrossRef
  17. Kim, C
• 作者单位：R. R. Shukurov (1)
  K. Yu. Kazachenko (1)
  D. G. Kozlov (2)
  A. A. Nurbakov (1)
  E. N. Sautkina (2)
  R. A. Khamitov (2)
  Yu. A. Seryogin (1)
  
  1. FARMAPARK Company, Moscow, 117246, Russia
  2. State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, 117545, Russia
  
• ISSN：1608-3024

文摘

Genetic constructs were designed in order to optimize darbepoetin production in CHO cells. They are characterized by a higher level of structural optimization of the darbepoetin gene, a higher gene dose, and the selection of promoter elements that have never been used before for this purpose. A transient transfection of CHO cells by the obtained constructs was performed. It was shown that each of the variable factors in the constructs influenced darbepoetin gene expression. A construct containing a doubled dose of the darbepoetin synthetic gene with optimized codon composition under the control of the CMV-EF1α chimerical promoter was proved to be the most efficient.