Abstract
Changes in protein glycosylation owing to changes in environmental conditions are not well understood. To better understand these relationships, methods to quantify controlling factors are needed. Because enzymes are translated from genes, the ability to quantify gene expression levels for glycosylation-related enzymes would be advantageous. We developed quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays to monitor gene expression in Chinese hamster ovary (CHO) cells for five terminal glycosylation genes. The five enzymes were sialidase, a putative α2,3-sialyltransferase, β1,4-galactosyltransferase, cytosine monophosphate-sialic acid transporter, and uracil diphosphate-galactosyl transporter. Four of these CHO cell genes were publicly available from GenBank; however, the α2,3-sialyltransferase gene for Cricetulus griseus (CHO cell species) was not available and, therefore, was sequenced as a part of this work. The qRT-PCR primers and probes (based on the TaqManTM chemistry) were designed and validated for these five genes. The gene expression profiles were obtained for CHO cells producing the recombinant interleukin-4/13 cytokine trap molecule in batch reactors.
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Hooker, A. D., Goldman, M. H., Markham, N. H., et al. (1995), Biotechnol. Bioeng. 48, 639–648.
Hayter, P. M., Curling, E. M., Gould, M. L., et al. (1993), Biotechnol. Bioeng. 42, 1077–1085.
Hu, W. S., Zhou, W. C., and Europa, L. F. (1998), J. Microbiol. Biotechnol. 8, 8–13.
Kotani, N., Asano, M., Iwakura, Y., and Takasaki, S. (2001), Biochem. J. 357, 827–834.
Hinton, D. A., Evans, S. C., and Shur, B. D. (1995), Exp. Cell. Res. 219, 640–649.
Rajput, B., Shaper, N. L., and Shaper, J. H. (1996), J. Biol. Chem. 271, 5131–5142.
Harduin-Lepers, A., Vallejo-Ruiz, V., Krzewinski-Recchi, M.A., et al. (2001), Biochimie 83, 727–737.
Masibay, A. S., Damewood, G. P., Boeggeman, E., and Qasba, P. K. (1991), Biochim. Biophys. Acta 1090, 230–234.
Wen, D. X., Svensson, E. C., and Paulson, J. C. (1992), J. Biol. Chem. 267, 2512–2518.
Baum, L. G., Derbin, K., Perillo, N. L., et al. (1996), J. Biol. Chem. 271, 10,793–10,799.
Taniguchi, A. and Matsumoto, K. (1999), Biochem. Biophys. Res. Commun. 257, 516–522.
Chung, M. I., Lim, M. H., Lee, Y. J., et al. (2003), J. Microbiol. Biotechnol. 13, 217–224.
Grahn, A. and Larson, G. (2001), Glycoconjugate J. 18, 759–767.
Wang, H., Tachibana, K., Zhang, Y., et al. (2003), Biochem. Biophys. Res. Commun. 300, 738–744.
Ferrari, J., Gunson, J., Lofgren, J., Krummen, J., and Warner, T. G. (1998), Biotechnol. Bioeng. 60, 589–595.
Wang, M. D., Yang, M., Huzel, N., and Butler, M. (2002), Biotechnol. Bioeng 77, 194–203.
Puig-Kroger, A., Sanz-Rodriguez, F., Longo, N., et al. (2000), J. Immunol. 165, 4338–4345.
Klein, J. M. and McCarthy, T. A. (2002), Pediatr. Res. 51, 62A.
Yang, M. and Butler, M. (2000), Cytotechnology 34, 83–99.
Sears, H. J., Sawers, G., Berks, B. C., Ferguson, S. J., and Richardson, D. J. (2000), Microbiology 146, 2977–2985.
Yang, M. and Butler, M. (2002), Biotechnol. Prog. 18, 129–138.
Jeong, Y., Jinwoo, K., Suhyun, K., et al. (2003), Plant Dis. 87, 890–895.
Lunge, V. R., Miller, B. J., Livak, K. J., and Batt, C. A. (2002), J. Microbiol. Methods 51, 361–368.
Puig, S. and Perez-Ortin, J. E. (2000), Syst. Appl. Microbiol. 23, 300–303.
Steinbach, D., Lengemann, J., Voight, A., et al. (2003), Clin. Cancer Res. 9, 1083–1086.
Proudnikov, D., Yuferov, V., Laforge, K. S., Ho, A., and Kreek, M. J. (2003), Mol. Brain Res. 112, 182–185.
Proudnikov, D., Yuferov, V., Zhou, Y., et al. (2003), J. Neurosci. Methods 123, 31–45.
Berg, W., Gutschkergdaniec, G., and Schauer, R. (1985), Anal. Biochem. 145, 339–342.
Warner, T. G., Chang, J., Ferrai, J., et al. (1993), Glycobiology 3, 455–463.
Gramer, M. J., Goochee, C., Chock, V. Y., Brousseau, D. T., and Sllwkowski, M. B. (1995), Biotechnology 13, 692–698.
Ferrari, J., Harris, R., and Warner, T. G. (1994), Glycobiology 4, 367–373.
Anumula, K. R. (1997), in Techniques in Glycobiology, Townsend, R. R., Arland, J., and Hotchkiss, T., eds., Marcel Dekker, New York, pp. 349–357.
Gawlitzek, M., Ryll, T., Lofgren, J., and Sliwkowski, M. B. (2000), Biotechnol. Bioeng. 68, 637–646.
Suzuki T., Higgins, P., and Crawford, D. (2000), Bio Techniques 29, 332–337.
Freeman, W. M., Walker, S. J., and Vrana, K. E. (1999), BioTechniques 26, 112–125.
Mackay, I. M. (2004), Clin. Microbiol. Infect. 10, 190–212.
Livak, K. J. and Schmittgen, T. D. (2001), Methods 25, 402–408.
Gramer, M. J. and Goochee, C. F. (1993), Biotechnol. Prog. 9, 366–373.
Munzert, E., Muthing, J., Buntemeyer, H., and Lehmann, J. (1996), Biotechnol. Prog. 12, 559–563.
Goochee, C. F. and Gramer, M. J. (1994), Biotechnol. Bioeng. 43, 423–428.
Goochee, C. F., Gramer, M. J., Schaffer, D. V., and Sliwkowski, M. B. (1994), J. Cell. Biochem. (S18D), 263.
Hasegawa, T., Carnero, C. F., Wada, T., Itoyama, Y., and Miyagi, T. (2001), Biochem. Biophys. Res. Commun. 280, 726–732.
Sasaki, K., Watanabe, E., Kawashima, K., et al. (1993), J. Biol. Chem. 268, 22,782–22,787.
Kono, M., Ohyama, Y., Lee, Y. C., et al. (1997), Glycobiology 7, 469–479.
Kitagawa, H. and Paulson, J. C. (1994), J. Biol. Chem. 269, 17,872–17,878.
Endo, T., Koizumi, S., Tabata, K., and Ozaki, A. (2000), Appl. Microbiol. Biotechnol. 53, 257–261.
Taniguchi, A., Morishima, T., Tsujita, Y., Matsumoto, Y., and Matsumoto, K. (2003), Biochem. Biophys. Res. Commun. 300, 570–576.
Hirooka, T., Suganuma, N., Furuhashi, M., et al. (1996), Endocr. J. 43, 423–428.
Paulson, J. C., Colley, K., Lee, E. U., and Roth, J. (1988), Glycoconjugate J. 5, 330.
Taniguchi, A., Hioki, M., and Matsumoto, K. (2003), Biochem. Biophys. Res. Commun. 301, 764–768.
Eckhardt, M., Muhlenhoff, V., Bethe, A., and Gerardyschahn, R. (1996), Proc. Natl. Acad. Sci. USA 93, 7572–7576.
Eckhardt, M., Gotza, B., and Gerardy-Schahn R. (1999), J. Biol. Chem. 274, 8779–8787.
Bill, R. M., Revers, L., and Wilson, I. B. H. (1998), Protein Glycosylation, Kluwer Academic, Boston.
Berninsone, P. M. and Hirschberg, C. B. (2000), Curr. Opin. Struct. Biol. 10, 542–547.
Kumamoto, K., Goto, Y., Sekikawa, K., et al. (2001), Cancer Res. 61, 4620–4627.
Harduin-Lepers, A., Vallejo-Ruiz, V., Krzewinski-Recchi, M. A., et al. (2000), Biochem. J. 352, 37–48.
Lee, J. H., Sundaram, S., Shaper, N. L., Raju, T. S., and Stanley, P. (2001), J. Biol. Chem. 276, 13,924–13,934.
Gasteiger, E., Gattiker, A., Hoogland, C., et al. (2003), Nucleic Acids Res. 31, 3784–3788.
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Clark, K.J.R., Harcum, S.W., Griffiths, J. et al. Development of a method to quantify gene expression levels for glycosylation pathway genes in Chinese hamster ovary cell cultures. Appl Biochem Biotechnol 125, 159–173 (2005). https://doi.org/10.1385/ABAB:125:3:159
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DOI: https://doi.org/10.1385/ABAB:125:3:159