Development of a method to quantify gene expression levels for glycosylation pathway genes in chinese hamster ovary cell cultures.
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 alpha2,3-sialyltransferase, beta1,4-galactosyltransferase, cytosine monophosphate-sialic acid transporter, and uracil diphosphate-galactosyl transporter. Four of these CHO cell genes were publicly available from GenBank; however, the alpha2,3-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 TaqMan 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.
Clark
Kevin J-R
KJ
Department of Chemical Engineering, Clemson University, 112 Biosystems Research Complex, 51 New Cherry Street, Clemson, SC 29634-0905, USA.
Harcum
Sarah W
SW
Griffiths
Jennifer
J
Bailey
Kevin M
KM
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000502:physiology; D006031:Glycosylation; D006801:Humans; D018793:Interleukin-13 / Q000096:biosynthesis* / Q000235:genetics; D015847:Interleukin-4 / Q000096:biosynthesis* / Q000235:genetics; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D015398:Signal Transduction / Q000502:physiology*; D014166:Transferases / Q000378:metabolism*
D018793:Interleukin-13; D011993:Recombinant Fusion Proteins; D015847:Interleukin-4; D014166:Transferases; D019158:N-Acetylneuraminic Acid