Overexpression of Trehalose Synthase and Accumulation of Intracellular Trehalose in 293H and 293FTetR:Hyg Cells

• Published in: Cryobiology 2001-09, Vol.43 (2), p.106-113
• Main Authors: Lao, Guifang, Polayes, Deborah, Xia, Jiu Lin, Bloom, Fredric R., Levine, Fred, Mansbridge, Jonathan
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Cell Line; Cell physiology; Cytomegalovirus - genetics; Desiccation; Effects of physical and chemical agents; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression - drug effects; Genes, Bacterial; Genetic Vectors; Glucosyltransferases - genetics; Glucosyltransferases - metabolism; Humans; Molecular and cellular biology; Phosphoric Monoester Hydrolases - genetics; Phosphoric Monoester Hydrolases - metabolism; Promoter Regions, Genetic; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Tetracycline - pharmacology; Tetracycline Resistance - genetics; Transfection; trehalose; Trehalose - metabolism; trehalose synthase
• ISSN: 0011-2240; 1090-2392
• DOI: 10.1006/cryo.2001.2354

A humanized clone containing the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase (otsA/B) has been constructed. Using the Gateway Cloning System (Invitrogen, Inc.), the otsA/B genes have been placed under the control of the CMV promoter (pEXPcmv-otsA/B) or the CMV promoter and the tet operator (pEXP cmv TetO-otsA/B). The pEXPcmv-otsA/B clone has been introduced into 293H cells using LIPOFECTAMINE 2000 and the intracellular concentration of trehalose has been evaluated. The 293H cells accumulate 4–5 μg trehalose/mg dry weight and this concentration increases to 7–10 μg trehalose/mg dry weight if trehalose is included in the growth medium. The pEXPcmv TetO-otsA/B clone has been transfected into 293FTetR:Hyg cells which contain the tet repressor integrated into the genome. When these transfected cells are grown in the absence of tetracycline, no intracellular trehalose is detected. Inclusion of 0.3 μg/ml tetracycline in the growth medium results in the accumulation of 11–14 μg trehalose/mg dry weight, a value which increases to 19–20 μg trehalose/mg dry weight if trehalose is included in the growth medium. The data for the 293FTetR:Hyg cells indicate that intracellular trehalose accumulates in response to the addition of tetracycline. This system will allow us to manipulate the intracellular concentration of trehalose and to evaluate the desiccation tolerance of these cells as a function of intracellular trehalose concentration.