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A novel vector platform for vitamin H-inducible transgene expression in mammalian cells

Paper ID Volume ID Publish Year Pages File Format Full-Text
25157 43556 2007 9 PDF Available
Title
A novel vector platform for vitamin H-inducible transgene expression in mammalian cells
Abstract

Inducible transgene control systems have been instrumental to gene therapy, biopharmaceutical manufacturing, drug discovery, synthetic biology and functional genomic research. The most widely used heterologous gene regulation systems are responsive to antibiotics of the tetracycline, streptogramin and macrolide classes. Although these antibiotics are clinically licensed, concerns about the emergence of resistant bacteria, side-effects in animal studies, and economic considerations associated with clearance of antibiotics in biopharmaceutical manufacturing, have limited the use of heterologous transgene control modalities to basic research activities. We have therefore designed a strategy to convert antibiotic-responsive transcription factors into gene regulation systems responsive to non-toxic biotin, also known as vitamin H. Constitutive ligation of biotin to the Avitag-containing VP16 transactivation domain by the Escherichia coli biotin ligase BirA enables heterodimerization with tetracycline- (TetR), streptogramin- (Pip), and macrolide- (E) dependent repressors fused to streptavidin, which creates synthetic transactivators able to activate specific promoters (PhCMV*−1, PPIR, PETR). We have demonstrated (i) that exogenous biotin (40 nM) can induce heterologous transgene expression in a biotin- (serum-) free culture environment (biotin-dependent heterodimerization of transactivator); (ii) that excess biotin (above 200 μM) gradually represses transgene expression in a biotin- (serum-) containing environment (saturation of streptavidin by excess biotin prevents heterodimerization of the transactivator); and (iii) that avidin can sequestrate endogenous biotin in serum-containing cultures and so repress transgene expression in a dose-dependent manner. In addition, by engineering all off the components required for biotin-controlled transgene expression (Avitag-VP16, repressor-streptavidin, BirA) into a tricistronic (lenti)vector configuration, it was possible to transfect (transduce) a variety of mammalian cell lines and primary cells and enable biotin-controlled transgene expression in a simple and straightforward manner. The conversion of generic antibiotic-responsive transcription control modalities into systems adjustable by non-toxic vitamin H may foster novel advances in reprogramming of mammalian cells and production of difficult-to-produce protein pharmaceuticals.

Keywords
Gene regulation; Expression control; Biotin; Macrolide; Streptogramin; Mammalian cells; Biopharmaceutical manufacturing
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A novel vector platform for vitamin H-inducible transgene expression in mammalian cells
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 131, Issue 2, 31 August 2007, Pages 150–158
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us