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New-generation multicistronic expression platform: pTRIDENT vectors containing size-optimized IRES elements enable homing endonuclease-based cistron swapping into lentiviral expression vectors.

Cornelia Fux·Dominik Langer·Jens M Kelm·Wilfried Weber·Martin Fussenegger
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Abstract

Capitalizing on a proven multicistronic expression vector platform we have designed novel pTRIDENT vectors which (1). enable coordinated expression of three desired transgenes, (2). are size-optimized, (3). take advantage of small highly efficient internal ribosome entry sites of the GTX or Rbm3 type, (4). harbor various sites specific for homing endonucleases facilitating promoter/multicistronic expression unit/polyadenylation site swapping as well as (5). straightforward integration into human HIV-l-based lentiviral expression vectors tailored to contain compatible homing endonucleases. Multicistronic expression profiles of novel pTRIDENT vectors engineered for different tricistronic expression configurations encoding human low-molecular-weight urokinase-type plasminogen activator (u-PA(LMW)) or Bacillus stearothermophilus-derived alpha-amylase (SAMY), human vascular endothelial growth factor (hVEGF), and human placental secreted alkaline phosphatase (SEAP) have been quantified in Chinese hamster ovary cells (CHO-K1), mouse fibroblasts (NIH/3T3), and/or human fibrosarcoma (HT-1080) cells. In addition, a pTRIDENT-derived SAMY-VEGF-SEAP expression cassette transferred into a compatible lentiviral expression vector enabled simultaneous high-level transgene expression following transduction of transgenic lentiviral particles into primary human chondrocytes.

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Cloning, MolecularEndonucleasesForkhead Box Protein M1Forkhead Transcription FactorsGene Expression Regulation, EnzymologicGene Transfer TechniquesGenesGenetic VectorsLentivirusPromoter Regions, GeneticProtein EngineeringTranscription FactorsComparative StudyEvaluation StudyJournal ArticleResearch Support, Non-U.S. Gov'tValidation Study

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Cornelia Fux, Dominik Langer, Jens M Kelm, Wilfried Weber, Martin Fussenegger. 2004-04-20. New-generation multicistronic expression platform: pTRIDENT vectors containing size-optimized IRES elements enable homing endonuclease-based cistron swapping into lentiviral expression vectors.. https://doi.org/10.1002/bit.20028

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