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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
Author(s) -
Fux Cornelia,
Langer Dominik,
Kelm Jens M.,
Weber Wilfried,
Fussenegger Martin
Publication year - 2004
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20028
Subject(s) - expression cassette , biology , transgene , internal ribosome entry site , chinese hamster ovary cell , expression vector , viral vector , microbiology and biotechnology , homing (biology) , recombinant dna , messenger rna , cell culture , vector (molecular biology) , genetics , gene , translation (biology) , ecology
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 α‐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. © 2004 Wiley Periodicals, Inc.