New-generation multicistronic expression platform: pTRIDENT vectors containing size-optimized IRES elements enable homing endonuclease-based cistron swapping into lentiviral expression vectors.
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.
Fux
Cornelia
C
Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Hoenggerberg, HPT D74, CH-8093 Zurich, Switzerland.
Langer
Dominik
D
Kelm
Jens M
JM
Weber
Wilfried
W
Fussenegger
Martin
M
D003001:Cloning, Molecular / Q000379:methods*; D004720:Endonucleases / Q000235:genetics*; D000072278:Forkhead Box Protein M1; D051858:Forkhead Transcription Factors; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D018014:Gene Transfer Techniques; D005796:Genes / Q000235:genetics*; D005822:Genetic Vectors; D016086:Lentivirus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods; D014157:Transcription Factors / Q000235:genetics*
D000072278:Forkhead Box Protein M1; D051858:Forkhead Transcription Factors; C491524:Foxm1 protein, mouse; D014157:Transcription Factors; D004720:Endonucleases