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-1-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. (C) 2004 Wiley Periodicals, Inc.