Fux Cornelia, Langer Dominik, Kelm Jens M, Weber Wilfried, Fussenegger Martin
Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Hoenggerberg, HPT D74, CH-8093 Zurich, Switzerland.
Biotechnol Bioeng. 2004 Apr 20;86(2):174-87. doi: 10.1002/bit.20028.
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.
利用经过验证的多顺反子表达载体平台，我们设计了新型pTRIDENT载体，这些载体：(1)能够使三个所需转基因协调表达；(2)经过大小优化；(3)利用GTX或Rbm3型高效小内部核糖体进入位点；(4)含有多种归巢内切核酸酶特异性位点，便于启动子/多顺反子表达单元/聚腺苷酸化位点交换，以及(5)直接整合到经改造以包含兼容归巢内切核酸酶的基于人类HIV-1的慢病毒表达载体中。已对为编码人类低分子量尿激酶型纤溶酶原激活剂（u-PA(LMW)）或嗜热脂肪芽孢杆菌来源的α淀粉酶（SAMY）、人类血管内皮生长因子（hVEGF）和人类胎盘分泌碱性磷酸酶（SEAP）的不同三顺反子表达配置设计的新型pTRIDENT载体的多顺反子表达谱在中国仓鼠卵巢细胞（CHO-K1）、小鼠成纤维细胞（NIH/3T3）和/或人类纤维肉瘤（HT-1080）细胞中进行了定量分析。此外，将源自pTRIDENT的SAMY-VEGF-SEAP表达盒转移到兼容的慢病毒表达载体中，在将转基因慢病毒颗粒转导到原代人软骨细胞后能够实现同时的高水平转基因表达。
