Swiss Federal Institute of Technology, ETH Zurich, Institute of Biotechnology, CH-8093, Zurich, Switzerland.
Cytotechnology. 1998 Nov;28(1-3):229-35. doi: 10.1023/A:1008014706196.
Based on internal ribosomal entry sites (IRES) of picornaviral origin we constructed a novel family of mammalian expression vectors. pQuattro vectors contain quattrocistronic artificial eukaryotic operons which link, in a single transcript, the simultaneous and coordinated as well as adjustable expression of up to three independent genes of interest to a terminal neomycin (neo) resistance marker. Due to the strict genetic linkage of the transgenes and the terminal selection marker, this genetic configuration enables, by the selection on neomycin, multigene metabolic engineering of mammalian cells in a single step (one-step metabolic engineering). Furthermore, selection on the terminal cistron of multicistronic expression units enforces cocistronic expression of all upstream encoded genes and maximises genetic integrity of the eukaryotic operon in stable mammalian cell lines, since clones harbouring damaged multicistronic expression units become neomycin-sensitive and are automatically counterselected (auto-selection). The modular set-up and the abundance of restriction sites in pQuattro vectors facilitate the movement of individual genes between multicistronic expression vectors and guarantees high compatibility with genetic elements of a wide variety of existing mammalian expression vectors.
基于小核糖核酸病毒起源的内部核糖体进入位点，我们构建了一个新型哺乳动物表达载体家族。pQuattro 载体包含四顺反子人工真核操纵子，在单个转录本中，将多达三个独立的感兴趣基因的同时、协调和可调节表达与末端新霉素（neo）抗性标记连接起来。由于转基因和末端选择标记的严格遗传连锁，这种遗传结构通过新霉素选择，能够在单个步骤中对哺乳动物细胞进行多基因代谢工程（一步代谢工程）。此外，多顺反子表达单元的末端顺式元件的选择强制所有上游编码基因的共顺式表达，并使稳定的哺乳动物细胞系中的真核操纵子的遗传完整性最大化，因为携带受损多顺反子表达单元的克隆对新霉素敏感，并自动被反选择（自动选择）。pQuattro 载体的模块化设计和丰富的限制酶切位点促进了单个基因在多顺反子表达载体之间的转移，并保证了与各种现有哺乳动物表达载体的遗传元件的高度兼容性。
