Subcellular trafficking of the cytoplasmic expression system

Cationic liposomes have provided many advantages over viral vector formulat ions; however, the problem of inefficient gene expression remains. This is due in part to the nuclear membrane, which limits DNA entry into the nucleu s. Cytoplasmic expression systems using T7 RNA polymerase have been develop ed to express genes in the cytoplasm and avoid the need for nuclear import of DNA, Although these systems show improved transgene expression, little i s known about how they function in transfected cells. Direct comparisons be tween a cytoplasmic and nuclear expression system were carried out with a 2 93 cell line stably expressing T7 RNA polymerase, A formulation for optimal reporter gene expression was developed and used in conjunction with a vari ety of subcellular trafficking inhibitors to study the process of DNA endoc ytosis. Transfected cells were also studied at different stages of the cell cycle to determine the dependence of each system on mitosis, These results showed that cytoplasmic and nuclear expression systems utilize similar end ocytosis pathways to the point of endosomal release, Once DNA is released i nto the cytoplasm, the cytoplasmic expression system shows immediate expres sion that is proportional to the amount of DNA released, In contrast, DNA t argeted for nuclear expression requires additional time for nuclear entry, The level of nuclear expression is also restricted by the limited amount of DNA that is imported into the nucleus. Finally, mitosis is required for ef fective nuclear expression but not for cytoplasmic expression. Therefore, t he cytoplasmic expression system has considerable advantages over tradition al nuclear expression systems and may be an effective method for transfecti ng nondividing cells.