A novel T7 RNA polymerase autogene for efficient cytoplasmic expression oftarget genes

Inefficient nuclear transport of plasmid DNA continues to be a problem in n onviral vector-mediated gene transfer. This has made the cytoplasmic expres sion system an increasingly attractive idea. We have developed a new T7 RNA polymerase autogene for cytoplasmic expression containing both a CMV and a T7 promoter The pCMV/T7-T7pol autogene does not encounter the problems ass ociated with previously used autogenes. For instance, pCMV/T7-T7pol is easi ly amplified and purified from bacteria. Furthermore, the CMV promoter is u sed to drive the first round of synthesis of T7 RNA polymerase, thus negati ng the use of purified enzyme in the transfection complex. The endogenous T 7 RNA polymerase produced from the CMV promoter could then act on the T7 pr omoter of pCMV/T7-T7pol in an autoregulatory mechanism. pCMV/T7-T7pol induc es higher, more sustained levels (>7 days) of reporter gene expression than that observed with the previously used autogene pT7 AUTO 2C(-) or with the nuclear expression system pCMV-CAT. This seems to be due to the high level s of T7 RNA polymerase protein that are detected in cells transfected with pCMV/T7-T7pol. This vector also functions as an efficient autogene since at least 50 times more mRNA is transcribed from the cytoplasmic T7 promoter a s compared with the nuclear CMV promoter in pCMV/T7-T7pol. Therefore, pCMV/ T7-T7pol could replace existing autogenes for regeneration of T7 RNA polyme rase and efficient target gene expression.