ASPECTS OF CELLULAR PHYSIOLOGY THAT INFLUENCE DNA-MEDIATED GENE-TRANSFER IN NIH3T3 CELLS

Cellular physiology has a significant influence on the efficiency of v arious gene transfer procedures, as shown by the fact that transfectio n efficiency varies dramatically among different cell lines. However, the aspects of cellular physiology which influence the transfection pr ocess remain substantially uncharacterized. In this study, NIH3T3 cell s were treated with inhibitors of protein synthesis, DNA synthesis, an d RNA synthesis to determine the importance of these processes in the calcium-phosphate transfection process. The results suggest that prote in synthesis during the first 4 h after DNA addition enhances transfec tion. In contrast, inhibition of RNA synthesis has no effect on transf ection during the first 24 h post-DNA addition. The DNA synthesis inhi bitor results remain inconclusive due to a secondary inhibition of an unknown cellular factor. Secondly, agents that destabilize microtubule s, microfilaments, and the golgi apparatus were used to determine whet her these elements play a role in the transfection process. The result s suggest that microtubules are not involved in the transfection proce ss, microfilaments are important but not necessary for the transfectio n process, and a functional golgi apparatus is essential early in the transfection process. These studies provide a foundation from which fu rther investigations into the cellular processes involved in the uptak e and expression of exogenous DNA can proceed.