Electroporation is one of the most common methods used to transfer exogenous gene into most prokaryotic or eukaryotic cells. The detailed mechanisms of DNA-uptake by cells, however, have not yet been clearly understood. In this thesis, we have summarized a series of experiment designed to examine the DAN- uptake mechanisms. In addition, cell treatments that can enhance the efficiency of electroporation are also discussed. The results show that after the application of electric field the additional treatment by using a recovery medium containing proper amounts of divalent ions can significantly increase the transfection efficiency in 3T6, Pt K2 and SK-N-SH cells. Several factors, including the strength of electric field, recovery time and concentrations of Ca⁺⁺ or Mg⁺⁺ were examined. The...[ Read more ]

Electroporation is one of the most common methods used to transfer exogenous gene into most prokaryotic or eukaryotic cells. The detailed mechanisms of DNA-uptake by cells, however, have not yet been clearly understood. In this thesis, we have summarized a series of experiment designed to examine the DAN- uptake mechanisms. In addition, cell treatments that can enhance the efficiency of electroporation are also discussed. The results show that after the application of electric field the additional treatment by using a recovery medium containing proper amounts of divalent ions can significantly increase the transfection efficiency in 3T6, Pt K2 and SK-N-SH cells. Several factors, including the strength of electric field, recovery time and concentrations of Ca⁺⁺ or Mg⁺⁺ were examined. The reason for increasing the transfection efficiency is believed to be mainly caused by promoting the entry of DNA through the cell membrane, rather than by enhancing the expression of the transferred gene. It has been suggested that DNA-uptake is a multiple- step process. (1) DNA may be driven into the cell by an electrophoretic force when the membrane pores were opened during the application of the electric field. (2) After the electric field was turned off, DNA may continue to diffuse into the cell before the membrane pores were closed. (3) Finally, DNA may be carried in the cell by endocytosis after the membrane pores were resealed. In this research, we have designed specific experiments to test each of these steps. Our results support steps (1) and (3), but not step (2). We found that the kinetics of membrane pore resealing depends strongly on the size of the transport molecules. Thus, uptake of large molecules such as FITC labeled dextran (70 kD) or DNA stopped almost within seconds after the electric field was turned off. In summary, we propose that the DNA-uptake mechanisms in electroporation involve a fast step of DNA entrance driven by the electric field, and a relatively slower process of DNA-uptake by endocytosis following the turn-off of the electric pulses. The experimental evidence will be discussed in detail in this thesis.