The introduction of biologically active foreign genes into human respiratory epithelial cells using electroporation.
A simple method for introducing genes into respiratory epithelial cells would assist molecular studies of a variety of pulmonary disorders. Several different techniques for introducing foreign DNA into cells have been described but have either not been useful for respiratory epithelial cells or are difficult and cumbersome to perform. Electroporation is a simple technique that consists of exposing a cell-DNA suspension to an electric shock. Although it has been used to introduce genes into a variety of cell types, it has not previously been applied to respiratory epithelial cells. Human nasal epithelial cells were transfected with the plasmid pRSVCAT, which is an expression vector containing the origin of replication of pBR322 coupled to the Rous sarcoma virus (RSV) long terminal repeat (LTR) region driving the coding sequence for the chloramphenicol acetyltransferase (CAT) gene. The CAT gene is useful for determining optimal conditions for electroporation since it is not normally present in eukaryotic cells, and CAT activity correlates with the level of CAT mRNA; this provides a measure of expression of introduced foreign genes. Successful expression of the CAT gene was demonstrated by electroporation, whereas calcium phosphate transfection resulted in very low CAT activity. Optimal conditions for electroporation of respiratory epithelial cells were determined. Electroporating nasal epithelial cells using 500 volts, a DNA concentration of 10 micrograms/ml, and a sucrose buffer yielded the highest CAT activity, which peaked at 48 h after electroporation.
Iannuzzi
M C
MC
Department of Internal Medicine, University of Michigan, Ann Arbor 48109.
Weber
J L
JL
Yankaskas
J
J
Boucher
R
R
Collins
F S
FS
D001358:Avian Sarcoma Viruses / Q000235:genetics*; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D005821:Genetic Techniques; D006801:Humans; D009297:Nasal Mucosa / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids; D014162:Transfection
D015500:Chloramphenicol O-Acetyltransferase