Transfection of a reporter plasmid into cultured cells by sonoporation in vitro.
Cultured Chinese hamster ovary cells were exposed to 2.25-MHz ultrasound in sterile 4.5-mL polyethylene chambers and tested for cell lysis, sonoporation and DNA transfection. Ten percent of Albunex, a gas-body-based ultrasound contrast agent, was added to ensure cavitation nucleation, and the chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures. Uptake of large fluorescent dextran molecules by some cells was observed for spatial peak pressure amplitudes as low as 0.1 MPa, which indicates transient permeabilization and resealing, i.e., sonoporation, of these cells during exposure. Significant lysis occurred for 0.2 MPa, and increased rapidly for exposures above the apparent cavitation threshold (using the H2O2 production test) of about 0.4 MPa spatial peak pressure amplitude. In the DNA transfection tests, 20 micrograms/mL luciferase reporter plasmid was added to the suspension during exposure, and cells were assayed for proliferation ability and luciferase gene expression 2 days after exposure. Cell proliferation was greatly reduced above the cavitation threshold. Luciferase production was significant for 0.20-MPa exposure, and reached 0.33 ng per 10(6) cells at 0.8-MPa exposure. The luciferase production was great for cells exposed in medium supplemented with serum than for cells exposed in serum-free medium. Cells harvested for exposure either in the log phase or in the stationary phase of culture gave similar proliferation and transfection results. The effects essentially disappeared when the Albunex was omitted from the suspension and the tube was not rotated. Thus, sonoporation by ultrasonic cavitation in the rotating tube system yields plasmid transfection with subsequent transient gene expression.
Bao
S
S
USTUR Washington State University, Richland, USA.
Thrall
B D
BD
Miller
D L
DL
D000818:Animals; D016466:CHO Cells / Q000000981:diagnostic imaging* / Q000201:enzymology; D002455:Cell Division; D002463:Cell Membrane Permeability; D002470:Cell Survival; D002478:Cells, Cultured / Q000000981:diagnostic imaging / Q000382:microbiology; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D013010:Sonication; D014162:Transfection / Q000379:methods; D014463:Ultrasonography
D004247:DNA; D008156:Luciferases