TRANSFECTION OF A REPORTER PLASMID INTO CULTURED-CELLS BY SONOPORATION IN-VITRO

Citation
Sp. Bao et al., TRANSFECTION OF A REPORTER PLASMID INTO CULTURED-CELLS BY SONOPORATION IN-VITRO, Ultrasound in medicine & biology, 23(6), 1997, pp. 953-959
Citations number
21
Categorie Soggetti
Radiology,Nuclear Medicine & Medical Imaging",Acoustics
ISSN journal
03015629
Volume
23
Issue
6
Year of publication
1997
Pages
953 - 959
Database
ISI
SICI code
0301-5629(1997)23:6<953:TOARPI>2.0.ZU;2-G
Abstract
Cultured Chinese hamster ovary cells were exposed to 2.25-MHz ultrasou nd in sterile 4.5-mL polyethylene chambers and tested for cell lysis, sonoporation and DNA transfection, Ten percent of Albunex(R), a gas-bo dy-based ultrasound contrast agent, was added to ensure cavitation nuc leation, and the chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures, Uptake of large fluorescent dext ran molecules by some cells was observed for spatial peak pressure amp litudes as low as 0.1 MPa, which indicates transient permeabilization and resealing, i.e., sonoporation, of these cells during exposure, Sig nificant lysis occurred for 0.2 MPa, and increased rapidly for exposur es above the apparent cavitation threshold (using the H2O2 production test) of about 0.4 MPa spatial peak pressure amplitude, In the DNA tra nsfection tests, 20 mu g/mL luciferase reporter plasmid was added to t he suspension during exposure, and cells were assayed for proliferatio n ability and luciferase gene expression 2 days after exposure, Cell p roliferation was greatly reduced above the cavitation threshold, Lucif erase 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 w as greater for cells exposed in medium supplemented with serum than fo r cells exposed in serum-free medium, Cells harvested for exposure eit her in the log phase or in the stationary phase of culture gave simila r proliferation and transfection results, The effects essentially disa ppeared when the Albunex was omitted from the suspension and the tube was not rotated, Thus, sonoporation by ultrasonic cavitation in the ro tating tube exposure system yields plasmid transfection with subsequen t transient gene expression. (C) 1997 World Federation for Ultrasound in Medicine & Biology.