Ultrasound-Mediated Transfection of Mammalian Cells

Abstract

Mammalian cells were successfully transfected with plasmid DNA in vitro using ultrasound transmitted through the walls of cell culture flasks or plates. Primary rat fibroblasts or chondrocytes were exposed to ultrasound in the presence of plasmids containing lacZ or neo genes. The transfection efficiency was evaluated by counting the number of beta-galactosidase (beta-Gal) positive cells or neomycin-resistant colonies. Transfection efficiency was optimized by varying ultrasound conditions, ambient temperatures (room temperature or 37 degrees C), plasmid concentrations, and initial cell populations. Additional experiments were performed performed to elucidate the mechanism of the ultrasound-mediated transfection. Maximal gene transfection was seen with two ultrasound conditions: 1-MHz carrier frequency 411 +/- 189 kPascal continuous wave with 20 or 30 sec of exposure time, and 1 MHz carrier frequency 319 +/- 157 kPascal continuous wave with 40 or 60 sec of exposure time. Gene expression was negligible when transfection procedures were performed at room temperature. The average stable transfection rate was 0.34% of surviving cells with a plasmid concentration of 40 micrograms/ml in primary fibroblasts. The transient transfection rate was 2.4% of surviving cells for primary chondrocytes. Data suggest that increasing plasmid concentration will increase efficiency. Identical treatment with 3.5 MHz produced no transfection, implying that cavitation produced by the ultrasound pressure wave appeared to play a critical role in mediating transfection. Ultrasound-mediated transfection was effective for suspended cells as well as for plated cells. This transfection method is simple, easy to keep sterile, and convenient. Ultrasound-mediated transfection appears to be a promising method for gene transfer into mammalian cells.

Synergistic effect of ultrasound and PEI on DNA transfection in vitro

“…We therefore conclude that ultrasound significantly enhanced the transfection efficiency of naked DNA, but also lead to a loss in cell viability. These finding are similar to previous results concerning transfection efficiency of naked DNA [31,33,34,35].…”

Transfection of NFκB-decoy oligodeoxynucleotides using efficient ultrasound-mediated gene transfer into donor kidneys prolonged survival of rat renal allografts

“…[31][32][33] Consistent with previous reports, the present study showed that ultrasound exposure induced transfection of decoy oligo deoxyribonucleic acid (ODN) in renal tissue, but its transfection efficiency was not high. We therefore modified the procedure using an echo-contrast agent, Optison, to increase the transfection efficiency, and demonstrated significantly improved transfection of decoy ODN in renal tissue.…”

Focused ultrasound (HIFU) induces localized enhancement of reporter gene expression in rabbit carotid artery

“…The optimal ultrasound parameters for transfection such as sonication time, pressure, frequency, averaged intensity, and cavitation dose have not been systematically characterized. 5,6,14,16,17,19,20,32 Sinusoidal focused ultrasound transfection can be applied at an intensity level without substantial side effects, and, unlike a number of viral gene transfer vectors, multiple, repeated ultrasound transfection applications are possible. Further improvements of efficiency are therefore possible, including the use of adjuncts such as liposomal sheltering of plasmids.…”

“…Accordingly, ultrasonic shockwaves 11 and low-dose sinusoidal ultrasound have been shown to permeabilize cell membranes and mediate the transfer of proteins, 12 and molecules such as dextranes into cells. 13 It has also been demonstrated that ultrasound enhances the transfer of plasmid DNA into cells in culture, [14][15][16][17][18][19] skeletal muscle in vivo, 20,21 and experimental tumors in vivo. 22 A wide range of ultrasonic waves have been used to augment gene transfer such as low-level diagnostic ultrasound, 20 ultrasonic shockwaves 22 and HIFU.…”