In vivotransfection of enhanced green fluorescent protein in rat retinal ganglion cells mediated by ultrasound-induced microbubbles*★

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Abstract

BACKGROUND:

Studies have demonstrated that ultrasound-mediated microbubble destruction significantly improves transfection efficiency of enhanced green fluorescent protein (EGFP) in in vitro cultured retinal ganglial cells (RGCs).

OBJECTIVE:

To investigate the feasibility of ultrasound-mediated microbubble destruction for EGFP transfection in rat RGCs, and to compare efficiency and cell damage with traditional transfection methods.

DESIGN, TIME AND SETTING:

In vivo, gene engineering experiment. The study was performed at the Central Laboratory, Institute of Ultrasonic Imaging, Chongqing Medical University from March to July 2008.

MATERIALS:

Eukaryotic expression vector plasmid EGFP and microbubbles were prepared by the Institute of Ultrasonic Imaging, Chongqing Medical University. The microbubbles were produced at a concentration of 8.7 × 1011/L, with a 2–4 μm diameter, and 10-hour half-life in vitro.

METHODS:

A total of 50 Sprague Dawley rats were randomly assigned to four groups. Normal controls (n = 5) were infused with 5 μL normal saline to the vitreous cavity; the naked plasmid group (n = 15) was infused with 5 μL EGFP plasmid to the vitreous cavity; in the plasmid with ultrasound group (n = 15), the eyes were irradiated with low-energy ultrasound wave (0.5 W/cm2) for a total of 60 seconds (irradiated for 5 seconds, at 10-second intervals) immediately following infusion of EGFP plasmids to the vitreous cavities. In the microbubble-ultrasound group (n = 15), the eyes were irradiated with the same power of ultrasonic wave immediately following infusion of microbubbles containing EGFP plasmids to the vitreous cavities.

MAIN OUTCOME MEASURES:

After 7 days, retinal preparations and EGFP expression in RGCs were observed by fluorescence microscopy. RGC quantification in the retinal ganglion cell layer was performed. In addition, EGFP mRNA expression was semi-quantitatively determined by RT-PCR.

RESULTS:

The transfection efficiency of EGFP to RGCs by microbubbles with ultrasound was significantly greater than the other groups, and no obvious damage was detected in the RGCs.

CONCLUSION:

Under irradiation of low-frequency ultrasound waves, ultrasound-mediated microbubble destruction was effective and resulted in safe transfection of the EGFP gene to the RGCs.

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