siRNA-mediated gene silencing in the salivary gland using in vivo microbubble-enhanced sonoporation.
OBJECTIVE
siRNA-induced gene silencing in the salivary gland using microbubble-enhanced sonoporation was used to develop an in vivo gene knockdown technique.


METHODS
siRNA targeting rat glyceraldehyde-3-phosphate dehydrogenas (GAPDH) was mixed with echo-enhanced microbubbles and reverse-injected into rat parotid glands using transdermal ultrasound. To compare direct and transdermal ultrasound efficiencies, an incision was made on the lateral neck to expose the parotid glands for direct application. The efficiency of gene suppression was determined using quantitative reverse transcription-polymerase chain reaction 24-72 h after siRNA delivery. Cytotoxicity was assessed using histological analysis.


RESULTS
Expression of rat GAPDH in the parotid glands was silenced 48 h after siRNA was delivered by ultrasound (frequency: 1 MHz; intensity: 2 W cm(-2); exposure time: 2 min). High-intensity ultrasound induced tissue damage and apoptotic change. Echo-enhanced microbubbles significantly improved siRNA-induced gene silencing by 10-50%. Compared with transdermal application, direct-exposure ultrasound was only slightly effective, and no significant difference in gene expression was observed.


CONCLUSIONS
The results indicate that microbubble-enhanced sonoporation can yield in vivo siRNA gene silencing in the rat parotid gland. This technique could be applied to provide gene knockdown organs for functional genomic analyses and to develop siRNA-based gene therapy.
Sakai
T
T
Department of Pharmacology, Oral Health Science Center HRC7, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba, Chiba, 261-8502, Japan. takasakai@tdc.ac.jp
Kawaguchi
M
M
Kosuge
Y
Y
D000818:Animals; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000096:biosynthesis / Q000235:genetics; D057086:High-Intensity Focused Ultrasound Ablation; D020287:In Situ Nick-End Labeling; D008297:Male; D045423:Microbubbles; D010306:Parotid Gland / Q000201:enzymology* / Q000293:injuries; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar
D034741:RNA, Small Interfering; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)