Gene introduction into cells by means of disintegration of DNA-containing microfoam under exposare to ultrasound

1999 Fiscal Year Final Research Report Summary

• Project/Area Number: 09470137
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Gastroenterology
• Research Institution: KYOTO UNIVERSITY
• Principal Investigator: MORIYASU Fuminori School of Medicine, Kyoto University assistant professor, 医学研究科, 助教授 (80191055)
• Co-Investigator(Kenkyū-buntansha): TABATA Yasuhiko Institute for Frontier Medical Sciences, Kyoto University assistant professor, 再生医科学研究所, 助教授 (50211371) ; KISHI Kiyohiko School of Medicine, Kyoto University assistant, 医学研究科, 助手 (20273774)
• Project Period (FY): 1997 – 1999
• Keywords: microfoams / Introduction of DNA / c-myc gene / ultrasound treatment / liver / polylactate microfoams / cationized gelatin / hepatocytes

Research Abstract

Establishment of a method to adsorb DNA on microfoams : We prepared a complex of gelatin cationized using DNA and hexamethylene diamine and established a method to consistently adsorb DNA/cationized gelatin on the surface of polylactate microfoams by means of the electric potential of the complex.
Introduction of DNA by sonoporation : We attemped to introduce a plasmid containing luciferase-gene into RGM-1. A complex of this DNA and cationized gelatin was prepared, mixed with a medium, and exposed to ultrasound at a relatively low sound pressure level used for clinical diagnosis.
The luciferase activity 48 hours after exposure was about 500 times higher than in the control group. From these results, DNA introduction was confirmed to be promoted by sonoporation when a DNA/cationized gelatin complex is exposed to ultrasound.
Promotion of DNA introduction in animals : A complex was prepared by coating the above microfoams with cationized gelatin and binding them with c-myc gene. This complex was injected i.v. into mice, their liver was extracorporeally irradiated by ultrasound, excised after apredetermined period, and the expression of the target gene was examined by immunostaining. By comparison among the DNA alone group. DNA/gelatin complex group(no microfoams), and microfoams/cationized gelatin/DNA group, the expression of c-myc protein in hepatocytes was confirmed to be the strongest in the last group. These results suggest accumulation of this microfoam/cationized gelatin/DNA complex in the liver and furthen promotion of gene introduction into hepatocytes by ultrasound treatment.
Our study demonstrated that efficiency of gene introduction into animal hepatocytes can be markebly improved by the use of microfoams and ultrasound, and clinical application of this technique is anticipated.