Gene Transfer into Limb Bud of mammalian Embryos Using Electropolation Technique

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 15591905
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Plastic surgery
• Research Institution: Jikei University School of Medicine
• Principal Investigator: KISHI Yoko Jikei University School of Medicine, Lecturer, 医学部, 講師 (70257921)
• Co-Investigator(Kenkyū-buntansha): KURIHARA Kunihiro Jikei University School of Medicine, Professor, 医学部, 教授 (70133387) ; UCHIDA Mitsuru Jikei University School of Medicine, Professor, 医学部, 教授 (00176695) ; WATANABE Toshiaki Jikei University School of Medicine, Assistant, 医学部, 助手 (50266706)
• Project Period (FY): 2003 – 2004
• Keywords: Gene transfer / The whole embryo culture system / Electropolation

Research Abstract

After the decording of human gene was almost finished, Next big theme is how each gene interrlate to the other genes, and how to make the complex human structure and function. In this study, the whole embryo culture(WEC)system which is able to reproduce the development completely has been used, we talk about the establishment of the way to induce gene to the mouse embryo bud.
Using Std-ddt mouse(E12), Embryo was dissected out at the state of remaining of the york sac, amnion, chorioallantoic placenta. Plasmid(CMV enhancer+β-actin promoter) added GFP 0.1μl was injected by microcapillery tube into the york sac Immediately after injection of material, the embryo was pinched by the pincet type element.
In the Tyrode's solution Electric shock(30.40. and 50V,50ms,3pulse) was applied to the embryo. Then Embryo was put into the bottle within which the mouse serum with ampicirin 5 μg/ml and glucose 2mg/ml was infused. The rotating bottles were incubated at 37℃, fresh 95% O2,5% N2 was supplied to the bottle at 2 times a day. The rotator bottle were turened at 30rpm. After 48 hours the frozen sample was resected, we observated them by fluoscent microscope.
Below was the results. At the power of 40v,50v, the GFP was detected in the whole body, but at the power of 30v the only bud portion the GFP was observed by the electric microscope.