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Study for gene transfection and intracellular distribution of transfected DNA.

基因转染及转染DNA细胞内分布的研究。

基本信息

批准号：
07558099
负责人：
NAKANISHI Mamoru
金额：
$ 8.32万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07558099/
关键词：
Cationic liposomes Gene transfection Cationic cholesterol Zeta potential Antisense DNA Atomic force microscopy Liposome DNA complex Plasmid DNA リポソームアンチロンスDNA プラスミドアンチセンス

项目摘要

DNA-mediated transfection has become an important tool in modern biology. We have studied the use of the cationic liposomes for gene transfection. The results showed that the cationic liposomes containing a cationic cholesterol derivative, cholesteryl-3 beta -carboxyamido ethylenedimethylamine, were most effective among eight derivatives of cationic cholesterol. At the same time we measured zeta potential of the liposomes by laser Doppler spectroscopy. Values of zeta potential of the liposomes were well consistent with those of transfection efficiency of pSV2CAT into the cells. The results showed that zeta potential of cationic liposomes was one of the important factors which control gene transfection.Next, we investigated that a novel cationic cholesterol derivative with a hydroxyethylamino head group. The transfection efficiency by the new cholesterol derivative was much higher than those by the cationic liposomes containing cationic derivatives with a dialkylamino head group described above. Further, the efficiency by the liposomes was not so much decreased in the presence of serum. This suggested that a novel cationic cholesterol derivative should be very promising in liposome-mediated gene transfection of plasmid and antisense DNA into target cells.Further, atomic force microscopy (AFM) was used for studying gene transfection mediated by cationic liposomes which contain a cationic cholesterol derivative. AFM images showed that vesicles made of the liposome/DNA complex had various diameters depending on each cationic cholesterol derivative with a different spacer arm. The results showed that the diameter of the complex was well related to the transfection activity of plasmid DNA to cultured cell lines. From the results it was found that the vesicles with moderate diameters (from 0.4 to 1.4 mum) were most effective for gene transfection of plasmid DNA into the target cells.

dna介导的转染已成为现代生物学的重要工具。我们研究了阳离子脂质体在基因转染中的应用。结果表明，在8种阳离子胆固醇衍生物中，含有胆固醇-3 β -羧胺-乙二甲胺的阳离子脂质体效果最好。同时用激光多普勒光谱法测定了脂质体的ζ电位。脂质体的zeta电位值与pSV2CAT转染细胞的效率值一致。结果表明，阳离子脂质体的zeta电位是控制基因转染的重要因素之一。接下来，我们研究了一种具有羟基乙胺头基团的新型阳离子胆固醇衍生物。新胆固醇衍生物的转染效率远高于含有上述二烷基胺头基阳离子衍生物的阳离子脂质体。此外，脂质体的效率在血清的存在下没有那么大的下降。这表明，一种新的阳离子胆固醇衍生物在脂质体介导的质粒和反义DNA基因转染靶细胞中具有很大的应用前景。此外，原子力显微镜（AFM）用于研究含有阳离子胆固醇衍生物的阳离子脂质体介导的基因转染。AFM图像显示，脂质体/DNA复合物制成的囊泡具有不同的直径，这取决于每个具有不同间隔臂的阳离子胆固醇衍生物。结果表明，该复合物的直径与质粒DNA转染培养细胞系的活性密切相关。结果表明，中等直径的囊泡（0.4 ~ 1.4 μ m）最适合将质粒DNA转染到靶细胞中。