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用途。结果表明，含有阳离子胆固醇衍生物胆固醇-3β-羧酰胺乙二甲胺的阳离子脂质体是八种阳离子胆固醇衍生物中最有效的。同时我们通过激光多普勒光谱测量了脂质体的zeta电位。脂质体的zeta电位值与pSV2CAT转染细胞的效率一致。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，我们研究了一种新型的带有羟乙基氨基头基的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具有二烷基氨基头基的阳离子衍生物的阳离子脂质体的转染效率。此外，在血清存在的情况下，脂质体的效率并没有降低太多。这表明一种新型的阳离子胆固醇衍生物在脂质体介导的质粒和反义DNA基因转染至靶细胞中应该是非常有前途的。此外，原子力显微镜（AFM）被用于研究含有阳离子胆固醇的阳离子脂质体介导的基因转染衍生物。 AFM 图像显示，由脂质体/DNA 复合物制成的囊泡具有不同的直径，具体取决于具有不同间隔臂的每种阳离子胆固醇衍生物。结果表明，复合物的直径与质粒DNA对培养细胞系的转染活性有很好的相关性。结果发现，中等直径（0.4至1.4μm）的囊泡对于将质粒DNA基因转染至靶细胞中最有效。