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Development of efficient gene transfection procedure for animal cells

开发动物细胞高效基因转染程序

基本信息

批准号：
10650783
负责人：
KAMIHIRA Masamichi
金额：
$ 2.56万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10650783/
关键词：
Artificial virus Gene carrier Targeting Gene transfer Animal cells Lipid vesicle Genome integration Retrovirus integrase プロタミンリピッドベシクルレセプターリガンド

项目摘要

We studied gene transfer for animal cells using cationic lipid vesicles. In order to enhance transfection efficiency, we attempted (1) addition of DNA binding proteins to protect DNA degradation and to promote nuclear transfer, (2) introduction of ligands to lipid vesicles for cell-specific targeting, and (3) combination with retrovirus integrase to enhance host genome in integration.By adding to protamine to DNA solution before the formation of DNA/cationic lipid vesicle complexes, transfection efficiency and expression level were enhanced for all the cell lines and all the plasma tested. The enhancement in both transfection efficiency and expression level was at most 20-fold compared with that using only lipid vesicles.We also examined modification of lipid vesicles with a ligand such as insulin and galactose-residues to realize receptor-mediated gene transfer. By insulin-and galactose-modified lipid vesicles, transfection efficiency increased 3-4 fold in all the cell lines tested and selectively in hepatoma cell lines, respectively.Then, we attempted to incorporate the retrovirus integration machinery in lipid vesicle-mediated gene transfection with the aim of achieving efficient stable transfection. A DNA fragment, in which a target gene was flanked between partial LTR sequences, was transfected with integrase expression vectors by means of lipid vesicle-mediated gene transfection. Under optimal conditions, the stable transfection efficiency showed a 16-fold improvement over that without integrase.

我们研究了利用阳离子脂质囊泡进行动物细胞的基因转移。为了提高转染效率，我们尝试（1）加入DNA结合蛋白以保护DNA降解并促进核转移，（2）将配体引入脂质囊泡以实现细胞特异性靶向，（3）与逆转录病毒整合酶结合以增强宿主基因组的整合。对于所有测试的细胞系和所有血浆，转染效率和表达水平都得到提高。与单独使用脂质囊泡相比，转染效率和表达水平最多提高20倍。我们还研究了用配体如胰岛素和半乳糖残基修饰脂质囊泡以实现受体介导的基因转移。经胰岛素和半乳糖修饰的脂质囊泡在所有检测的细胞系中的转染效率提高了3-4倍，在肝癌细胞系中的转染效率也有选择性提高。通过脂质囊泡介导的基因转染，用整合酶表达载体转染其中靶基因侧接在部分LTR序列之间的DNA片段。在最佳条件下，稳定的转染效率显示了16倍的提高比没有整合酶。