用质粒DNA对悬浮培养的小鼠FM3A乳腺癌细胞进行高频转染。

High-frequency transfection of mouse FM3A mammary carcinoma cells in suspension culture with plasmid DNA.

作者信息

Ogawa T, Hyodo M, Ihara S, Takekoshi M, Minato K, Suzuki K

机构信息

Cosmetics Laboratory, Kanebo, Ltd., Kanagawa, Japan.

出版信息

Cell Struct Funct. 1987 Dec;12(6):567-74. doi: 10.1247/csf.12.567.

DOI:10.1247/csf.12.567

PMID:3435918

Abstract

High-frequency transfection of mouse FM3A cells, grown in suspension, with plasmid pSV2neo DNA was achieved by incubation of the cells with DNA plus polybrene for 6 h followed by an osmotic shock with a hypertonic NaCl solution. When incubated for 20 min at 34 degrees C, FM3A cells showed resistance to the osmolarity change from 0.1 to 9.0% NaCl in the medium. Within this concentration range, 5-7% gave the highest efficiency of transfection. Both linear and circular forms of plasmid DNA produced transformants with equal efficiency. This method was simple, reproducible, and carrier DNA was not required. The efficiency was about 100 times higher than that of the method with DNA-calcium phosphate precipitates. Transformed cells were stable and different numbers of plasmid DNA copies were detected.

摘要

通过将悬浮培养的小鼠FM3A细胞与DNA加聚凝胺一起孵育6小时，随后用高渗NaCl溶液进行渗透压休克处理，实现了用质粒pSV2neo DNA对其进行高频转染。当在34℃孵育20分钟时，FM3A细胞对培养基中NaCl渗透压从0.1%变为9.0%表现出抗性。在此浓度范围内，5 - 7%的NaCl浓度产生最高的转染效率。质粒DNA的线性和环状形式产生转化体的效率相同。该方法简单、可重复，且不需要载体DNA。其效率比DNA - 磷酸钙沉淀法高约100倍。转化细胞稳定，且检测到不同数量的质粒DNA拷贝。