一种高效省时利用磷酸钙转染DNA改进方法.docVIP

一种高效省时的利用磷酸钙转染DNA的改进方法 　　摘 要 对传统磷酸钙转染方法进行改进，即在经胰酶消化处理成悬浮状态的293T细胞悬液中直接加入磷酸钙转染试剂，而不需待其贴壁后再加转染试剂进行转染.通过比较悬浮和贴壁状态下293T细胞的转染效率、细胞增殖和荧光强度等指标，发现这两种转染方法的各项指标没有显著性差异.结果表明，293T细胞在悬浮状态下进行磷酸钙转染与传统的贴壁转染能够达到同等效果，在用胰酶消化的单细胞悬液中直接加入磷酸钙转染试剂进行转染，不影响细胞的转染效果，节省了操作步骤，缩短了操作时间. 　　关键词 293T细胞；磷酸钙；贴壁状态；悬浮状态；细胞转染 　　中图分类号 Q813.1 文献标识码 A 文章编号2016 　　An Efficient and TimeSaving Method for DNA Transfection Using Calcium Phosphate 　　HUANG Jina， Fu Jiqiangb， ZHANG Qinghuaa* 　　（a.School of Life Science and Technology， b.School of Medicine， Tongji University， Shanghai 200092， China） 　　Abstract The use of calcium phosphate for the transfection of adherent cells was a traditional and popular yet complicated and timeconsuming method. In this paper， this traditional method is further improved through the direct addition of calcium phosphate transfection reagent into the trypsinized 293T cell suspension solution， instead of adding transfection reagent after the cells were adhered to the bottom. By comparison with such indicators as transfection efficiency， cell proliferation and fluorescence intensity of 293T cells in suspended status and adherent status， we found no significant difference between the two transfection methods with regard to these indicators. Based on the results from this work， the use of calcium phosphate in transfecting 293T cells in suspended status could be same efficient and effective as the traditional adherent transfection method. With the transfection efficiency unaffected， our method of directly adding calcium phosphate transfection reagent into the trypsinized singlecell suspension solution is not only experimentally simplified in procedure but also timesaving in operation. 　　Key words 293T cells； calcium phosphate； adherent status； suspended status； cell transfection 　　目前有多种细胞转染方法，例如脂质体转染法[1]、基因鸟枪法、电穿孔法[2]、显微注射法[3]和磷酸钙共沉淀法等.脂质体转染法虽然操作简单，转染效率较高，但脂质体价格昂贵，且细胞毒性较大；基因鸟枪法速度快，简单易行，成本较低，但不适用于真核细胞；电穿孔法转染效率高，但所需仪器较贵，对细胞损伤较大；显微注射法整合效率高，不需载体，但需精密仪器，技术难度大，且价格昂贵；磷