Brewer C B
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
Methods Cell Biol. 1994;43 Pt A:233-45. doi: 10.1016/s0091-679x(08)60606-8.
Several versions of plasmid vectors that incorporate CMV immediate early promoters are now in use. Of particular utility and convenience for making permanently transfected polarized cell lines are those that also direct expression of a selectable marker. Several methods of transfecting cells are available, but the polybrene method is recommended for MDCK cells because it is effective, easy, and inexpensive. After transfection, cells are replated in a selective drug for 10-14 days to kill untransfected cells; then surviving colonies are cloned with cloning rings. Screening of these colonies for expression of the desired protein ordinarily yields 10-15% cell lines with sufficiently high expression to be useful. It should not be assumed that every clone of a polarized cell line will be properly polarized, particularly in the case of MDCK cells. However, assays for correct sorting of endogenous markers can be used to verify proper polarity of transfectants or to identify well-polarized untransfected clones to be transfected. Using these methods and CMV vectors, one can easily establish one or more permanently transfected polarized cell lines within about 1 mo.
目前有几种包含巨细胞病毒（CMV）立即早期启动子的质粒载体版本在使用。对于构建永久转染的极化细胞系而言，特别实用且方便的是那些还能指导选择标记物表达的载体。有几种细胞转染方法可供选择，但对于MDCK细胞，推荐使用聚凝胺方法，因为它有效、简便且成本低廉。转染后，将细胞重新接种到含有选择性药物的培养基中培养10 - 14天，以杀死未转染的细胞；然后用克隆环克隆存活的菌落。对这些菌落进行所需蛋白质表达的筛选，通常会得到10 - 15%表达水平足够高且有用的细胞系。不应假定极化细胞系的每个克隆都会正确极化，尤其是对于MDCK细胞而言。然而，对内源标记物正确分选的检测可用于验证转染子的正确极性，或鉴定待转染的极化良好的未转染克隆。使用这些方法和CMV载体，大约1个月内就能轻松建立一个或多个永久转染的极化细胞系。
