Molecular Medicine and Renal Units, Beth Israel Hospital, Depts. of Medicine and of Cell Biology, Harvard Medical School, 02215, Boston, Massachusetts, USA.
Cytotechnology. 1995 Jan;17(2):71-82. doi: 10.1007/BF00749394.
We have evaluated transient transfection of MDCK cells by the DEAE-dextran/chloroquine method as a rapid method for study of heterologous plasma membrane protein polarity. Transiently transfected cells reseeded onto permeable supports formed confluent monolayers with normal tight junctions and normal distribution of endogenous apical and basolateral surface markers. Transfected monolayers reseeded onto opaque polycarbonate filters attained cell heights 3 times greater than on transparent filters. Conventional and confocal immunofluorescence microscopy were used to assess polarity of transient expression of heterologous proteins previously defined in stably transfected cell lines as apical (DAF-CD55), basolateral (VSV-G), and nonpolarized (CD7) in distribution. Through each transiently expressed protein exhibited a polarity phenotype in most cells which resembled the stable phenotype, consistency of polarized localization was less than in stably transfected cells. Similar results were obtained by lipofection. We conclude that transient transfection of MDCK cells may be useful as a rapid screen, but is not sufficiently reliable for definitive assessment of heterologous membrane proein polarity.
我们评估了 DEAE-葡聚糖/氯喹法转染 MDCK 细胞作为研究异源质膜蛋白极性的快速方法。重新接种到可渗透载体上的瞬时转染细胞形成了具有正常紧密连接和正常分布的内源性顶端和基底外侧表面标记物的连续单层。重新接种到不透明聚碳酸酯过滤器上的转染单层细胞的高度比接种到透明过滤器上的高度增加了 3 倍。传统和共聚焦免疫荧光显微镜用于评估先前在稳定转染细胞系中定义为顶端（DAF-CD55）、基底外侧（VSV-G）和非极化（CD7）的异源蛋白瞬时表达的极性。通过每种瞬时表达的蛋白在大多数细胞中表现出类似于稳定表型的极性表型，但是与稳定转染细胞相比，极化定位的一致性较低。脂质体转染也得到了类似的结果。我们得出结论，MDCK 细胞的瞬时转染可能作为快速筛选有用，但不足以可靠地确定异源质膜蛋白的极性。
