Assessment of heterologous membrane protein polarity in transiently transfected MDCK cells |
| |
Authors: | Haller C Alper S L |
| |
Institution: | (1) Molecular Medicine and Renal Units, Beth Israel Hospital, Depts. of Medicine and of Cell Biology, Harvard Medical School, 02215 Boston, Massachusetts, USA;(2) Present address: Abt. III, Medizinische Universitätskinik, Bergheimerstr. 58, 6900 Heidelberg, Germany |
| |
Abstract: | 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.Abbreviations CD55-DAF
CD55-Decay-accelearating factor
- DMSO
Dimethylsulfoxide
- FBS
Fetal bovine serum
- FITC
Fluorescein isothiocyanate
- MDCK
Madin Darby canine kidney cells
- PBS
Phosphate-buffered saline
- TER
Transepithelial resistance
- VSVG
Vesicular stomatis virus G protein |
| |
Keywords: | MDCK cells epithelial polarity transient transfection CD7 CD55 Decay Accelerating Factor Vesicular Stomatitis Virus G protein chloroquine |
本文献已被 PubMed SpringerLink 等数据库收录! |
|