Cytomegalovirus plasmid vectors for permanent lines of polarized epithelial cells.
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.
Brewer
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CB
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D016764:Cell Polarity; D002522:Chlorocebus aethiops; D003587:Cytomegalovirus / Q000235:genetics* / Q000502:physiology; D004285:Dogs; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D007668:Kidney; D018374:LLC-PK1 Cells; D008969:Molecular Sequence Data; D010957:Plasmids; D013552:Swine