Immunoselection of stably transformed MDCK cells expressing the vesicular stomatitis virus G-protein at the basolateral surface.
We have used immunoisolation on a magnetic solid support for the positive selection of stable MDCK transformants which express VSV-G protein via genomic integration of a cloned cDNA. This method is a simple, inexpensive alternative to selection with a fluorescence-activated cell sorter. The G-protein is synthesized in the absence of other viral proteins and is transported to the plasma membrane. The G-positive cells were enriched by immunoselection during normal passage of the transformed population. Using sterile conditions, antibodies to G were incubated with a suspension of transformed cells at 4 degrees C, unbound antibodies were then removed, and the cells were incubated with the immunoabsorbent (3 micron magnetic beads; J. Ugelstad et al. (1983) Nature (London) 303, 95) containing bound IgG molecules against the Fc portion of rabbit IgG. The magnetic properties of the beads were used to retrieve and further wash the immunoselected population. The cells are then removed from the beads by the same trypsinization conditions used for routine passaging and returned to culture. Using this selection scheme we have been able to increase the number of G-expressing cells five- to sevenfold per round; with repeated rounds enrichment from 2 to 74% was obtained. When grown on filters the immunoselected cells were shown to have the same morphology and electrical resistance (150-200 ohm.cm2) as untransformed MDCK II cells. Indirect immunofluorescence staining and [125I]protein A binding assays carried out on these cells demonstrated that G protein was localized exclusively to the basolateral surface as is observed with viral infection.
Roman
L M
LM
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Scharm
A
A
Howell
K E
KE
D000818:Animals; D002461:Cell Line, Transformed / Q000032:analysis / Q000502:physiology*; D002462:Cell Membrane / Q000032:analysis; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D007163:Immunosorbent Techniques; D008562:Membrane Glycoproteins; D008863:Microspheres; D011401:Promoter Regions, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*
D004279:DNA, Viral; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins