Tetracycline regulated expression of vimentin in fibroblasts derived from vimentin null mice.
Fibroblast cell lines were derived from vim-/- mice that express a mouse vimentin transgene in a tetracycline regulatable manner. Vimentin null mouse primary embryo fibroblasts were transformed with SV-40 early genes and vim- cell lines were isolated. A vim- cell line was then serially transfected with an expression plasmid encoding the tetracycline regulatable transactivator (tTA) and a mouse vimentin cDNA expression plasmid under the regulation of Escherichia coli tet operator and minimal CMV promoter sequences. Two stable cell lines were obtained that contained little or no vimentin in the presence of low concentrations of tetracycline but rapidly expressed abundant vimentin filaments after removal of tetracycline. The vimentin content of one cell line was similar to that of control vim+/+ fibroblasts. The level of transgene expression could be regulated by the concentration of tetracycline in a dose dependent fashion. Induction of vimentin expression in these cells did not observably affect cell growth, the distribution of microfilaments or microtubules, or the shape of the nucleus. Enucleation studies indicated that while disassembly of microfilaments significantly increased the sensitivity of the cells to enucleation, the presence or absence of vimentin had no detectable effect on the degree of enucleation with increasing sedimentation force. Monolayer wounding experiments demonstrated that vimentin expression did not alter the mobility of polarized cells at the edge of the wound. Experiments to more directly test the effect of vimentin expression on the capacity of these fibroblasts to survive mechanical trauma indicated that vimentin expression had no obvious effect on the survival of suspension cells subjected to nitrogen cavitation or the fraction of cells that survived the mechanical scraping of monolayer culture. These studies indicate that vimentin expression in a single population of cells does not have an obvious effect on cytoplasmic organization and provides a useful system to study the effects of IFs on the capacity of individual cells to resist mechanical injury.
Holwell
T A
TA
Department of Pathology, University of Colorado Health Sciences Center, Denver 80262, USA.
Schweitzer
S C
SC
Evans
R M
RM
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D011994:Recombinant Proteins / Q000096:biosynthesis; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection; D014746:Vimentin / Q000096:biosynthesis* / Q000172:deficiency / Q000235:genetics
D011994:Recombinant Proteins; D014746:Vimentin; D013752:Tetracycline