Use of a recombinant parvovirus to facilitate screening for human melanomacell clones expressing tetracycline-responsive transactivators

The tetracycline regulatory (TET) system provides a useful means of control ling foreign gene expression in mammalian cells. Exploiting this system in cultured cells requires the prior isolation, from the cells of interest, of transfectant clones expressing the necessary TET transactivator, tTA, or r everse transactivator, rtTA. We describe a simple screening procedure for i dentifying transfectant clones expressing a properly regulated transactivat or, and the application of this method to isolating clones of human melanom a cells expressing either tTA or rtTA. Clones in multi-well plates are tran sduced by exposure to a recombinant parvovirus containing a luciferase repo rter, under control of a promoter responsive to the TET system transactivat ors. Transactivation of reporter expression in the presence or absence of d oxycycline (DOXY) is determined after one to two days, using a rapid lucife rase assay. Screening is easier and more reproducible with this transductio n method than with conventional transient transfection of analogous reporte r plasmids. Clones of two human melanoma cell lines showing >100-200-fold t ransactivation after transfection with either tTA or rtTA were readily iden tified using this method. (C) 1999 Elsevier Science B.V. All rights reserve d.