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

The tetracycline regulatory (TET) system provides a useful means of controlling 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...

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Bibliographic Details
Published in:Gene 1999-03, Vol.229 (1), p.125-129
Main Authors: Pacheco, Theresa R., Maxwell, Françoise, Wu, Ming-Fang, Na, Sopheap, Maxwell, Ian H.
Format: Article
Language:English
Subjects:
Clone Cells
Doxycycline - pharmacology
Gene Expression Regulation, Neoplastic - drug effects
Genes, Reporter - genetics
Humans
Melanoma
Melanoma - genetics
Parvovirus - genetics
Parvovirus vector
Screening clones
Tetracycline - pharmacology
Tetracycline regulation
Trans-Activators - genetics
Transcriptional Activation - genetics
Transduction, Genetic
Transfection
Tumor Cells, Cultured
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Summary:The tetracycline regulatory (TET) system provides a useful means of controlling 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 reverse transactivator, rtTA. We describe a simple screening procedure for identifying transfectant clones expressing a properly regulated transactivator, and the application of this method to isolating clones of human melanoma cells expressing either tTA or rtTA. Clones in multi-well plates are transduced by exposure to a recombinant parvovirus containing a luciferase reporter, under control of a promoter responsive to the TET system transactivators. Transactivation of reporter expression in the presence or absence of doxycycline (DOXY) is determined after one to two days, using a rapid luciferase assay. Screening is easier and more reproducible with this transduction method than with conventional transient transfection of analogous reporter plasmids. Clones of two human melanoma cell lines showing >100–200-fold transactivation after transfection with either tTA or rtTA were readily identified using this method.
ISSN:0378-1119
1879-0038
DOI:10.1016/S0378-1119(99)00034-7