Creation of a stable human reporter cell line suitable for FACS-based, transdominant genetic selection.
Quality bioassays are central to all approaches directed at understanding or perturbing the function of proteins. One type of cell-based bioassay involves an engineered reporter whose transcriptional activity serves as a readout for upstream signals of a biochemical pathway(s) that feeds into the reporter. We describe a general strategy for creating a mammalian reporter line with attributes suitable for a high complexity, en masse transdominant genetic screen. The basic criteria required of the mammalian cells engineered with the reporter include ease of maintenance, ease of sorting by FACS, ability to be transduced by retroviruses, and high expression of transduced peptides or cDNAs. For maximal enrichment during selection, the reporter line should have a relatively homogeneous response and a high signal-to-background ratio. We use a melanoma cell line transduced with a retinoic-acid-responsive promoter coupled to a GFP reporter as a case study to demonstrate the strategy. We characterize an optimized retinoic-acid-responsive reporter clone to determine the kinetics of reporter induction and decay in the presence and absence of retinoids. Dose-response studies reveal that the reporter responds to all-trans retinoic acid with an EC50 of approximately 1 nM. The strategy described is general and may be applied to create other reporter lines that respond to a specific stimulus.
Richards
B
B
Arcaris, Inc, 615 Arapeen Drive, Suite 300, Salt Lake City, Utah 84108, USA.
Karpilow
J
J
Dunn
C
C
Zharkikh
L
L
Maxfield
A
A
Kamb
A
A
Teng
D H
DH
D001483:Base Sequence; D002469:Cell Separation; D004247:DNA; D017931:DNA Primers; D005434:Flow Cytometry; D005799:Genes, Dominant; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008545:Melanoma / Q000235:genetics / Q000473:pathology; D012190:Retroviridae / Q000235:genetics; D012641:Selection, Genetic; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA