Positive selection of growth-inhibitory genes.
We have isolated a limited set of cDNAs that limit cell proliferation using a unique assay based on the dilution of a lipophilic fluorescent dye as transfected cells divide. The identification of growth-inhibitory factors has been limited by the lack of a strong assay for growth inhibitors. A growth-inhibited cell does not grow and so is at a selective disadvantage in vitro when compared with any growing cell. Several assays have been used to screen for growth-inhibitory genes; however, these approaches are either very difficult to implement, leaky, or not comprehensive. We have developed an assay that selects for cDNAs capable of inhibiting proliferation in which cells are nonspecifically labeled with a lipophilic fluorescent dye, PKH-2, and subsequently transfected with a cDNA library made from growth-inhibited cells. With each cell division, the amount of dye per cell is reduced by one-half. Over time, growth-inhibited cells will retain more dye per cell relative to actively growing cells. The population is then analyzed by fluorescence-activated cell sorting, and the brightest cells in the population are isolated. This assay has allowed us to select pools of cDNAs enriched for growth-inhibitory activities and may provide a general method for identifying growth-inhibitory genes active in varying biological contexts. We report here the successful application of the dye retention assay to the selection of cDNAs that inhibit epithelial cell proliferation.
Maines
J Z
JZ
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455, USA.
Sunnarborg
A
A
Rogers
L M
LM
Mandavilli
A
A
Spielmann
R
R
Boyd
F T
FT
D000818:Animals; D002455:Cell Division / Q000235:genetics*; D002460:Cell Line; D002469:Cell Separation; D018076:DNA, Complementary / Q000235:genetics; D004734:Energy Metabolism; D004798:Enzymes / Q000235:genetics; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D015723:Gene Library; D006131:Growth Inhibitors / Q000235:genetics*; D007201:Indicator Dilution Techniques; D008168:Lung; D008907:Mink; D009930:Organic Chemicals; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics; D012641:Selection, Genetic; D014162:Transfection
D018076:DNA, Complementary; D004798:Enzymes; D005456:Fluorescent Dyes; D006131:Growth Inhibitors; D009930:Organic Chemicals; C068241:PKH-2; D011994:Recombinant Proteins