A modified procedure for replica plating of mammalian cells allowing selection of clones based on gene expression.
The polyester cloth replica-plating technique for selection of mammalian cell clones was modified by growing cells in colonies on a flexible polytetrafluoroethylene membrane and then transferring them completely to polyester cloth (27-microns mesh), from which a replica was made by allowing cells to transfer to a cloth of smaller pore size (17-microns mesh). Using this technique, two phenotype selection methods are demonstrated here: in situ hybridization for detection of a specific mRNA and a photographic film assay for detection of luciferase expression. Cells were transfected with pSV2AL-A delta 5' in which firefly luciferase cDNA is under the control of the simian virus 40 promoter. The luciferase assay was adapted for colonies on polyester cloth; cells were permeabilized with digitonin to allow access of ATP and luciferin to the cell without disruption of colonies. Clones selected for expression or nonexpression of luciferase by the photographic film assay were positive or negative for expression after isolation from the cloth replica and subsequent growth under conventional culture conditions. The replica-plating procedure described here should be generally applicable to most mammalian cell types. The ability to produce replicas of colonies, combined with in situ hybridization or assays that can be adapted to in situ detection, provides phenotype selection for clones based on gene expression independent of growth characteristics.
Hornsby
P J
PJ
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912.
Yang
L
L
Lala
D S
DS
Cheng
C Y
CY
Salmons
B
B
D000818:Animals; D002415:Cats; D002417:Cattle; D002478:Cells, Cultured; D002999:Clone Cells / Q000737:chemistry*; D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D008322:Mammals / Q000235:genetics; D009693:Nucleic Acid Hybridization; D010641:Phenotype; D010781:Photography / Q000295:instrumentation; D011091:Polyesters; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012588:Scintillation Counting
D011091:Polyesters; D011993:Recombinant Fusion Proteins; D008156:Luciferases