A genetic screen to identify latent transforming growth factor beta activators.
The mechanisms by which latent transforming growth factor beta (TGFbeta) is converted to the active cytokine are largely unknown. Here we present a genetic screen that combines retroviral mutagenesis and cDNA expression cloning to reveal proteins involved in the extracellular regulation of latent TGFbeta activation. The screen employs a cell line engineered to express green fluorescent protein (GFP) in response to TGFbeta. The cells produce their own latent TGFbeta. Therefore, after transduction with a retroviral cDNA library that contains an insert for an activator of latent TGFbeta, cells expressing the activator are GFP-bright. These cells are enriched by fluorescence-activated cell sorting and grown as individual clones. The isolated clones are cocultured with a second TGFbeta reporter cell line that produces luciferase in response to TGFbeta. Cells that have acquired the ability to activate latent TGFbeta induce luciferase expression in the absence but not in the presence of neutralizing antibodies to TGFbeta. The activator expressed by the positive clones can be identified by retrieval of the retrovirus cDNA insert.
Annes
Justin
J
Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA.
Vassallo
Melinda
M
Munger
John S
JS
Rifkin
Daniel B
DB
D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D003001:Cloning, Molecular; D005434:Flow Cytometry / Q000379:methods; D015723:Gene Library; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins; D039641:Integrin beta Chains / Q000235:genetics; D008156:Luciferases / Q000032:analysis; D008164:Luminescent Proteins / Q000032:analysis; D051379:Mice; D016296:Mutagenesis; D051381:Rats; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D039641:Integrin beta Chains; D008164:Luminescent Proteins; D016212:Transforming Growth Factor beta; C072170:integrin beta6; D049452:Green Fluorescent Proteins; D008156:Luciferases