Capture of cytokine-responsive genes (NACA and RBM3) using a gene trap approach

We have developed a gene trap approach to select specific cytokine receptor /ligand responsive genes in the cell line TF-1, This cell line exhibits a d ependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or i nterleukin-3 (IL-3) and responds to interleukin-5 (IL-5), In an attempt to detect genes modulated by one of these factors, cells were infected with th e Rosa beta geo retrovirus in the presence of GM-CSF, IL-3, or IL-5 and clo nes were selected for retroviral integration on the basis of G418 resistanc e, Housekeeping and cytokine-regulated trapped genes were then differentiat ed on the basis of G418 resistance versus sensitivity in the presence of th e different cytokines. To determine the reliability of this screen, DNA seq uences upstream of the proviral integration site were identified by 5' rapi d amplification of DNA ends polymerase chain reaction (RACE PCR) from selec ted GM-CSF-treated and -infected clones. Comparison of the sequences with t hose in the Genbank database revealed that 2 sequences correspond to known genes: NACA and RBM3, NACA was recently defined as a coactivator of c-jun-m ediated transcription factors in osteoblasts, and RBM3 as a protein from th e heterogeneous nuclear ribonucleoprotein family. Data from transcriptional analysis of these 2 genes in TF-1 cells showed a specific up-regulation by GM-CSF, Both transcripts were also found to be upregulated in purified CD3 4(+) cells, suggesting their involvement in proliferative processes during hematopoiesis. Interestingly, down-regulation was observed during monocytic differentiation of TF-1 cells, suggesting their extinction could contribut e to monocytic lineage development. This study demonstrates that this gene trap approach is a useful method for identifying novel, specific cytokine-r esponsive genes that are involved in the regulation of hematopoiesis. (C) 2 000 by The American Society of Hematology.