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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3750-3757
Capture of cytokine-responsive genes (NACA and RBM3) using a
gene trap approach
Sandrine Baghdoyan,
Patrice Dubreuil,
Frédéric Eberlé, and
Sophie Gomez
From the Laboratoire de Cancérologie expérimentale,
Marseille, France.
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 dependency on granulocyte-macrophage colony-stimulating
factor (GM-CSF) or interleukin-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 the Rosa geo retrovirus in the
presence of GM-CSF, IL-3, or IL-5 and clones were selected for
retroviral integration on the basis of G418 resistance. Housekeeping
and cytokine-regulated trapped genes were then differentiated on the
basis of G418 resistance versus sensitivity in the presence of the
different cytokines. To determine the reliability of this screen, DNA
sequences upstream of the proviral integration site were identified by
5' rapid amplification of DNA ends polymerase chain reaction (RACE PCR)
from selected GM-CSF-treated and -infected clones. Comparison of the
sequences with those 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-mediated transcription
factors in osteoblasts, and RBM3 as a protein from the
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 up-regulated in purified CD34+ 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
contribute to monocytic lineage development. This study demonstrates
that this gene trap approach is a useful method for identifying novel,
specific cytokine-responsive genes that are involved in the regulation
of hematopoiesis.
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