Dna microarray analysis of g-csf dependent, lyn-dependent genes

We have developed a model to study the singular contribution of a Src kinase to cytokine receptor signaling. The DT40 B cell line expresses only one, Lyn, of the nine members of the avian Src family. A mutagenized cell line, lacking Lyn, has been developed by classic gene targeting. We have ectopically expressed the human G-CSF receptor in these cell lines G-CSF-induced proliferation requires the presence of Lyn, as the activation of neither Jak1 nor Jak2 is sufficient. Downstream events of Lyn which are involved in proliferation include Cbl-P1 3-kinase and Shc. In order to identify additional components of Lyn-dependent proliferative pathways, we have used a chicken cDNA microarray. Total mRNA was harvested from wild type and lyn-deficient DT40 cells expressing the G-CSF receptor, After cells were stimulated with 100 ng/ml G-CSF for 6 hrs. Next, mRNA was isolated and alphaP32dCTP incorporated into reverse transcribed cDNA. Two filters, with > 1500 cDNAs each, were hybridized in parallel with labeled cDNA probes from control and experimental cells. Genes which were found to be up-regulated by G-CSF and Lyn were other signaling molecules, such as protein phosphatases, cell surface proteins, such as the B Cell Receptor, and cell growth factors, such as elongation factor alpha. Additional data will be presented from studies looking at only G-CSF or only Lyn.