|Budget Amount *help
¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 1999 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1998 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Identification of transforming genes has been largely relied on the transformation assay in mouse 3T3 fibroblast cells with the genomic DNA isolated from various cancer specimens. Although these studies have revealed the activation of ras, raf and myc oncogenes, they have been hampered by the fact that this kind of assay only allows the detection of genes, expression of which is active in 3T3 fibroblasts. Therefore, oncogenes in hematopoietic malignancies may not be transcriptionally active in 3T3 fibroblasts, and can not be identified in these types of assay.
To overcome this limit, we have developed a novel screening method using mouse interleukin-3 (IL-3)-dependent BA/F3 cells and retrovirus. mRNAs were isolated from the bone marrow mononuclear cells in a patient with acute lymphoid leukemia (ALL), and converted into double-stranded cDNAs, which were subsequently ligated into pMX retrovirus vector in a uni-direction. With a help of BOSC23 packaging cell line, we could obtain a retroviral stock with a titer exceeding lx 10ィイD16ィエD1/mI. BAIF3 cells were infected with this retrovirus under the presence of engineered fibronectin (Takara Shuzo, Shiga, Japan) with an infection efficiency 【greater than or equal】90%.
After the infection of retrovirus stock, BA/F3 cells were cultured in the absence of IL-3, generating a few IL-3-independent clones. Expression of leukemia-derived transforming genes were expected to render these cells factor-independency. Recovery of retroviral inserts from these transformed cells have identified a few cDNAs which can encode novel peptides. Studies are currently in progress to characterize the nature of these putative candidates of novel ALL oncogenes.