The hematopoietic cell line, BL3, carries a rearranged N2-IL-3 retroviral genome. The cells were derived from a mouse which developed a myeloproliferative disease and lymph node enlargement after its marrow cells were infected with a retroviral vector containing the neomycin resistance and the IL-3 genes. The cells resemble immature blast cells and the deletion of a portion of the neomycin resistance gene, the IL-3 gene, and the 3' LTR accounts for the rearrangement. The rearranged viral genome, therefore, also provides a genetic tag for BL3 cells. With this tag, the Principal Investigator has studied whether BL3 cells could reconstitute lethally irradiated mice. DNA from organs of clinically normal recipients were analyzed by Southern blot analysis. An LTR-neo/neo specific band pattern identical to that in the parental BL3 cell line was observed in the thymus, spleen, marrow, and lung, but not in the liver or kidney. Methylcellulose assays of marrow cells from the recipients revealed the presence of factor-independent colonies composed of differentiated granulocyte and macrophage cells. These data suggested that the immortalized BL3 cells could differentiate in vivo and give rise to functional and mature hematopoietic cells of lymphoid and myeloid lineages. To determine whether BL3 cells had long term repopulating potential, the Principal Investigator repeated the reconstitution experiments and analyzed the mice 7 months later. He demonstrated that as few as 100 BL3 cells could rescue a lethally- irradiated recipient. Spleen cells were isolated and were enriched for lymphoid or myeloid cells with a percoll gradient. Also, cells were stimulated with PHA or WEHI-3-conditioned media. BL3-specific rearranged bands were present in each enriched cell population. With a series of molecular genetic analyses, the Principal Investigator has begun to identify the gene accounting for the immortalization of BL3 cells. Northern blot RNA analysis of these cells revealed the presence of a novel MRNA of approximately 3 kb. He constructed an Okyama-Borg CDNA expression library from RNA of BL3 cells and obtained one clone that contained a 2.75 kb insert which similarly hybridized with the LTR-neo probe. Cloning of this hybrid gene is underway with the intent that it may relate to the immortalization of BL3 cells with the proposal. The Principal Investigator plans to characterize BL3 cells more fully to identify the gene accounting for the immortalization of BL3 cells, and to examine the genomic organization of this gene once identified.
