The lymphohematopoietic progenitors represent <0.01% of nucleated marrow cells. Here, we describe the immortalization of the murine lymphohematopoietic progenitors by a retroviral vector harboring a dominant-negative retinoic acid receptor. The immortalized progenitors proliferate as a stem-cell-factor-dependent clonal line EML that spontaneously generates pre-pro-B lymphocytes and erythroid and myeloid progenitors. Upon stimulation with intedeukin-7 and stromal cells, the pre-pro-B lymphocytes express RAG-1 and undergo D-[ rearrangements of the immunoglobulin heavy-chain genes. With erythropoietin the erythroid progenitors proliferate and differentiate into red cells. Generation of the common progenitors for neutrophils and macrophages is suppressed in EML but is inducible by high concentrations of retinoic acid. An additional block in neutrophil differentiation occurs at the promyelocyte stage but can also be overcome by high concentrations of retinoic acid. These studies demonstrate a reproducible way to immortalize lymphohematopoietic progenitors and implicate specific roles for retinoic acid receptors at two distinct stages of hematopoiesis.
The retinoic acid receptor (RARot) is expressed in virtually all hematopoietic lineages, but the role of this transcription factor in regulating the growth and differentiation of hematopoietic progenitors is unknown. We have constructed a mutant RARer that both exhibits dominant-negative activity against the normal RARer in transient expression assays in mouse fibroblasts and inhibits retinoic acid-induced neutrophilic differentiation of the HL-60 human promyelocytic leukemia cell line. When this dominant-negative RARa construct is introduced into the multipotent intedeukin-3-dependent FDCP mix A4 murine hematopoietic cell line, there is a rapid switch from spontaneous neutrophil/monocyte differentiation to basophil/mast cell development. Thus, in this multipotent hemopoietic cell line the normal RARa transcription factor and/or related molecules appear to promote the differentiation of neutrophils and monocytes but suppress the development of basophils/mast cells.
E12 and E47 are two non-tissue-specific helx-oop-helix (HLH) (100 pLg/ml) was used for hygromycin phosphotransferase (Hygro); 120 FM hypoxanthine/0.4 ,uM aminopterine/20 ,AM thymidine was used for hypoxanthine phosphoribosyltransferase (HPRT); 2 ,AM gancyclovir was used for thymidine kinase (TK).PCR and Southern Analyses. PCR was used to screen the targeting events. Individual clones were picked out with cloning cylinders, and half of the cells were used for quick PCR analysis (11). The PCR-positive clones were then further analyzed by Southern blotting.Reverse Transcription (RT)-PCR Assay. PCR was used to determine the expression pattern ofthe E2A gene in knockout clones. Primers (see Fig. 1) that are specific to certain exons of the E2A gene and the marker genes were used to detect the common as well as the specific splicing patterns of the wild-type and mutant alleles. The assays were done essentially as described (12)
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To study the regulation of the early stages of hematopoiesis, cDNA representational difference analysis was used to isolate genes that were differentially expressed in primitive hematopoietic progenitors. The reasoning was that such genes were more likely to provide functions important to hematopoietic stem cells and progenitors. One of the genes identified through this approach encodes mouse Jagged2(mJagged2). Using quantitative reverse transcription–polymerase chain reaction, it was shown that mJagged2 was differentially expressed in c-kit+ hematopoietic progenitors, including those with the phenotypes of Lin− c-kit+Rhlo Holo and Lin−c-kit+ Rhhi Holo, and that they have been shown to be highly enriched for long-term and short-term repopulating hematopoietic stem cells, respectively. Western blot analyses showed that endothelial cells also expressed high levels of Jagged2, but stromal fibroblasts did not. Using a coculture system we found that exogenous, full-length mJagged2 promoted the survival and proliferation of hematopoietic progenitors, including the high-proliferative potential colony-forming cells. Direct cell-to-cell contact was required for this effect. Taken together, these findings indicate that both c-kit+ hematopoietic progenitors and endothelial cells express Jagged2 and that exogenous, full-length Jagged2 promotes the survival and proliferation of hematopoietic progenitors.
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