The Regulation Of Erythroid Differentiation Related Factor (EDRF) On Erythroid Differentiation

EDRF (erythroid differentiation-related factor) is a 472 bp Splenic erythroblasts from anemic strain of Friend virus infected musculus cDNA,mRNA sequence. which is belong to CNC transcriptional family. EDRF is critical for the globin expression. The binding of EDRF to the MARES in the DNase I hypersensitivity site HS2 which is located in the LCR (locus control region) of globin induce the high expression of globin. There is also EDRF binding site in the HS40 site which is the enhancer ofαglobin. EDRF is a heterozimer which is formed by 2 subunit p18 andEDRF. EDRF subunit is tissue restricted protein which is almost exclusively found only in hematopoeitic processor, erythroid cell, megakyrocyte, granμle cell. In addition to the function on the erythroid differentiation, it is also essential for normal platelet production. It leads to severe thrombocytopenia but little if any defect in erythropoiesis, indicating that other molecμles can compensateEDRF's function in red cell maturation in vivo.1. Breeding and F-MuLV inoculation of newborn mice. EDRF heterozygou (1/2) mice of the 129/Sv strain were mated with BALB/c mice (Jackson Laboratories) for six generations in order to confer upon them sensitivity Friend virus-induced erythroleukemias. The offspring were genotyped by Southern blot analysis of tail DNA as described previously. 1/2 newborn mice from the BALB/c cross were then tested for sensitivity to F-MuLV by single intraperitoneal injection at birth. It was found that these mice were susceptible to F-MuLV-induced erythroleukemia. Accordingly, two breeding pairs of the 1/2 mice were used to generate offspring for viral inoculation.2. Tumor induction and establishment of cell lines. Newborn pups were injected intraperitoneally with F-MuLV clone B as described elsewhere. They were monitored for (i) enlarged abdomens causing hunched postures, a symptom of splenomegaly, and (ii) a lack of movement, reflecting low energy due to severe anemia, and were sacrificed when moribund. Mice displaying these marked symptoms rarely survive for over a day. For genotyping, tail tissues and tumor cells were processed for Southern analysis. The spleen cells from these erythro-leukemias were cultured in a-minimum essential medium supplemented with 15% heat-inactivated fetal bovine serum (Gibco BRL). Cells were cultured in this medium alone or supplemented with either1Uof erythropoietin (Epo)/ml,10% stem cell factor (SCF) conditioned medium, or both growth factors as described elsewhere . SCF was obtained from SCF-producing BHK-MKL cells. These conditions were maintained until cells were established inculture, at which time fetal bovine serum was reduced to10% and growth factors were removed in order to determine the growth factor dependency of the cell lines. To examine cellular growth rates, erythroleuke mice cells were cultured in triplicate in the presence of Epo, SCF, or Epo plus SCF and viable cells were determined by trypan blue dye exclusion at various times.3. DNA isolation and Southern analysis. High-molecular-weight DNA was iso-lated from homogenized spleen tissue, tail samples, or cell lines. Genomic DNA from tumors or cell lines was digested overnight with appropriate restriction enzymes and separated on 1% agarose gels. And transferred onto nylon membranes .The membranes were hybridized with EDRF,Globin DNA probe. RNA isolation and Northern analysis. Find that Tumor cell genotype is EDRF+/-,it's EPH cell line genotype is EDRF-/-,EDRF-/- cell line could not express Globin mRNA. EDRF+/- cell line could express Globin mRNA. It tell us EDRF is very important for Globin express.4. EDRF viral vector and cell infection. The 0.6-kbp EcoRI fragment corre-sponding to full-length EDRF cDNA was cloned into the EcoRI site of pMX-puro retroviral expression vector and designated pMX-EDRF. To generatethe retroviruses, pMX-puro and pMX- EDRF constructs were transfected into G418 selected PT67 packaging cells by the Lipofectin transfection method (Life Technologies). Cells resistant to puromycin (2 mg/ml) were pooled and cocultured with erythroleukemia cell lines for two days. Resistant cells were used for expression and cell growth analyses.We found that pMX- EDRF infected cell line can express globin while others can not.And pMX- EDRF infected cell line growth slowly, so EDRF can inhibit tumor growth.At last, we conclusion that EDRF is very important for cell growth forever. Lack of EDRF gene, the leukemia cell growth faster. EDRF is very important for globin express and erythroid differentiation.