Differentially expressed genes during in vitro differentiation of murine embryonic stem cells transduced with a human erythropoietin receptor cDNA.
Our previous study demonstrated that transduction of murine embryonic stem (ES) cells with a human erythropoietin (Epo) receptor (R) cDNA resulted in enhanced erythropoiesis in developing embryonic bodies (EBs). To address possible mechanisms of gene regulation, we compared gene expression between hEpoR cDNA-transduced ES (ES-hEpoR) cells and parental ES cells during in vitro differentiation induced by withdrawal of leukemia inhibitory factor (LIF) and cultured in the absence of Epo using differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR). A total of 48 differentially expressed cDNA fragments were found; 12 were sequenced and five were confirmed by Northern blot analysis to be up- or down-regulated in ES-hEpoR cells during differentiation compared to parental ES cells. In a GenBank search of the five putatively regulated cDNA fragments, two fragments shared high sequence homology to two known genes: the Surf-6 gene and the gene for calcyclin binding protein. Northern blot analysis demonstrated that 2.5-kb and 0.3-kb transcripts of the Surf-6 gene were expressed in undifferentiated ES-hEpoR and parental ES cells at a low level, but this expression was enhanced from day 2 to 14 of differentiation after withdrawal of LIF and culture in the presence of Epo. Furthermore, the enhanced expression of these two transcripts was also noticed in EML-C1 cells, a murine multipotential hematopoietic cell line that has erythroid differentiation potential in response to Epo. In summary, our results demonstrate that Surf-6 gene expression is regulated during differentiation of hematopoietic stem/progenitor cells in response to Epo, suggesting a possible role for Surf-6 gene in erythropoiesis.
Xia
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Department of Microbiology/Immunology, Indiana University School of Medicine, Indianapolis 46202-5254, USA.
Dai
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MS
Magoulas
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Broxmeyer
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HE
Lu
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D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000502:physiology; D002454:Cell Differentiation; D018076:DNA, Complementary; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology / Q000502:physiology*
D018076:DNA, Complementary; D009687:Nuclear Proteins; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; C484378:SURF6 protein, human; C098938:Surf6 protein, mouse