Isolation And Characterization Of Genes Associated With Human Erythroid Development

Erythroid development is an important physiological process controlled by multiple gene products throughout individual development. Up to now, only few of these genes have been identified. Investigation of genes expressed during this process can not only contribute to insight into molecular basis of normal erythrogenesis, but also help elucidate pathogenesis of hematopoietic disorders, such as erythroleukemia, hereditary persistence of fetal hemoglobin (HPFH), lethal p-thalassemia caused by inactivation of EKLF in transgenic mice and erythroblast immortalization e.g. caused by over-expression of PU.1.To isolate genes differentially expressed during human erythroid development from fetal to adult stage, subtractive hybridization was employed. The target was human adult bone barrow BFUe-derived cells (BFUeDCs) antisense ss cDNA pool, containing abundant α-, β- and γ-globin species, while the driver was hemin-induced K562 cells (K562-H) mRNA with abundant α- and γ-globins but without β-globin transcripts. The subtractive hybridization may efficiently enrich genes dominantly expressed in BFUeDCs, but it still is time-consuming to separate new, interesting sequences from the remainder of the abundant copies such as globin transcripts. A novel strategy established by us was used to remove the abundant globin species from the target cDNA pool prior to perform subtractive hybridization. This method also facilitate study in other highly differentiated cell or tissue types.The subtracted cDNA was cloned into pGEM-T Easy vector and about 1.1 ×10~3 clones were given. 37 clones randomly picked out were sequenced, in which 12 were novel expressed sequence tags (ESTs).