The Cloning of Erythropoiesis-related Genes with the Innovated Model System  

用改进的模式系统克隆红细胞发育相关基因(英文)

作　　者：阚晟[1] 李文雍[1] 刘桂生[1] 陈清轩[1] 

机构地区：[1]中国科学院遗传与发育生物学研究所,北京100080

出　　处：《Developmental and Reproductive Biology》2002年第2期101-111,共11页发育与生殖生物学报（英文版）

基　　金：TheprojectwassupportedbytheNationalNaturalScienceFoundationofChina (No.3 0 0 70 42 3 7)

摘　　要：Most studies of erythropoiesis have been performed either with the virally transformed MEL cells or with the scarce erythroblasts within unhomogeneous cell populations.MEL cells are the Friend virus transformed mouse erythroleukemia cells and have lost the capacity to respond to erythropoietin.Moreover,the research in the field of erythropoiesis has been severely hampered by the paucity of erythroblasts obtained from chick embryos,human,mouse,rat fetal liver,mouse spleen,or the bone marrow.In our paper,the fetal blood from newly slaughtered pregnant sheep meets the needs of studies as abundant staring biological materials at the nucleotide level and also reflects in vivo proliferative and differential status of erythroblasts.Appling the technique of mRNA differential display with minor modification,we directly compared cDNA fragmerts from the fetal blood erythroblasts and adult pregnant sheep leukocytes by PCR on DNA sequencing gels and revealed two differentially expressing bands.One is a novel gene fragment, which has homology with UV-sensitive cone opsin or rhodopsin by examinating the predicted amino acid sequence.Since the blood cells are sensitive to radiation,it can be safely inferred that this fragment participate in the process of cytocidal effect of the radiation.The other fragment is glucose induced gene /elongation factor 2 3′end noncoding region,which was first cloned from glucose induced bovine aortic smooth muscle cells.Using Northern blot hybridization method,authentic specific expression was confirmed.红细胞发生的研究主要依赖于MEL细胞系和分离到的少量的小鼠成红细胞。MEL细胞由Friend病毒转化而来 ,对促红细胞生成素不敏感 ;而成红细胞主要来源于鸡、人、小鼠、大鼠胎肝、小鼠肾脏或骨髓 ,因量少 ,所以严重困扰着红细胞发生在分子水平上的研究。本文采用现宰杀的胎羊血液 ,不仅满足了分子生物学起始材料量的要求 ,又能直接反映出红细胞发生在体内的真实状况。通过对mRNA差异显示技术的改进 ,在DNA测序胶上比较胎羊成红细胞和怀孕母羊淋巴细胞扩增的cDNA片段 ,发现两条差异条带 :其中一个片段是新基因 ,进行蛋白质序列预测发现与UV敏感的锥状视蛋白、视紫红质有一定的同源性 ,由于其血细胞对辐射很敏感 ,故推测该片段与辐射造成的细胞杀伤作用有关 ;另一个片段为葡萄糖诱导基因 /延伸因子 2的 3′非编码区 ,该片段最早在葡萄糖诱导后的牛主动脉平滑肌细胞被克隆到。通过反向Northern杂交证明 ,所发现的新基因具有真实性。

关 键 词：fetal sheep ERYTHROPOIESIS ERYTHROBLAST 

分 类 号：Q344.5[生物学—遗传学] Q953