Study On Expression And Regulation Of Human Thrombopoietin Gene

Thrombopoietin(TPO),also known as c-mplligand, is a key regulator of the proliferation and maturation of megakaryocytes as well as platelet production. In order to study on regulational role of TPO gene 5 UTR and intron on TPO expression, TPO eDNA, TPO genomic DNA and all intron of TPO gene were isohted by PCR and LD-PCR techniques. Sequencing analysis indicates that all coding sequence, exon/intron splice site are the same with as. that reported by Sohma, Foster and Gurrey. To study the role of 5UTR and intron in expression and regulation of TPO gene,a number of expression vector for TPO gene fused to the regulatory elements of the CMV. rat WAP gene and goat β-casein gene were constructed. These expression vector were introduced into cultured cos-1 cells and HC-11 cells for transient expression of TPO gene using lipofectin method. The supernatant of cells transfeetion with above gene at 48h was analysized by sandwich ELISA, the results showed that (a)the expression level of the recombinant plasmids containing CMV-TPO fusion gene was the highest in cos-1 cells; (b) the expression level of the recombinant plasmids containing β-casein TPO fusion gene was the highest in HC-11 cells; (c)the trend of TPO gene expression level was TPO intron v> TPOcDNA>ATPO intron I> TPO intron I> TPO 3 gLINA in two cell lines. These results suggested that 5IJTR of TPO gene may inhibit expression of TPO gene and intron of TPO gene may play a different role in the expression and regulation of TPO gene. The WAP/TPO and β-casein/TPO fusion gene were also microinjected into fertilized eggs of mouse to generate transgenic mice. The total of 546 young mice (F0) were produced , 1 of them were eaten by their mothers. 3?8% integration rate of Fa individuals was estimated based on PCR and/or southern blot. The human TPO was detectable in milk of the 5 lactating transgenic mice containing WAP/TPO fusion gene, the expression level is approximately 400-2000pg/ml and the level in transgenic mice containing IAP-TPO intronV-TPO cDNA fusion gene are the highest. The milk of 7 lactating transgenic mice containing P 梒asein/TPO fusion gene also was analysed, the expression level of human TPO is approximately lng?46ng/ml and the level in transgenic mice containing P-TPO intronV-TPO cDNA fusion gene are also the highest in all transgenic mice detected. These resultes suggested that (a)the last intron of TPO gene can improve expression level of TPO gene; (b) 5~UTR of TPO gene may inhibit expression of TPO gene; (c) the regulatory elemenit of goat β-casein gene can specifically control the high-expression of TPO gene in mammary gland of transgenic animal.