Study Of Vectors Of Human Thrombopoietin Gene Expressed In The Mammary Gland Specifically

Human thrombopoietin (TPO) is the all-important regulatory factor which can regulate the generation of thrombocyte in human body. It can specifically improve the level of thrombocyte in human body, prevent and cure thrombocytopenia caused by chemotherapy, and also prevent and cure idiopathic thrombocytopenic purpura. Furthermore, the latest research shows, as a cell regulatory factor, TPO can be used to accelerate the growth of hemopoietic stem cells. The objective and meaning of this research are: In order to produce transgene cloning cow which can express human TPO high efficiently in its milk, eucaryon expressed vectors piloted by various promoters are constructed, and specifically expressed in the mammary gland, the quantity of expression of TPO are measured both in cell and individual levels respectively, which could provide accurate data for the regulation of gene expression of TPO and promoters. The research and experiment method are as following: Three vectors specifically expressed in the mammary gland, namely pB3.9T, pB1.9T and pPT are constructed, after the transient transfection and the stably integration, the expression of TPO are measured in both transcription and translation levels.The results shows: Firstly, the TPO minigene with length of about 1.3kb adopted in the experiment can be accurately splicing after transcription, and can be translated to compound human TPO. Secondly, three regulating and regulatory element, namely BLG3.9, BLG1.9 and P1A3, can all be used to promote the expression of human TPO in the mammary gland-specific. Thirdly, the expression quantity of pB3.9T is markedly higher than that of pB1.9T. Fourthly, after the expressing vectors are stably integrated into the cell genome, the expression quantity of TPO increases steadily, and higher than that in transient transfection. Fifthly, during the early stage of transient transfection and stable transfection periods, the expression level of TPO eucaryon expressed vectors piloted by P1A3 promoter is markedly higher than that of TPO eucaryon expressed vectors piloted by BLG3.9 and BLG1.9 promoters. But with the time lapse and the continually increase of the number of the cells, the expression level of pB3.9T exceeds that of pPT out and away. Sixthly, the monoclone of fibroblast cells line that stably integrated with three different vectors are acquired.The following conclusion can be educed: Firstly,β-lactoglobulin gene andβ-casein promoter of goat can all be used to instruct the expression of TPO in the mammary gland specific. Secondly, there may be a special enhanced sequence which should be located between -3.9kb and -1.9 kb in the upstream of 5'end of the BLG gene. Thirdly, with the continually increase of the number of the cells, BLG 3.9 promoter may activate some kind of enhancing mechanism, or weaken some kind of restraining mechanism. Fourthly, the existence of Derepression and activation mechanisms of BLG transcripton is testified, which is initially concluded to be located between -3.9kb and -1.9kb in the upstream of BLG 5'end. Fifthly, the three vectors constructed in the research can all be used to produce transgene animals, and also can be used to provide transgene nuclear donor cell for cloning cows.