In order to overcome the interference of the internal promoter in retroviral vector with a foreign DNA expression, we used the double-copy vector(DC vector) system to improve the effect of transduced gene expression.H...In order to overcome the interference of the internal promoter in retroviral vector with a foreign DNA expression, we used the double-copy vector(DC vector) system to improve the effect of transduced gene expression.Human G-CSF cDNA was inserted into the Bgl Ⅱ site of the polyclonal sites within the U3 region of the 3'long terminal repeat(3'-LTR) in the vector(N2A). After being identified, the gene was transduced into Ψ-2 packaging cell line by using the electroporation method. Consequently, the gene was duplicated in the infected cells, and transferred to the 5'-LRT, and then placed outside the retroviral transcriptional unit. After two weeks the neomycin resistance positive colonies were grown in the G-418 medium. The supernatant virus was titred and then transferred to NIH3T3 cells. The G-CSF value of positive clone N2AG7-4 was up to 53. 3 U/106 cells. Southern blot and Northern blot analysis showed that the chimeric gene faithfully duplicated in the cells infected with the corresponding virus and generated two copies with one in each LTR.展开更多
文摘In order to overcome the interference of the internal promoter in retroviral vector with a foreign DNA expression, we used the double-copy vector(DC vector) system to improve the effect of transduced gene expression.Human G-CSF cDNA was inserted into the Bgl Ⅱ site of the polyclonal sites within the U3 region of the 3'long terminal repeat(3'-LTR) in the vector(N2A). After being identified, the gene was transduced into Ψ-2 packaging cell line by using the electroporation method. Consequently, the gene was duplicated in the infected cells, and transferred to the 5'-LRT, and then placed outside the retroviral transcriptional unit. After two weeks the neomycin resistance positive colonies were grown in the G-418 medium. The supernatant virus was titred and then transferred to NIH3T3 cells. The G-CSF value of positive clone N2AG7-4 was up to 53. 3 U/106 cells. Southern blot and Northern blot analysis showed that the chimeric gene faithfully duplicated in the cells infected with the corresponding virus and generated two copies with one in each LTR.
