摘要
Intron was found to play an important role in improving gene expression. To improve the human factor IX(hFIX) expression level in hemophilia B gene therapy study, the retroviral vector containing intron 1 of hFIX gene was constructed in forwarded configuration, but the intron 1 was found spliced in virus particles by RT_PCR detection. So the inverted configuration vector G1NaPAi′IX was suggested and constructed on the basis of SNMBAIXm and transfected into PA317. Then C2C12 cells were transfected using the above virus supernatant and the G418_resistant clones were selected. PCR and RT_PCR detection found that intron 1 structure existed in C2C12 clones and retroviral particles. And the expression level of inverted vector was 3 times higher than that of forwarded vector. These results showed that the inverted configuration vector was in deed able to avoid splicing of intron 1 during the process of retroviral packaging and improved the expression level of hFIX protein.
Intron was found to play an important role in improving gene expression. To improve the human factor IX(hFIX) expression level In hemophilia B gene therapy study, the retroviral vector containing intron 1 of hFIX gene was constructed in forwarded configuration, but the intron 1 was found spliced in virus particles by RT-PCR detection. So the inverted configuration vector G1NaPAi’IX was suggested and constructed on the basis of SNMBAIXm and transfected into PA317. Then C2C12 cells were transfected using the above virus supernatant and the G418-resistant clones were selected. PCR and RT-PCR detection found that intron I structure exlsted in C2C12 clones and retroviral particles. And the expression level of inverted vector was 3 times higher than that of forwarded vector. These results showed that the inverted configuration vector was in deed able to avoid splicing of intron 1 during the process of retroviral packaging and improved the expression level of hFIX protein.
基金
ThisworkwassupportedbyState 86 3HighTechnologyDevelopmentProject (GrantNo .86 3_BH_0 3_0 2_0 1)andShanghaiScience&TechnologyDevelopmentProject (GrantNo .95JC14 0 0 9)
