Integration of a target gene into chromosomal genome of BF-2 cells using UV-inactivated snakehead retrovirus (SnRV)
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Integration and expression of a target gene into chromosomal genomes of host cell by retrovirus mediated gene transfer system usually require complicate and laborious procedures. In the present study, we investigate a simple method to integrate a target gene into genome of BF-2 cells using ultraviolet (UV)-inactivated snakehead retrovirus (SnRV), a fish retrovirus. First of all, an optimization of transfection condition was determined with BF-2 cells using Lipofectamine 2000 and Transome. Using 0.5 ㎕ Lipofectamine 2000 resulted in 33.8, 40.6 and 40.2% of transfection efficacy with high survival rate (minimum 80%) in 0.5, 1 and 2 ㎍ DNA, respectively, and those of Transome were all less than 5%. It was confirmed that UV-treatment for 5 min was enough to inactivate infectivity of SnRV. Next, a cassette composed of GFP (green fluorescent protein) gene flanked by LTR (long terminal repeats) sequences derived from SnRV was constructed and transfected into BF-2 cells followed by treatment with UV-inactivated SnRV for optimization of integration and expression of the cassette gene. As the results, the fluorescence was expressed in BF-2 cells treated with UV-inactivated SnRV 3 and 5 times, while there was no expression in BF-2 cells with once and non treatment. Accordingly, it was confirmed that GFP gene was integrated into chromosomal genome of BF-2 cells with UV-inactivated SnRV.
Abstract
Integration and expression of a target gene into chromosomal genomes of host cell by retrovirus mediated gene transfer system usually require complicate and laborious procedures. In the present study, we investigate a simple method to integrate a target gene into genome of BF-2 cells using ultraviolet (UV)-inactivated snakehead retrovirus (SnRV), a fish retrovirus. First of all, an optimization of transfection condition was determined with BF-2 cells using Lipofectamine 2000 and Transome. Using 0.5 ㎕ Lipofectamine 2000 resulted in 33.8, 40.6 and 40.2% of transfection efficacy with high survival rate (minimum 80%) in 0.5, 1 and 2 ㎍ DNA, respectively, and those of Transome were all less than 5%. It was confirmed that UV-treatment for 5 min was enough to inactivate infectivity of SnRV. Next, a cassette composed of GFP (green fluorescent protein) gene flanked by LTR (long terminal repeats) sequences derived from SnRV was constructed and transfected into BF-2 cells followed by treatment with UV-inactivated SnRV for optimization of integration and expression of the cassette gene. As the results, the fluorescence was expressed in BF-2 cells treated with UV-inactivated SnRV 3 and 5 times, while there was no expression in BF-2 cells with once and non treatment. Accordingly, it was confirmed that GFP gene was integrated into chromosomal genome of BF-2 cells with UV-inactivated SnRV.
참고문헌 (12)
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