To construct a lentiviral shRNA vector targeting rat CD40 gene and detect its effectiveness of gene silencing in dendritic cells（DCs）, specific siRNA targets with short hairpin frame were designed and synthesized ac...To construct a lentiviral shRNA vector targeting rat CD40 gene and detect its effectiveness of gene silencing in dendritic cells（DCs）, specific siRNA targets with short hairpin frame were designed and synthesized according to the mRNA sequence of rat CD40 gene. DNA oligo was cloned into lentiviral expression vector, and then PCR and sequencing analyses were conducted to verify the constructs. The verified plasmids were transfected into 293T cells that over-express recombinant CD40 in order to select the most effective siRNA targets, shRNA lentiviruses from the selected constructs were propagated and harvested with a virus packaging system, and the virus titers were determined. Western blot and Real-time PCR were performed to determine CD40 expression level in the virusinfected dendritic cells. PCR and sequencing analyses reveal that shRNA plasmids of four targets were successfully constructed. The optimal interfering target was selected, and the virus with a titer of 5 × 10^7 TU/mL was successfully packaged. CD40 expression in rat DCs was knockdown at both mRNA and protein levels by virus infection. In comparison to that of control groups, CD40 mRNA expression and protein expression were decreased by 60.9% and 61.2%, respectively. We have successfully constructed recombinant lentiviral shRNA expression vector targeting rat CD40 gene that can effectively down-regulate CD40 gene expression at mRNA and protein levels in rat DC.展开更多
基金Supported by the National Natural Science Foundation of China(No.30670301)Science and Technology Services of Jilin Province, China(No.20050408-1)+1 种基金PhD Scientific Research Foundation of Ministry of Education of China(No.20050183069)the Jilin Province Talent Development Foundation(No.JRJB2007-2)
文摘To construct a lentiviral shRNA vector targeting rat CD40 gene and detect its effectiveness of gene silencing in dendritic cells（DCs）, specific siRNA targets with short hairpin frame were designed and synthesized according to the mRNA sequence of rat CD40 gene. DNA oligo was cloned into lentiviral expression vector, and then PCR and sequencing analyses were conducted to verify the constructs. The verified plasmids were transfected into 293T cells that over-express recombinant CD40 in order to select the most effective siRNA targets, shRNA lentiviruses from the selected constructs were propagated and harvested with a virus packaging system, and the virus titers were determined. Western blot and Real-time PCR were performed to determine CD40 expression level in the virusinfected dendritic cells. PCR and sequencing analyses reveal that shRNA plasmids of four targets were successfully constructed. The optimal interfering target was selected, and the virus with a titer of 5 × 10^7 TU/mL was successfully packaged. CD40 expression in rat DCs was knockdown at both mRNA and protein levels by virus infection. In comparison to that of control groups, CD40 mRNA expression and protein expression were decreased by 60.9% and 61.2%, respectively. We have successfully constructed recombinant lentiviral shRNA expression vector targeting rat CD40 gene that can effectively down-regulate CD40 gene expression at mRNA and protein levels in rat DC.
