Construction of recombinant lentivirus vector expressing small hairpin RNA against human Bax inhibitor-1 gene expression / 中国组织工程研究

ABSTRACT

BACKGROUND: Lentivirus-mediated RNA interference technology has been widely used in the study of gene function because of its specificity, high inhibition efficiency and persistent effects. Virus packaging, transfection, as well as small hairpin RNA (shRNA)-coding sequence could affect the inhibition efficiency. Therefore, impact factors of RNA interference for human Bax inhibitor 1 (BI-1) was studied in this experiment.OBJECTIVE: To find the valid small interference RNA (siRNA) targeting human BI-1 gene by Lentivirus.DESIGN, TIME AND SETTING: Single sample observation was completed in Department of Medical Genetics, Basic Medical College, Peking University Health Science Center from September 2007 to December 2008.MATERIALS 293T cell and SH-SY5Y cells were preserved in this laboratory; 4 shRNAs were designed through a use of online design tool by Ambion (www.ambion.com). METHODS: Several recombinant plasmids which expressed shRNAs targeting different regions of the BI-1 gene and labeled enhanced green fluorescent protein as a fusion gene were constructed. Four types of shRNA-expressing virions were obtained by cotransfection of shRNA-expressing plasmid and package protein-expressing plasmids into 293T cells. Green fluorescent protein expression was determined using flow cytometry to search the optimal package conditions. Real-time PCR and beta actin mRNA was used as an internal control, four types of recombinant viral supernatant and control viral supernatant were added on the SH-SY5Y cells, knock-down efficacies of the endogenous BI-1 gene were determined, and the RNA interference effective sequences were optimized. MAIN OUTCOME MEASURES: Intracellular green fluorescent protein expression rate of the cells infected with different packaging virus. RESULTS: The optimal infective packaging virus could be obtained when the ratio of plasmids pLentiLox3.7, REV, VSVG and RRE was 2111 and the supernatants were collected 48 hours after transfection. Replacement of culture media 24 hours after transfection could increase the infection efficacy of the virions. shRNA targeting-2-17nt (start coding region) of BI-1 gene was most valid interference efficiency and could decrease the gene expression to 40%. CONCLUSION: Influencing factors of Lentivirus-mediated RNA interference technology virus packages were investigated in this study, which could be a foundation for constructing stable BI-1 knocked down neuronal cell model and for studying the abnormal expression of BI-1 related to the neuron apoptosis disease.