Application Of Dual-function Lentiviral Expression Vector In The Study Of TFIIA? Mutation

Objective:This study is to construct a dual-function lentiviral expression vector that can express both sh RNA and protein simultaneously,and study the effect of TFIIA? mutation on gene transcription with the obtained vector.Methods:1.The original vector p LVsh RNA-EGFP-Puro was reconstruted by gene cloning to obtain a lentiviral expression vector capable of simultaneously expressing sh RNA and protein.2.The sh RNA interfering with beta-tubulin expression and HA-beta-tubulin fusion gene were respectively cloned into different sites within a dual-function lentiviral expression vector,and the function of the vector was identified.3.The sh RNA interfering with TFIIA? expression and the genes encode HA-tagged TFIIA? wild-type or its derivatives were cloned on the same dual-function vector.The obtained vector was used to determine whether it can express exogenous protein and meanwhile reduce the expression of endogenous genes.Using the stable cell lines that express exogenous TFIIA? wild-type or its mutants,the effect of TFIIA? mutation on gene transcription was examined and the regulatory mechanism mediated by TFIIA? mutation was exploredResults:1.The results from Western blot and fluorescence microscopy assays showed that a series of modification to original vector including mutation of the restriction sites and insertion of multiple cloning sites(MCS)and CMV promoter sequence did not affect the basic function of the original vector,but the vector obtained a new function-protein expression.It suggests that the establishment of a dual-function lentiviral expression vector is successful.2.The data from the assays with the vectors expressing both TFIIA? shRNA and HA-TFIIA? fusion gene indicated that this vector can express exogenous HA-TFIIA? proteins and silence the expression of endogenous TFIIA? in 293 T cells.3.Reporter gene assays were performed using the stable cell lines expressing both exogenous HA-TFIIA?(or its mutants)and TFIIA? sh RNA.The results showed that mutations of TFIIA? enhanced the basal activity of Ad ML promoter but not the activated activity.The data from the transfection assays with the reporter vectors respectively driven by human natural promoters and the stable cell lines revealed that TFIIA? mutations differentially regulated the activities of different promoters.4.Protein-DNA binding assays showed that mutation of TFIIA? enhanced TFIIA? binding to Ad ML promoter.Conclusions:The dual-function lentiviral expression vector developed in this study can simultaneously express sh RNA and protein,and it can be successfully applied to study the effect of TFIIA? point mutation on transcription.Therefore,this novel method provides a simple and fast approach for the study of site-directed mutagenesis of a gene.