A preliminary study on construction of a random RNAi library based on RNAi expression vector with convergent U6 and H1 promoters

Objective:To construct a random RNAi library by using a RNAi expression vector with convergent U6 and H1 promoters.Methods:Firstly,RNAi plasmids targeting EGFP gene and p53 gene were constructed respectively based on pRHU vector in order to determine the efficiency of the vector and assayed their interfering effects at cellular level.Secondly,a DNA template containing random sequence and two linkers suited for PCR amplification was designed and synthesized.After PCR amplification and HindⅢ/ BamHⅠ digestion,the DNA fragment was cloned into pRHU vector and transformed into high efficient competent cells.Twenty clones were randomly selected and sequenced.Results:pRHU Vector was determined to be an efficient RNAi expression vector based on the fact that it could interfere expressions of exogenous EGFP gene and endogenous p53 gene.A random RNAi library with the capacity of 4×106 clones derived from 50 μg of initial pRHU vector was constructed in a relatively short cycle.DNA Sequencing results showed that all the twenty randomly selected clones contained GN17-19 sequence and their sequences were not identical one another.Conclusion:A simple and rapid method for constructing a random RNAi library based on RNAi expression vector with convergent U6 and H1 promoters was established and laid foundation for further genomic screening of functional genes.