High-throughput Construction Of Lentiviral RNA Interference Vectors

As the accomplishment of human genome project and the launching of proteomic research, the study of gene function is no longer confined to a single gene, but the whole genome. Systematic research methods are invented and applied to analyze the function of the whole genome. Thus, in recent years, more effective, high-throughput gene functional analysis tools get more and more attention. Large-scale RNAi library has emerged as a powerful tool to systematic study gene functions on a genome-scale. RNAi library is an artificial combinatorial library which silence many different genes through RNAi mechanisms simultaneously by inducing the expression of siRNA. The function of multiple genes can be studied at the same time, because RNAi library can simultaneously reduce the expression of a number of different genes. Current methods for the construction of shRNA expression vectors usually require the synthesis of long oligonucleotides for its specific stem-loop structure, which cause a high rate of mutation and high cost. Furthermore, most of these methods include complicated procedures for identifying the desired recombinants because the inserts are extremely short.We have developed an advanced Ligation Independent Cloning(LIC) method for the generation of shRNA expression vectors. First, LIC cloning vectors were first nicked with enzyme Nb.BbvCI, and then digested by restriction endonuclease Bfu I to produce specific long overhangs. Second, we synthesized primers to generate a shRNA expression structure by primer annealing. The primers have additional12-nt tails complementary to the tails of linear vectors. Then the shRNA expression structure incubated with the the linear vectors in vitro conditions and, following transformation, the resulting circular recombinant molecules formed without vitro ligation for efficient bacterial transformation. This approach is independent of purification, restriction enzyme digestion, ligation of target DNAs and the synthesis of long primers(about60bp). In this study,20shRNA expression vectors were constructed with a cloning efficiency of above90%and the mutation rate of positive shRNA clones was less than15%. In addition, by using shRNA vectors directly against expression of exogenous p53and EGFP genes, we have demonstrated that this shRNA cloning system is both effective and faithful.Base on this study, we constructed shRNA lentiviral vectors by using the same approach. Six shRNA lentiviral vectors were successfully constructed. In further studies, In order to establish shRNA lentiviral expression vector librarirs, we plan to construction more shRNA lentiviral vectors and and test their interference ability.