Alternative methods for the efficient construction of short hairpin RNA expression vectors.

Abstract Short hairpin RNA (shRNA)-mediated RNA interference has become a basic technique in modern molecular biology and biochemistry for studying gene function and biological pathways. Here, we report two alternative and efficient methods to construct shRNA expression vectors based respectively on multiple-step sequential PCR and primer extension–homologous recombination (PE-HR). Neither method requires synthesizing long oligonucleotides containing hairpin sequences as used in traditional approaches. The hairpin sequences may produce mutations during oligo synthesis, pose problems in annealing, and lead to inefficient cloning. The PE-HR method further provides rapid and economical construction of shRNA expression vectors without needing the ligation procedure.