title: 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.
authors: College of Animal Science & Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors: Kun
authors: K
authors: Xu
authors: College of Animal Science & Technology, Northwest A&F University, Yangling, Shaanxi 712100, China; Research Institute of Applied Biology, Shanxi University, Taiyuan, Shanxi 030006, China.
authors: Tingting
authors: T
authors: Zhang
authors: College of Animal Science & Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors: Lijun
authors: L
authors: Guo
authors: College of Animal Science & Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors: Ying
authors: Y
authors: Xin
authors: College of Animal Science & Technology, Northwest A&F University, Yangling, Shaanxi 712100, China; Frontier Institute of Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, China.
authors: Long
authors: L
authors: Zhang
authors: College of Animal Science & Technology, Northwest A&F University, Yangling, Shaanxi 712100, China. Electronic address: zhangzhy@nwsuaf.edu.cn.
authors: Zhiying
authors: Z
authors: Zhang
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D059765:Homologous Recombination; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics; D051379:Mice; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D000097603:Dyrk Kinases
chemical_list: D007335:Insulin-Like Growth Factor II; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D034741:RNA, Small Interfering; D000097603:Dyrk Kinases; C513121:IGF2 protein, mouse
keywords: Homologous recombination; Multiple-step sequential PCR; Primer extension; RNA interference; Short hairpin RNA vector construction