An advanced blue-white screening method for construction of shRNA expression vectors.
Short hairpin RNA (shRNA) encoded within an expression vector is an effective tool for exploration of gene function in mammalian cells. Many of the current methods for constructing shRNA expression vectors require cumbersome and time-consuming procedures for identification of the desired recombinants. We have developed a highly efficient and less labor-intensive cloning method that allows the construction of shRNA expression vectors in one day and with minimal effort. This advanced blue-white screening technique was developed by combining the reconstitution of ideal lacO with TA cloning. The DNAs are simply ligated into the destination vectors and, following transformation, a desired recombinant event will give a typical blue colony. In addition, we have used this cloning method for the construction of targeting reporter expression vectors to measure the efficacy of the corresponding shRNA. We constructed 122 functional shRNA expression vectors and sequencing of the positive cloning vectors confirmed a high degree of accuracy. Only three short DNA primers are needed for constructing both shRNA and targeting reporter expression vectors. This advanced blue-white screening system is a powerful tool for the high-throughput assay of RNAi libraries.
Yang
Ju
J
Institute of Biochemistry and Molecular Biology, Biology Faculty of Hubei University, Wuhan, Hubei Province 430062, PR China.
Xu
Jun
J
Zhong
Xing
X
Zhang
Yun
Y
Wu
Qiong
Q
Li
Wenjuan
W
Zhang
Xing
X
Zhen
Peng
P
Yang
Xiaosong
X
Chen
Liang
L
Wang
Yaping
Y
Zhu
Dewu
D
Zhai
Chao
C
Ma
Lixin
L
D003001:Cloning, Molecular / Q000379:methods*; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D007763:Lac Operon; D009875:Operator Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*
D034741:RNA, Small Interfering