Construction of equalized short hairpin RNA library from human brain cDNA.
Short hairpin RNA (shRNA) library is a powerful new tool for high-throughput loss-of-function genetic screens in mammalian cells. An shRNA library can be constructed from synthetic oligonucleotides or enzymatically cleaved natural cDNA. Here, we describe a new method for constructing equalized shRNA libraries from cDNA. First, enzymatically digested cDNA fragments are equalized by a suppression PCR-based method modified from suppression subtractive hybridization. The efficiency of equalization was confirmed by quantitative real-time PCR. The fragments are then converted into an shRNA library by a series of enzymatic treatments. With this new technology, we constructed a library from human brain cDNA. Sequence analysis showed that most of the randomly selected clones had inverted repeat sequences converted from different cDNA. After transfecting HEK 293T cells and detecting gene expression, three out of eight clones were demonstrated to significantly inhibit their target genes.
Xu
Lei
L
Department of Biochemistry, Basic Medical School, Tongji University, No. 1239, Siping Road, Shanghai 200092, China. xvleii@mail.china.com
Li
Jingqi
J
Liu
Li
L
Lu
Lixia
L
Gao
Jingxia
J
Li
Xueli
X
D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry*; D015723:Gene Library; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000138:chemical synthesis*
D018076:DNA, Complementary; D034741:RNA, Small Interfering