PCR-based generation of shRNA libraries from cDNAs |
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Authors: | Cheng Du Baosheng Ge Zhongfeng Liu Kai Fu Wing C Chan Timothy W McKeithan |
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Affiliation: | (1) Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198-0766, USA;(2) Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198-7680, USA |
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Abstract: | Background The use of small interfering RNAs (siRNAs) to silence target gene expression has greatly facilitated mammalian genetic analysis by generating loss-of-function mutants. In recent years, high-throughput, genome-wide screening of siRNA libraries has emerged as a viable approach. Two different methods have been used to generate short hairpin RNA (shRNA) libraries; one is to use chemically synthesized oligonucleotides, and the other is to convert complementary DNAs (cDNAs) into shRNA cassettes enzymatically. The high cost of chemical synthesis and the low efficiency of the enzymatic approach have hampered the widespread use of screening with shRNA libraries. |
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