Alternative approach to generate shRNA from cDNA.
Short hairpin RNA (shRNA) synthesized from vector-based expression is as effective as short interfering RNA (siRNA) synthesized in vitro for suppressing the expression of their corresponding genes. Recently, three groups independently reported a new technology to construct an shRNA library from cDNA, providing great hope for genome-wide functional screens in many biological systems. In the present study, we report an alternative approach to generate shRNA from cDNA. A major improvement was to use a nicking enzyme to open up the double-stranded DNA so that the loop region remains single-stranded while the rest of the DNA fragment is double-stranded at an elevated temperature (e.g., 72 degrees C). The single-stranded DNA was then converted into double-stranded DNA by Taq DNA polymerase using the existing strand in the double-stranded region as a primer. Thus, the extended product carried a palindromic structure of 19 bp separated by a loop. Finally, the DNA fragment was cloned into a vector that carries an H1 promoter at the upstream region and ends with 5Ts, a terminator for the Pol III polymerase, at the downstream region. To prove the principle, we constructed shRNA from green fluorescent protein (GFP) cDNA and successfully suppressed GFP expression. Consequently, this simplified approach provides a better alternative to generate shRNA libraries from cDNA. Such shRNA libraries can be used to identify potential siRNA target sequences and study gene functions by a variety of selection methods.
Dinh
Anh
A
Southern Illinois University School of Medicine, Springfield, IL 62794, USA.
Mo
Yin-Yuan
YY
D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D020868:Gene Silencing; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D018076:DNA, Complementary; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins