Small interfering RNA production by enzymatic engineering of DNA (SPEED).
Small interfering RNAs (siRNAs) potently silence expression of target genes. In principle siRNA libraries can be used to perform effective genome-scale functional genetic screens in mammalian cells, but their development has been hampered by the need to chemically synthesize thousands of oligonucleotides and to incorporate them into expression vectors. We have developed a technology to efficiently convert a double-stranded cDNA library into a retroviral siRNA library in which 21-base siRNAs are produced in infected cells at high levels and efficiently block expression of their target genes. The key steps are the generation of random cDNA fragments that are fused to a hairpin linker, cleavage with the MmeI endonuclease that creates 20- to 21-bp cDNA fragments, conversion to a double-stranded DNA that contains two copies of the cDNA insert in a head-to-head palindrome, and insertion of the construct downstream of a polymerase III promoter. We constructed a siRNA library with 3 x 10(6) clones from a mouse embryo cDNA library; siRNAs were found against many different genes; and multiple siRNAs can be generated from a single mRNA. We further showed that specific siRNAs were efficiently produced in stably infected mammalian cells and resulted in significant and specific reduction of their target mRNAs. Because no prior knowledge about target transcripts is needed, a cDNA-derived siRNA library will generate siRNAs against unknown transcripts and genes. Finally, cDNA-derived siRNA libraries can be readily generated from any cell type or species, enabling genome-wide functional screens in many biological systems.
Luo
Biao
B
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.
Heard
Amanda D
AD
Lodish
Harvey F
HF
D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D020868:Gene Silencing; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D012320:RNA Polymerase III / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics; D012190:Retroviridae / Q000235:genetics
D018076:DNA, Complementary; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012320:RNA Polymerase III; C049760:endodeoxyribonuclease MmeI; D015252:Deoxyribonucleases, Type II Site-Specific