A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs.
BACKGROUND
Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems.


RESULTS
We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription.


CONCLUSIONS
We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells.
Zhu
Xiaocui
X
The Alliance for Cellular Signaling, Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA. xiaocui_zhu@hotmail.com
Santat
Leah A
LA
Chang
Mi Sook
MS
Liu
Jamie
J
Zavzavadjian
Joelle R
JR
Wall
Estelle A
EA
Kivork
Christine
C
Simon
Melvin I
MI
Fraser
Iain Dc
ID
D000818:Animals; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D016086:Lentivirus / Q000235:genetics; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs; D008840:Microfilament Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012319:RNA Polymerase II / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D007527:Isoenzymes; D035683:MicroRNAs; D008840:Microfilament Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000098662:Vasodilator-Stimulated Phosphoprotein; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D012319:RNA Polymerase II