RNA interference screen to identify pathways that enhance or reduce nonviral gene transfer during lipofection.
Some barriers to DNA lipofection are well characterized; however, there is as yet no method of finding unknown pathways that impact the process. A druggable genome small-interfering RNA (siRNA) screen against 5,520 genes was tested for its effect on lipofection of human aortic endothelial cells (HAECs). We found 130 gene targets which, when silenced by pooled siRNAs (three siRNAs per gene), resulted in enhanced luminescence after lipofection (86 gene targets showed reduced expression). In confirmation tests with single siRNAs, 18 of the 130 hits showed enhanced lipofection with two or more individual siRNAs in the absence of cytotoxicity. Of these confirmed gene targets, we identified five leading candidates, two of which are isoforms of the regulatory subunit of protein phosphatase 2A (PP2A). The best candidate siRNA targeted the PPP2R2C gene and produced a 65% increase in luminescence from lipofection, with a quantitative PCR-validated knockdown of approximately 76%. Flow cytometric analysis confirmed that the silencing of the PPP2R2C gene resulted in an improvement of 10% in transfection efficiency, thereby demonstrating an increase in the number of transfected cells. These results show that an RNA interference (RNAi) high-throughput screen (HTS) can be applied to nonviral gene transfer. We have also demonstrated that siRNAs can be co-delivered with lipofected DNA to increase the transfection efficiency in vitro.
Barker
Gregory A
GA
Penn Center for Molecular Discovery, Institute for Medicine and Engineering, Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Diamond
Scott L
SL
D001011:Aorta / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D018274:Electroporation; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000494:pharmacology*; D015894:Genome, Human; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008156:Luciferases / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014162:Transfection
D008055:Lipids; C086724:Lipofectamine; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D008156:Luciferases