Highly efficient small interfering RNA delivery to primary mammalian neurons induces MicroRNA-like effects before mRNA degradation.
The study of protein function in neurons has been hindered by the lack of highly efficient, nontoxic methods of inducing RNA interference in such cells. Here we show that application of synthetic small interfering RNA (siRNA) linked to the vector peptide Penetratin1 results in rapid, highly efficient uptake of siRNA by entire populations of cultured primary mammalian hippocampal and sympathetic neurons. This treatment leads to specific knock-down of targeted proteins within hours without the toxicity associated with transfection. In contrast to current methods, our technique permits study of protein function across entire populations with minimal disturbance of complex cellular networks. Using this technique, we found that protein knock-down (evident after 6 hr) precedes any decrease in targeted message (evident after 24 hr), suggesting an early, translational repression by perfectly targeted siRNAs.
Davidson
Thomas J
TJ
Department of Pathology, Taub Institute for the Study of Alzheimer's Disease and the Aging Brain, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Harel
Sivan
S
Arboleda
Valerie A
VA
Prunell
Giselle F
GF
Shelanski
Michael L
ML
Greene
Lloyd A
LA
Troy
Carol M
CM
D000818:Animals; D002352:Carrier Proteins / Q000008:administration & dosage; D053148:Caspase 3; D020169:Caspases / Q000032:analysis / Q000235:genetics; D057846:Cell-Penetrating Peptides; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D018390:Gene Targeting / Q000379:methods*; D006624:Hippocampus / Q000166:cytology; D051379:Mice; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology*; D051381:Rats; D017783:Superior Cervical Ganglion / Q000166:cytology; D013482:Superoxide Dismutase / Q000032:analysis / Q000235:genetics; D000072105:Superoxide Dismutase-1; D014162:Transfection
D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D016895:Culture Media, Serum-Free; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C414409:penetratin; C000606291:Sod1 protein, mouse; C000606292:Sod1 protein, rat; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; C505409:Casp3 protein, mouse; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases