RNAi-induced gene silencing by local electroporation in targeting brain region.
Genetic manipulation for "knockout" (KO) is a useful tool for characterizing a target gene. However, its shortcomings that need to be overcome hinder its easy and ready usage in ordinary laboratories. Here we describe a knockdown technique termed the RNA interference (RNAi)-induced gene silencing by local electroporation (RISLE). Small interfering RNA (siRNA) introduction by electroporation into a specific brain region results in a marked reduction in the expression levels of both the mRNA and protein of the target genes such as GluR2 and Cox-1 without affecting the expression levels of proteins other than that of the target protein or causing pathological changes in the target tissues. The effective electrical pulses are relatively weak, consisting of a strong short pulse and a weak long pulse applied in tandem. RISLE can knock down a gene at the target region, for example, the visual cortex and the CA1 region of the hippocampus, without affecting other regions. Moreover, the knockdown models constructed using this technique have physiological functions consistent with previous findings, that is, glutamate release from presynaptic sites, long-term potentiation (LTP), and long-term depression (LTD). These results suggest that this technique is applicable and characterized by spatial flexibility, temporal accessibility, and ease of establishment of knockdown models. The intactness of the tissue subjected to RISLE is due to the weak electrical pulses applied and the limited area of gene silencing. Thus RISLE may be applicable to disease therapy in the future.
Akaneya
Yukio
Y
Division of Neurophysiology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita 565-0871, Japan. akaneya@nphys.med.osaka-u.ac.jp
Jiang
Bin
B
Tsumoto
Tadaharu
T
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000033:anatomy & histology / Q000187:drug effects* / Q000502:physiology; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D051545:Cyclooxygenase 1; D004558:Electric Stimulation / Q000379:methods; D004566:Electrodes; D018274:Electroporation / Q000379:methods; D005071:Evoked Potentials / Q000528:radiation effects; D015870:Gene Expression / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects*; D005829:Geniculate Bodies / Q000528:radiation effects; D018698:Glutamic Acid / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D066298:In Vitro Techniques; D008565:Membrane Proteins; D008845:Microinjections / Q000379:methods; D011189:Potassium Chloride / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors
D019208:Brain-Derived Neurotrophic Factor; D008565:Membrane Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018091:Receptors, AMPA; D018698:Glutamic Acid; D011189:Potassium Chloride; D051545:Cyclooxygenase 1; D011451:Prostaglandin-Endoperoxide Synthases; C496513:Ptgs1 protein, rat; C104722:glutamate receptor ionotropic, AMPA 2