title: Optimized transfection of diced siRNA into mature primary human osteoclasts: inhibition of cathepsin K mediated bone resorption by siRNA.
abstract: Osteoclasts are large multinucleated cells responsible for bone resorption. Bone resorption is dependent on the liberation of calcium by acid and protease destruction of the bone matrix by proteinases. The key proteinase produced by the osteoclast is cathepsin K. Targeted knock-down of cathepsin K was performed using small inhibitory RNA (siRNA). siRNA is a method that introduces short double-stranded RNA molecules that instruct the RNA-induced silencing complex (RISC) to degrade mRNA species complementary to the siRNA. Transfection of siRNA by lipid cations allows for short-term inhibition of expression of the targeted gene. We show that transfection of primary human osteoclasts with siRNA to cathepsin K reduces expression by > or = 60% and significantly inhibits bone resorption with a reduction of both resorption pit numbers (P = 0.018) and resorbed area (P = 0.013). We also show that FuGENE 6 is an effective lipid transfection reagent with which to transfect primary human osteoclasts, that does not produce off-target effects.
authors.affiliation: School of Medical Sciences, Griffith University, Gold Coast campus, Southport, Qld, 4215, Australia.
authors.forename: Christina I
authors.initials: CI
authors.lastname: Selinger
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Day
authors.forename: Nigel A
authors.initials: NA
authors.lastname: Morrison
mesh_terms: D001862:Bone Resorption; D002118:Calcium / Q000378:metabolism; D056657:Cathepsin K; D002403:Cathepsins / Q000378:metabolism*; D002412:Cations; D018929:Cell Culture Techniques / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D015233:Models, Statistical; D020411:Oligonucleotide Array Sequence Analysis; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012330:RNA, Double-Stranded / Q000737:chemistry; D034741:RNA, Small Interfering / Q000378:metabolism*; D014162:Transfection
chemical_list: D002412:Cations; D008055:Lipids; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D002403:Cathepsins; C534130:CTSK protein, human; D056657:Cathepsin K; D002118:Calcium