title: A Convenient In Vivo Model Using Small Interfering RNA Silencing to Rapidly Assess Skeletal Gene Function.
abstract: It is difficult to study bone in vitro because it contains various cell types that engage in cross-talk. Bone biologically links various organs, and it has thus become increasingly evident that skeletal physiology must be studied in an integrative manner in an intact animal. We developed a model using local intraosseous small interfering RNA (siRNA) injection to rapidly assess the effects of a target gene on the local skeletal environment. In this model, 160-g male Sprague-Dawley rats were treated for 1-2 weeks. The left tibia received intraosseous injection of a parathyroid hormone 1 receptor (Pth1r) or insulin-like growth factor 1 receptor (Igf-1r) siRNA transfection complex loaded in poloxamer 407 hydrogel, and the right tibia received the same volume of control siRNA. All the tibias received an intraosseous injection of recombinant human parathyroid hormone (1-34) (rhPTH (1-34)) or insulin-like growth factor-1 (IGF-1). Calcein green and alizarin red were injected 6 and 2 days before euthanasia, respectively. IGF-1R and PTH1R expression levels were detected via RT-PCR assays and immunohistochemistry. Bone mineral density (BMD), microstructure, mineral apposition rates (MARs), and strength were determined by dual-energy X-ray absorptiometry, micro-CT, histology and biomechanical tests. The RT-PCR and immunohistochemistry results revealed that IGF-1R and PTH1R expression levels were dramatically diminished in the siRNA-treated left tibias compared to the right tibias (both p<0.05). Using poloxamer 407 hydrogel as a controlled-release system prolonged the silencing effect of a single dose of siRNA; the mRNA expression levels of IGF-1R were lower at two weeks than at one week (p<0.01). The BMD, bone microstructure parameters, MAR and bone strength were significantly decreased in the left tibias compared to the right tibias (all p<0.05). This simple and convenient local intraosseous siRNA injection model achieved gene silencing with very small quantities of siRNA over a short treatment period (≤7 days).
authors.affiliation: Department of Orthopaedics, Peking University Third Hospital, Beijing, China.|Beijing Key Laboratory of Spinal Diseases, Beijing, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Orthopaedics, Peking University Third Hospital, Beijing, China.|Beijing Key Laboratory of Spinal Diseases, Beijing, China.
authors.forename: Can
authors.initials: C
authors.lastname: Liu
authors.affiliation: Department of Orthopaedics, Peking University Third Hospital, Beijing, China.|Beijing Key Laboratory of Spinal Diseases, Beijing, China.
authors.forename: Bao
authors.initials: B
authors.lastname: Hai
authors.affiliation: Department of Orthopaedics, Peking University Third Hospital, Beijing, China.
authors.forename: Guohong
authors.initials: G
authors.lastname: Du
authors.affiliation: Department of Orthopaedics, Peking University Third Hospital, Beijing, China.|Beijing Key Laboratory of Spinal Diseases, Beijing, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Orthopaedics, Peking University Third Hospital, Beijing, China.|Beijing Key Laboratory of Spinal Diseases, Beijing, China.
authors.forename: Huijie
authors.initials: H
authors.lastname: Leng
authors.affiliation: Department of Neurology, Peking University Third Hospital, Beijing, China.
authors.forename: Yingsheng
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Department of Orthopaedics, Peking University Third Hospital, Beijing, China.|Beijing Key Laboratory of Spinal Diseases, Beijing, China.
authors.forename: Chunli
authors.initials: C
authors.lastname: Song
mesh_terms: D015502:Absorptiometry, Photon; D000818:Animals; D015519:Bone Density; D001842:Bone and Bones / Q000378:metabolism*; D008297:Male; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017526:Receptor, IGF Type 1 / Q000037:antagonists & inhibitors* / Q000235:genetics; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000037:antagonists & inhibitors* / Q000235:genetics; D013977:Tibia / Q000166:cytology / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; D044168:Receptor, Parathyroid Hormone, Type 1; D017526:Receptor, IGF Type 1