title: Development of a protocol for selection of genes fit for the in vivo knockdown method and its application to insulin receptor substrate genes in mice.
abstract: Prediabetes model mice in which more than one gene associated with diabetes is knocked down simultaneously are potentially useful for pharmaceutical and medical studies of diabetes. However, the effective conditions for sufficient knockdown in vivo are dependent on the intrinsic properties of the target genes. It is necessary to investigate which genes are applicable or not to the in vivo knockdown method. In this study, insulin receptor substrate 1 and 2 (Irs-1, Irs-2) were selected as target genes. Effective siRNAs against the respective genes were designed, and their efficacy was confirmed by cell-based experiments. Based on the results of siRNAs, shRNA expression vectors against Irs-1 and Irs-2 were constructed, respectively. Their efficacy was also confirmed by cell-based experiments. A hydrodynamic method was applied to the delivery of the vectors to mice. This method was found to be effective for predominant delivery to the liver by demonstrative delivery of an EGFP expression vector and successive histochemical analysis. Fifty micrograms of the shRNA expression vector was injected into the tail vein. After 24 h, the liver, pancreas, and muscle were isolated, and the expression levels of Irs-1 and Irs-2 were analyzed by quantitative RT-PCR. In the liver, Irs-2 was effectively knocked down to 60% of the control level, but Irs-1 was not influenced even under the same conditions. The protocol developed here is feasible for the selection of genes fit for in vivo knockdown method.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Mikako
authors.initials: M
authors.lastname: Saito
authors.forename: Yukari
authors.initials: Y
authors.lastname: Kakutani
authors.forename: Misako
authors.initials: M
authors.lastname: Kaburagi
authors.forename: Hisakage
authors.initials: H
authors.lastname: Funabashi
authors.forename: Hideaki
authors.initials: H
authors.lastname: Matsuoka
mesh_terms: D003920:Diabetes Mellitus / Q000235:genetics*; D005240:Feasibility Studies; D015870:Gene Expression; D055785:Gene Knockdown Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D049452:Green Fluorescent Proteins; D055504:Insulin Receptor Substrate Proteins / Q000032:analysis / Q000235:genetics*; D008099:Liver; D034622:RNA Interference; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012641:Selection, Genetic
chemical_list: D055504:Insulin Receptor Substrate Proteins; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins