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Animal Bioresource in Japan

.: Home > Animal Bioresource in Japan > 2013 > Volume 62 Number 2 > Mikako SAITO, Yukari KAKUTANI, Misako KABURAGI, Hisakage FUNABASHI, Hideaki MATSUOKAMikako SAITO, Yukari KAKUTANI, Misako KABURAGI, Hisakage FUNABASHI, Hideaki MATSUOKA

Development of a Protocol for Selection of GenesFit for the In Vivo Knockdown Method and its Application to Insulin Receptor Substrate Genesin Mice

Mikako SAITO, Yukari KAKUTANI, Misako KABURAGI, Hisakage FUNABASHI, Hideaki MATSUOKAMikako SAITO, Yukari KAKUTANI, Misako KABURAGI, Hisakage FUNABASHI, Hideaki MATSUOKA
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2–24–16 Naka- cho, Koganei, Tokyo 184-8588, Japan
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.

Keywords :
hydrodynamic delivery, in vivo knockdown method, Irs-1 , Irs-2 , shRNA expression vector

Date Deposited : 06 Apr 2015 11:33

Last Modified : 06 Apr 2015 11:33

Official URL: http://https://www.jstage.jst.go.jp/browse/expanim/62/1/_contents

Volume 62, Number 2, - 2013

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