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首页> 外文期刊>Experimental Animals >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
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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

机译：开发适合体内敲低方法的基因选择方案及其在小鼠胰岛素受体底物基因中的应用

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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.
机译：同时敲除多个与糖尿病相关的基因的糖尿病前期模型小鼠可能对糖尿病的药物和医学研究有用。然而，在体内充分敲低的有效条件取决于靶基因的内在特性。有必要研究哪些基因适用于或不适用于体内敲低方法。本研究选择胰岛素受体底物1和2（Irs-1、Irs-2）作为靶基因。设计了针对相应基因的有效siRNA，并通过基于细胞的实验证实了其功效。根据siRNAs的结果，分别构建了针对Irs-1和Irs-2的shRNA表达载体。它们的功效也得到了基于细胞的实验的证实。采用流体动力学方法将载体递送至小鼠。通过EGFP表达载体的示范递送和连续的组织化学分析，发现该方法可有效将主要递送至肝脏。将 50 μg 的 shRNA 表达载体注射到尾静脉中。24 h后，分离肝脏、胰腺和肌肉，定量RT-PCR分析Irs-1和Irs-2的表达水平。在肝脏中，Irs-2 被有效地敲低到对照水平的 60%，但即使在相同条件下，Irs-1 也没有受到影响。这里开发的方案对于选择适合体内敲低方法的基因是可行的。

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