Generation and Validation of Tissue-Specific Knockout Strains for Toxicology Research

Cherish A. Taylor, William Shawlot, Jin Xiang Ren, Somshuvra Mukhopadhyay
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Abstract

Tissue-specific knockout mice are widely used throughout scientific research. A principle method for generating tissue-specific knockout mice is the Cre-loxP system. Here, we give a detailed description of the steps required to generate and validate tissue-specific knockout mice using the Cre-loxP system. The first protocol describes how to use gene targeting in mouse embryonic stem cells to generate mice with conditional alleles. Subsequent protocols describe how to recover Cre transgenic mice from cryopreserved sperm using in vitro fertilization and present a breeding strategy for obtaining tissue-specific knockouts. Finally, methods are provided for validating the knockout mice using PCR of genomic DNA, reverse-transcription PCR and quantitative reverse-transcription PCR of mRNA, and immunoblot analysis of proteins. © 2019 by John Wiley & Sons, Inc.

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毒理学研究中组织特异性敲除菌株的产生与验证
组织特异性敲除小鼠在科学研究中被广泛使用。产生组织特异性敲除小鼠的主要方法是Cre-loxP系统。在这里,我们详细描述了使用Cre-loxP系统生成和验证组织特异性敲除小鼠所需的步骤。第一个方案描述了如何在小鼠胚胎干细胞中使用基因靶向来产生具有条件等位基因的小鼠。随后的协议描述了如何使用体外受精从冷冻保存的精子中恢复Cre转基因小鼠,并提出了获得组织特异性敲除的育种策略。最后,通过基因组DNA PCR、mRNA反转录PCR和定量反转录PCR、蛋白质免疫印迹分析等方法对敲除小鼠进行验证。©2019 by John Wiley &儿子,Inc。
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Issue Information Sperm-Binding Assay Using an In Vitro 3D Model of the Mammalian Cumulus-Oocyte Complex Using Human Primary Foreskin Fibroblasts to Study Cellular Damage and Mitochondrial Dysfunction An Open-Globe Porcine Injury Platform for Assessing Therapeutics and Characterizing Biological Effects Issue Information
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