Generation and Validation of Tissue-Specific Knockout Strains for Toxicology Research.
Generation and Validation of Tissue-Specific Knockout Strains for Toxicology Research.
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用于毒理学研究的组织特异性敲除菌株的生成和验证。
DOI：
10.1002/cptx.86
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发表时间：
2019
期刊：
影响因子：
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通讯作者：
Mukhopadhyay,Somshuvra
中科院分区：
文献类型：
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作者：
Taylor,CherishA;Shawlot,William;Ren,JinXiang;Mukhopadhyay,Somshuvra
组织特异性基因敲除小鼠在整个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。在此，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。随后的方案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，提供了使用基因组DNA的PCR、mRNA的逆转录PCR和定量逆转录PCR以及蛋白质的免疫印迹分析来验证敲除小鼠的方法。John Wiley & Sons，Inc._关于我们
Tissue‐specific knockout mice are widely used throughout scientific research. A principle method for generating tissue‐specific knockout mice is the Cre‐loxPsystem. Here, we give a detailed description of the steps required to generate and validate tissue‐specific knockout mice using the Cre‐loxPsystem. 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.