利用可诱导的ROSA26敲入转基因小鼠对癌基因表达的组织特异性调控

Q1 Agricultural and Biological Sciences

Current protocols in mouse biology Pub Date : 2015-06-01 DOI:10.1002/9780470942390.mo140150

Brandi L. Carofino, Monica J. Justice

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引用次数: 3

摘要

基因诱导小鼠模型常用于致癌基因的时空表达。随着大量的Cre重组酶系可用，诱导转基因代表了一种易于处理的方法来实现离散癌基因表达。在这里，我们描述了一种将re诱导基因靶向到普遍表达的ROSA26位点的方案。与标准的转基因技术相比，基因靶向有几个优势，包括已知的整合位点和先前表征的表达模式。从历史上看，ROSA26靶向载体的固有不稳定性阻碍了开发ROSA26敲入系的效率。在本协议中，我们提供了利用Gateway重组克隆的单个步骤以及筛选靶向ES细胞克隆的详细说明。通过遵循这一方案，可以在几个月内实现ROSA26敲入系的种系传播。©2015 by John Wiley &儿子,Inc。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

Tissue-Specific Regulation of Oncogene Expression Using Cre-Inducible ROSA26 Knock-In Transgenic Mice

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Tissue-Specific Regulation of Oncogene Expression Using Cre-Inducible ROSA26 Knock-In Transgenic Mice

Cre-inducible mouse models are often utilized for the spatial and temporal expression of oncogenes. With the wide number of Cre recombinase lines available, inducible transgenesis represents a tractable approach to achieve discrete oncogene expression. Here, we describe a protocol for targeting Cre-inducible genes to the ubiquitously expressed ROSA26 locus. Gene targeting provides several advantages over standard transgenic techniques, including a known site of integration and previously characterized pattern of expression. Historically, an inherent instability of ROSA26 targeting vectors has hampered the efficiency of developing ROSA26 knock-in lines. In this protocol, we provide individual steps for utilizing Gateway recombination for cloning as well as detailed instructions for screening targeted ES cell clones. By following this protocol, one can achieve germline transmission of a ROSA26 knock-in line within several months. © 2015 by John Wiley & Sons, Inc.

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Current protocols in mouse biology Agricultural and Biological Sciences-Animal Science and Zoology

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet, all too often, nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. The aim of Current Protocols in Mouse Biology is to provide the clearest, most detailed and reliable step-by-step instructions for protocols involving the use of mice in biomedical research. Written by experts in the field and extensively edited to our exacting standards, the protocols include all of the information necessary to complete an experiment in the laboratory—introduction, materials lists with supplier information, detailed step-by-step procedures with helpful annotations, recipes for reagents and solutions, illustrative figures and information-packed tables. Each article also provides invaluable discussions of background information, applications of the methods, important assumptions, key parameters, time considerations, and tips to help avoid common pitfalls and troubleshoot experiments. Furthermore, Current Protocols in Mouse Biology content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Mouse Biology brings together resources in mouse biology and genetics and provides a mouse protocol resource that covers all aspects of mouse biology. Current Protocols in Mouse Biology also permits optimization of mouse model usage, which is significantly impacted by both cost and ethical constraints. Optimal and standardized mouse protocols ultimately reduce experimental variability and reduce the number of animals used in mouse experiments.