Tissue-Specific Regulation of Oncogene Expression Using Cre-Inducible ROSA26 Knock-In Transgenic Mice.
Tissue-Specific Regulation of Oncogene Expression Using Cre-Inducible ROSA26 Knock-In Transgenic Mice.
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DOI：
10.1002/9780470942390.mo140150
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发表时间：
2015-06-01
影响因子：
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通讯作者：
Justice MJ
中科院分区：
文献类型：
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作者：
Carofino BL;Justice MJ
Cre诱导的小鼠模型经常用于癌基因的空间和时间表达。随着大量的Cre重组酶系的可用，诱导型转基因代表了实现离散癌基因表达的易处理的方法。在这里，我们描述了一个协议，用于靶向Cre诱导的基因，使用loxP-STOP-loxP的方法无处不在地表达的ROSA 26基因座。基因靶向提供了优于标准转基因技术的几个优点，包括已知的整合位点和先前表征的表达模式。历史上，ROSA 26靶向载体的固有不稳定性阻碍了开发ROSA 26敲入系的效率。在本方案中，我们提供了利用Gateway重组进行克隆的各个步骤，以及筛选靶向ES细胞克隆的详细说明。通过遵循该方案，可以在几个月内实现ROSA 26敲入系的种系传递。
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 using a loxP-STOP-loxP approach 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, and 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.