利用PhiC31整合酶通过靶向转基因整合在斑马鱼中对顺式调控元件进行检测

Testing of Cis-Regulatory Elements by Targeted Transgene Integration in Zebrafish Using PhiC31 Integrase.

作者信息

Hadzhiev Yavor, Miguel-Escalada Irene, Balciunas Darius, Müller Ferenc

机构信息

Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT, UK.

Department of Biology, Temple University, Philadelphia, PA, 19122, USA.

出版信息

Methods Mol Biol. 2016;1451:81-91. doi: 10.1007/978-1-4939-3771-4_6.

DOI:10.1007/978-1-4939-3771-4_6

PMID:27464802

Abstract

Herein we present several strategies for testing the function of cis-regulatory elements using the PhiC31 integrase system. Firstly, we present two different strategies to analyze the activity of candidate enhancer elements. Targeted integration of candidate enhancers into the same genomic location circumvents the variability-associated random integration and position effects. This method is suitable for testing of candidate enhancers identified through computational or other analyses a priori. Secondly, we present methodology for targeted integration of BACs into the same genomic location(s). By using additional reporters integrated into a BAC, this enables experimental testing whether cis-regulatory elements are functional in the sequence inserted in the BAC.

摘要

在此，我们展示了几种使用PhiC31整合酶系统测试顺式调控元件功能的策略。首先，我们提出了两种不同的策略来分析候选增强子元件的活性。将候选增强子靶向整合到相同的基因组位置可避免与随机整合和位置效应相关的变异性。该方法适用于测试通过计算或其他先验分析鉴定出的候选增强子。其次，我们展示了将BAC靶向整合到相同基因组位置的方法。通过使用整合到BAC中的额外报告基因，这能够进行实验测试顺式调控元件在BAC中插入的序列中是否具有功能。