Pronuclear Injection-Based Targeted Transgenesis.
Pronuclear Injection-Based Targeted Transgenesis.
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DOI：
10.1002/cphg.23
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发表时间：
2016-10-11
影响因子：
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通讯作者：
Gurumurthy CB
中科院分区：
文献类型：
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作者：
Schilit SLP;Ohtsuka M;Quadros RM;Gurumurthy CB
将DNA表达盒显微注射到受精卵中已经是产生转基因动物模型的标准方法。虽然有效，但注射的DNA随机整合到基因组中，导致潜在的问题，如内源基因或调控元件的破坏，拷贝数的变化，以及整合到抑制转基因表达的异染色质区域。最近开发的一种方法通过将转基因靶向到基因组中可以安全地容纳外源DNA的预定位点来解决传统转基因的这些缺陷。这种方法被称为基于原核注射的靶向转基因（PITT），采用酶促将外源DNA从供体载体转移到动物基因组中先前创建的着陆点。通过使用分子工具如Cre-LoxP重组酶和PhiC 31-attB/P整合酶系统实现DNA转移。在这里，我们提供了执行PITT的协议，并概述了目前的PITT工具提供给研究界。
Microinjection of DNA expression cassettes into fertilized zygotes has been a standard method for generating transgenic animal models. While efficient, the injected DNA integrates randomly into the genome, leading to potential problems such as disruption of endogenous genes or regulatory elements, variation in copy number, and integration into heterochromatic regions that inhibit transgene expression. A recently developed method addresses such pitfalls of traditional transgenesis by targeting the transgene to predetermined sites in the genome that can safely harbor exogenous DNA. This method, called Pronuclear Injection-based Targeted Transgenesis (PITT), employs an enzymatic transfer of exogenous DNA from a donor vector to a previously created landing pad site in the animal genome. The DNA transfer is achieved through the use of molecular tools such as Cre-LoxP recombinase and PhiC31-attB/P integrase systems. Here, we provide protocols for performing PITT and an overview of the current PITT tools available to the research community.