Ryu Junghyun, Chan William, Wettengel Jochen M, Hanna Carol B, Burwitz Benjamin J, Hennebold Jon D, Bimber Benjamin N
Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR 97006, USA.
Institute of Virology, Technical University of Munich/Helmholtz Zentrum München, München, 81675 Germany.
Mol Ther Methods Clin Dev. 2022 Jan 19;24:241-254. doi: 10.1016/j.omtm.2022.01.009. eCollection 2022 Mar 10.
Genome engineering is a powerful tool for research and the creation of novel model organisms and has growing clinical applications. Randomly integrating vectors, such as lentivirus- or transposase-based methods, are simple and easy to use but carry risks arising from insertional mutagenesis. Here we present enhanced-specificity tagmentation-assisted PCR (esTag-PCR), a rapid and accurate method for mapping transgene integration and copy number. Using stably transfected HepG2 cells, we demonstrate that esTag-PCR has higher integration site detection accuracy and efficiency than alternative tagmentation-based methods. Next, we performed esTag-PCR on rhesus macaque embryos derived from zygotes injected with piggyBac transposase and transposon/transgene plasmid. Using low-input trophectoderm biopsies, we demonstrate that esTag-PCR accurately maps integration events while preserving blastocyst viability. We used these high-resolution data to evaluate the performance of piggyBac-mediated editing of rhesus macaque embryos, demonstrating that increased concentration of transposon/transgene plasmid can increase the fraction of embryos with stable integration; however, the number of integrations per embryo also increases, which may be problematic for some applications. Collectively, esTag-PCR represents an important improvement to the detection of transgene integration, provides a method to validate and screen edited embryos before implantation, and represents an important advance in the creation of transgenic animal models.
基因组工程是一种用于研究和创建新型模式生物的强大工具，并且其临床应用也在不断增加。随机整合载体，如基于慢病毒或转座酶的方法，简单易用，但存在插入诱变带来的风险。在此，我们展示了增强特异性标签辅助PCR（esTag-PCR），这是一种用于绘制转基因整合位点和拷贝数的快速且准确的方法。利用稳定转染的HepG2细胞，我们证明esTag-PCR比其他基于标签的方法具有更高的整合位点检测准确性和效率。接下来，我们对来自注射了piggyBac转座酶和转座子/转基因质粒的受精卵的恒河猴胚胎进行了esTag-PCR。利用低输入量的滋养外胚层活检样本，我们证明esTag-PCR在保留囊胚活力的同时能准确绘制整合事件。我们利用这些高分辨率数据评估了piggyBac介导的恒河猴胚胎编辑的性能，证明增加转座子/转基因质粒的浓度可以增加具有稳定整合的胚胎比例；然而，每个胚胎的整合数量也会增加，这对于某些应用可能存在问题。总体而言，esTag-PCR代表了转基因整合检测的一项重要改进，提供了一种在植入前验证和筛选编辑胚胎的方法，并且代表了转基因动物模型创建方面的一项重要进展。
