Department of Medicine, Division of Cardiology, Stanford School of Medicine, Stanford, CA, USA.
Circ Res. 2012 Dec 7;111(12):1494-503. doi: 10.1161/CIRCRESAHA.112.274969. Epub 2012 Sep 11.
Molecular imaging has proven to be a vital tool in the characterization of stem cell behavior in vivo. However, the integration of reporter genes has typically relied on random integration, a method that is associated with unwanted insertional mutagenesis and positional effects on transgene expression.
To address this barrier, we used genome editing with zinc finger nuclease (ZFN) technology to integrate reporter genes into a safe harbor gene locus (PPP1R12C, also known as AAVS1) in the genome of human embryonic stem cells and human induced pluripotent stem cells for molecular imaging.
We used ZFN technology to integrate a construct containing monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase reporter genes driven by a constitutive ubiquitin promoter into a safe harbor locus for fluorescence imaging, bioluminescence imaging, and positron emission tomography imaging, respectively. High efficiency of ZFN-mediated targeted integration was achieved in both human embryonic stem cells and induced pluripotent stem cells. ZFN-edited cells maintained both pluripotency and long-term reporter gene expression. Functionally, we successfully tracked the survival of ZFN-edited human embryonic stem cells and their differentiated cardiomyocytes and endothelial cells in murine models, demonstrating the use of ZFN-edited cells for preclinical studies in regenerative medicine.
Our study demonstrates a novel application of ZFN technology to the targeted genetic engineering of human pluripotent stem cells and their progeny for molecular imaging in vitro and in vivo.
分子成像已被证明是在体研究干细胞行为的重要工具。然而，报告基因的整合通常依赖于随机整合，这种方法与不想要的插入突变和转基因表达的位置效应有关。
为了解决这个障碍，我们使用锌指核酸酶（ZFN）技术对人类胚胎干细胞和人类诱导多能干细胞的基因组进行基因编辑，将报告基因整合到基因组中的安全港基因座（PPP1R12C，也称为 AAVS1），用于分子成像。
我们使用 ZFN 技术将包含单体红色荧光蛋白、萤火虫荧光素酶和单纯疱疹病毒胸苷激酶报告基因的构建体整合到一个安全港基因座，分别由组成型泛素启动子驱动，用于荧光成像、生物发光成像和正电子发射断层扫描成像。在人类胚胎干细胞和诱导多能干细胞中均实现了 ZFN 介导的靶向整合的高效率。ZFN 编辑的细胞保持了多能性和长期报告基因表达。在功能上，我们成功地在小鼠模型中追踪了 ZFN 编辑的人类胚胎干细胞及其分化的心肌细胞和内皮细胞的存活情况，证明了 ZFN 编辑细胞可用于再生医学的临床前研究。
我们的研究展示了 ZFN 技术在体外和体内对人类多能干细胞及其后代进行靶向遗传工程以进行分子成像的新应用。
