Molecular Small Animal Imaging Centre, Nuclear Medicine and Molecular Imaging, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium.
Stem Cell Institute, KU Leuven, Leuven, Belgium.
J Nucl Med. 2017 Oct;58(10):1659-1665. doi: 10.2967/jnumed.117.189779. Epub 2017 Jun 8.
Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging.
分子成像对于确定移植干细胞的命运和持久性是不可或缺的。转基因诱导的标准策略包括使用易沉默和插入突变的病毒载体，或使用非人类基因。我们使用锌指核酸酶将人源成像报告基因诱导稳定表达至人胚胎干细胞的安全港腺相关病毒整合位点 1。构建了携带荧光、生物发光成像和人正电子发射断层扫描报告基因的质粒。体外实验证实了其功能，胚胎干细胞保留了分化能力。进行了畸胎瘤形成实验，并通过正电子发射断层扫描和生物发光成像对肿瘤进行了长期成像。本研究证明了基因组编辑在人胚胎干细胞中靶向整合人源成像报告基因进行长期分子成像的应用。
