携带Fluc和ZsGreen双报告基因小鼠iPS细胞系的建立

Generation of an induced pluripotent stem cell line harboring Fluc and ZsGreen dual reporter genes

背景与目的:诱导性多潜能干细胞(induced pluripotent stem cells,iPS细胞)在修复受损组织器官和治疗人类疾病方面已展示了良好的应用前景,但iPS细胞具有形成畸胎瘤的作用,这是其安全应用于临床的巨大障碍之一。为此本研究拟利用慢病毒体外感染的方法,建立携带萤火虫荧光素酶(firefly luciferase,Fluc)和绿色荧光蛋白(green fluorescent protein,ZsGreen)双报告基因的小鼠iPS细胞系,为活体动态监测畸胎瘤形成、定植和形成畸胎瘤所需最小细胞剂量等提供实验基础。方法:构建含Fluc和ZsGreen双报告基因的慢病毒载体。以pLH2BmRFP质粒为模板,PCR扩增获得Fluc基因片段,将其克隆至利用BamHⅠ酶切的pHAGE-fullEF1a-MCS-IZsGreen质粒中,挑选一个阳性克隆质粒进行测序,最终获得携带双报告基因的慢病毒载体pEF1a-Fluc-IRES-ZsGreen(pELZG)。采用脂质体介导的瞬时转染生产携带Fluc和ZsGreen基因的病毒,收集病毒上清,并感染iPS细胞,数天后荧光显微镜下观察是否有绿色荧光的iPS细胞克隆,不断挑取ZsGreen阳性的iPS细胞克隆以进一步纯化。将标记好的iPS细胞移植入裸鼠皮下,观察成瘤情况。结果:pELZG载体分别经XbaⅠ、PvuⅡ、SacⅠ单酶切,得到的片段大小分别为10.005、3.282和6.723 kb,2.441、3.401和6.604 kb,预示成功构建了Fluc和ZsGreen双报告基因的慢病毒载体。利用其生产的病毒上清成功感染iPS细胞,经过不断的纯化,最终获得含稳定表达Fluc和ZsGreen双报告基因的小鼠iPS细胞系。将标记好的iPS细胞植入裸鼠皮下后,观察到畸胎瘤的形成。结论:成功建立了携带Fluc和ZsGreen双报告基因的小鼠iPS细胞系,为相关后续研究打下了良好的基础。标记后的iPS细胞对畸胎瘤的形成和发展具有很好的示踪作用。

Background and purpose： Induced pluripotent stem cells （iPS cells） have demonstrated a good prospect, which includes repairing damaged tissues, organs and the treatment of human diseases, but iPS cells have the ability to form a teratoma, which is one of the huge obstacles involved in its security used in clinical trials. Thus, this study intended to establish mouse iPS cell lines carrying firefly luciferase （Fluc） and green fluorescent protein （ZsGreen） dual reporter genes to lay the foundation for in vivo monitoring teratoma formation and determining the minimum cell dose required for teratoma formation. Methods： To generate lentiviral expression vector harboring Fluc and zsGreen genes, Fluc gene was amplified by PCR from the template of pLH2BmRFP, and subsequently cloned into the plasmid of pHAGE-fullEFla-MCS-IzsGreen to obtain the final vector ofpEFla-Fluc-IRES-ZsGreen （referred to as pELZG） which was used to produce lentiviruses carrying Fluc and ZsGreen genes, followed by confirming that lentiviruses harboring Fluc and ZsGreen genes were successfully made through GFP assay under fluorescent stereo microscope after infecting 293T cells. Lentivirus supernatant harboring Fluc and ZsGreen genes were employed to infect iPS cells, followed by GFP assay under fluorescent stereo microscope several days after infection. The labeled iPS cells were transplanted into subcutaneous of nude mice. Results： As expected, the gel electrophoresis of pELZG vector respectively digested with Xba I, Pvu II and Sac 1, respectively resulted in a 10.005 kb, 3.282 kb, 6.723 kb, and 2.441 kb, 3.401 kb, 6.604 kb bands. It indicated that the lentiviral vector carrying the Fluc and ZsGreen dual reporter gene was successfully constructed. Mouse iPS cells were successfully infected by lentivirus supernatant, while the complete GFP-positive iPS cell colonies were obtained by picking clones. After iPS cells were transplanted into nude mice, teratoma formation was observed. Conclusion ： Mouse iPS cell line stably expressing Fluc and ZsGreen genes was successfully generated, and the Fluc- and ZsGreen-labeled iPS cells can trace the formation and development of the teratoma.