利用荧光素酶报告基因在体观察移植的人胚胎干细胞的动向

目的利用慢病毒载体携带荧光素酶报告基因并转导人胚胎干细胞，以实现在体观察移植细胞的增殖情况。方法酶切pGL3base质粒获得荧光素酶基因序列，酶切LTBR-GIP获得载体骨架，经过酶切产物热失活、平末端处理、CIP处理、胶纯化和连接，所构建慢病毒载体命名为ULIP，包括UBC启动子和IRES连接，能同时表达荧光素酶基因和Puromycin抗性基因。以ULIP转导人胚胎干细胞H9，并以Puromycin筛选稳定转导的细胞。随后移植于BALB／c小鼠和RAG^-/- γC^-/-小鼠，在XenogenIVIS（r）50成像系统进行荧光信号检测。结果稳定转导ULIP或LTBR—GIP的H9移植到BALB／c小鼠，ULIP组3d后荧光信号明显减弱，5d后基本上不能检出。而移植到RAG^-/-γC^-/-小鼠后，ULIP组的荧光信号在第3天稍有下降，随后荧光信号逐渐增强。LTBR-GIP-H9在移植BALB／c小鼠和RAG^-/-γC^-/-小鼠后均未能检测到荧光信号。结论荧光素酶报告基因可以用于观察移植细胞在体内的动向，实现实时观察移植细胞的存活情况。

Stable transduction of luciferase reporter gene in human embryonic stem cells and in vivo observation

Objective To observe the proliferation of transplanted human embryonic stem cells in vivo after transduction of lentiviral vector-mediated luciferase reporter gene. Methods pGL3 and LTBR-GIP vectors were digested by restriction enzyme. Then lentiviral vector named ULIP was constructed after the procedures of inactivation of the digestion product, blunt-end processing, CIP treatment, gel purification and ligation, which contains UBC promoter and IRES driving the expression of the luciferase gene and Purornycin resistance gene at the same time.The ULIP construct was then transduced into human embryonic stem cells H9 and screened with Puromyein. The stable transduced cells （ULIP-H9） were transplanted into BALB/c mice and RAG^-/- γC^-/- mice, and the fluorescence signals were detected by Xenogen IVIS（r） 50 imaging system. Results Three days after transplantation in BALB/c mice the fluorescent signal significantly decreased in the ULIP-H9 transplanted group, and no fluorescent signal could be detected on day 5. While in RAG^-/- γC%-/- mice, for the ULIP-H9 group, fluorescent signal decreased slightly on day 3, and then gradually increased. Conclution Luciferase reporter gene could be used for real-time observation of the survival of the transplanted cells in vivo.