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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pGL3 base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Xenogen IVIS(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。
Objective To use lentiviral vector to carry luciferase reporter gene and transduce human embryonic stem cells in order to observe the proliferation of transplanted cells in vivo. Methods The luciferase gene was digested by restriction endonuclease pGL3 base and the vector was digested by LTBR-GIP. After heat inactivation, blunt end treatment, CIP treatment, gel purification and ligation, the constructed lentiviral vector was named ULIP , Including UBC promoter and IRES, can express luciferase gene and Puromycin resistance gene simultaneously. Human embryonic stem cells H9 were transduced with ULIP and stably transduced cells were screened with Puromycin. Subsequently, BALB / c mice and RAG - / - γC - / - mice were transplanted for fluorescent signal detection on a Xenogen IVIS (R) 50 imaging system. Results The H9 cells transfected with ULIP or LTBR-GIP were transplanted into BALB / c mice. Fluorescence signal of the ULIP group decreased significantly after 3d and basically could not be detected after 5 days. After transplanted into RAG - / - γC - / - mice, the fluorescence signal of ULIP group decreased slightly on day 3, and then the fluorescence signal gradually increased. No fluorescence signal was detected in LTBR-GIP-H9 after transplanted BALB / c mice and RAG - / - γC - / - mice. Conclusion The luciferase reporter gene can be used to observe the transplanted cells in vivo and to observe the survival of transplanted cells in real time.