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背景与目的:活体动物体内光学成像(optical in vivo imaging)主要采用生物发光与荧光两种技术。生物发光是用荧光素酶(luciferase,Luc)基因标记细胞或DNA,而荧光技术则采用荧光报告基团(GFP、RFP、Cyt及dyes等)进行标记,利用一套非常灵敏的光学检测仪器,能够直接监控活体生物体内的细胞活动和基因行为,生物发光成像具有高的灵敏度和特异性,同时生物发光信号可用于精确定量,而荧光成像具有方便、便宜、直观、标记靶点多样和易于被大多数研究人员接受的优点。本研究基于慢病毒介导的转基因方法制备红色荧光蛋白(red fluorescent protein,RFP)和Luc双报告基因转基因小鼠(即RL转基因小鼠),将这两种技术融为一体。方法:制备携带RFP和Luc基因(简写RL基因)的慢病毒,然后将携带RL基因的慢病毒注入小鼠单细胞受精卵卵周隙以感染受精卵,胚胎移植进假孕母鼠以获得仔鼠,应用小动物活体成像仪、体视荧光显微镜和PCR等在蛋白和DNA水平上筛选和鉴定,并获得RL转基因小鼠。结果:移植卵周隙注射有慢病毒的胚胎125枚给6只假孕母鼠,其中4只假孕母鼠怀孕,共生仔鼠20只;利用小动物活体成像仪检测RFP和Luc表达,在蛋白水平证实20只F0代中,3只高表达RFP和Luc;DNA水平检测证实,3只RFP和Luc阳性的小鼠基因组中确实整合有外源转基因RL,预示基因型鉴定结果很好验证了小动物活体成像仪筛选和鉴定结果。此外,RL转基因首建鼠基因组中整合的RL转基因可稳定遗传至下一代,并能正常表达。RL转基因小鼠主要脏器均可见红色荧光和Luc信号,但不同脏器间荧光和Luc强度有差异。结论:成功制备RL双报告基因转基因小鼠,为后续研究干细胞在肿瘤发生、发展和转移中的作用和造血重构等提供双报告基因标记的各种移植用供体细胞,并对此供体细胞及其在体内衍生的细胞进行灵敏的非损伤、实时可视化体内跟踪。
BACKGROUND & OBJECTIVE: In vivo imaging of living animals mainly uses bioluminescence and fluorescence. Bioluminescence uses luciferase (Luc) gene to label cells or DNA. Fluorescence technology uses fluorescent reporter groups (GFP, RFP, Cyt and dyes, etc.) for labeling. Using a set of very sensitive optical detection instruments, Direct monitoring of cellular activity and gene behavior in living organisms, bioluminescence imaging with high sensitivity and specificity, and bioluminescent signals for precise quantitation, while fluorescence imaging is convenient, inexpensive, intuitive, has multiple targets and is easily Most researchers accept the advantages. In this study, red fluorescent protein (RFP) and Luc dual reporter transgenic mice (RL transgenic mice) were prepared based on the lentivirus-mediated transgene method and the two technologies were integrated. Methods: Lentivirus carrying RFP and Luc genes (abbreviated RL gene) was prepared, then the lentivirus carrying RL gene was injected into the perivitelline space of mouse single-cell zygotes to infect zygotes, and embryos were transplanted into pseudopregnant mice to obtain Mouse, small animal in vivo imager, stereo fluorescence microscopy and PCR and other protein and DNA screening and identification, and obtain RL transgenic mice. Results: 125 embryos injected with lentivirus were transplanted into perivitelline space to 6 fake pregnant rats, of which 4 fake pregnant rats were pregnant and 20 symbiotic pups were born. RFP and Luc expression were detected by live animal imager. Protein levels confirmed that of the 20 F0 generation, 3 were high expression of RFP and Luc; DNA level test confirmed that 3 RFP and Luc positive mouse genome does integrate exogenous gene RL, indicating that genotype identification results are well verified Screening and identification of small animal live imagers. In addition, the RL transgene, which has been integrated into the genome of the RL transgenic mouse, can stably inherit into the next generation and can be normally expressed. The main organs of RL transgenic mice showed red fluorescence and Luc signal, but there were differences in fluorescence and Luc intensity between different organs. CONCLUSION: Transgenic RL double reporter gene transgenic mice were successfully prepared, which can provide double reporter gene-labeled donor cells for follow-up study of the function of stem cells in tumorigenesis, development and metastasis and hematopoietic reconstruction. Cells and their in vivo-derived cells are sensitively non-invasive, real-time visualized in vivo tracking.