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目的:将转染了人突变dhfr基因的第二代小鼠骨髓,移植给经致死剂量照射的第三代小鼠,观察该基因对小鼠造血功能的长期保护作用.方法:分离存活的第二代小鼠骨髓有核细胞,直接移植给经致死剂量照射的同系小鼠,以MTX筛选,观察小鼠血象和生存率变化,PCR和Southem印迹杂交分析目的基因在小鼠染色体DNA中的整合与表达情况.结果:在大剂量MTX筛选下,实验组小鼠造血功能逐渐恢复,对照组3周内全部死亡.实验组生存率和生存期明显高于对照组,但较前两代生存率低.PCR和Southern印迹分析结果提示,实验组脾脏和肝脏组织中均检测到前病毒标志基因neo~R和dhfr基因的特异务带.结论:转染了人突变dhfr基因的第二代小鼠骨髓,能有效地重建经致死剂量照射的第三代小鼠造血功能.保护骨髓免遭大剂量MTX所致的严重骨髓抑制,dhfr基因在小鼠基因组DNA中的稳定整合是这种长期保护作用的物质基础.
OBJECTIVE: To transplant the second-generation mouse bone marrow transfected with the human mutated dhfr gene into a third-generation mouse exposed to lethal doses, and to observe the long-term protective effect of the gene on hematopoietic function in mice. Second-generation mouse bone marrow nucleated cells were directly transplanted into lethally irradiated syngeneic mice and screened with MTX to observe changes in blood counts and survival rates. PCR and Southern blot analysis were performed to analyze the integration of the target gene in mouse chromosomal DNA. Results of expression: Under high-dose MTX screening, the hematopoietic function of the experimental group gradually recovered, and all the control group died within 3 weeks. The survival rate and survival period of the experimental group were significantly higher than the control group, but the survival rate of the previous two generations. Low PCR and Southern blot analysis indicated that the pro-viral marker genes neo~R and dhfr were detected in the spleen and liver tissues of the experimental group. Conclusion: The second generation mouse with the human mutated dhfr gene was detected. Bone marrow can effectively reconstitute hematopoietic function in third-generation mice exposed to lethal doses. It protects bone marrow from severe myelosuppression caused by large doses of MTX. The stable integration of dhfr gene in mouse genomic DNA is such long-term protection. A material basis.