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目的为在体内研究MAN2C1的生物学意义而建立转hMan2c1基因的小鼠。方法构建pIRES2-EGFP-hMan2c1重组表达载体,经体外转染实验鉴定转染的基因能在COS-7细胞表达后,注射入ICR小鼠受精卵,以制备转基因小鼠。用基因组PCR鉴定目的基因在宿主基因组DNA的整合。用RT-PCR和Western blot分析hMan2c1在转基因小鼠的表达。结果在116只原代小鼠中,有7只hMan2cl基因组PCR阳性。在所检测的20只F1代小鼠中,有9只hMan2c1基因组PCR阳性。在所检测的21只F2代小鼠中,有16只基因组PCR阳性。用鼠尾组织RT-PCR和Western blot检测hMan2c1基因表达,确定基因组PCR阳性的7个系中有4个系阳性。结论建立了4个稳定表达hMan2c1的转基因小鼠系,为深入研究MAN2C1的生物学意义打下了基础。
The purpose of this study was to investigate the biological significance of MAN2C1 in vivo and to establish a mouse transgenic for hMan2c1. Methods The recombinant plasmid pIRES2-EGFP-hMan2c1 was constructed and transfected into COS-7 cells. The transfected cells were identified by in vitro transfection and injected into the fertilized eggs of ICR mice to prepare transgenic mice. Genome PCR was used to identify the integration of the target gene in the host genomic DNA. HMan2c1 expression in transgenic mice was analyzed by RT-PCR and Western blot. Results Among the 116 primary mice, 7 hMan2cl genotypes were PCR positive. Of the 20 F1 mice tested, 9 were positive for hMan2c1 genome. Of the 21 F2 mice tested, 16 were PCR positive. HMan2c1 gene expression was detected by RT-PCR and Western blotting in rat tail tissues, and 4 out of 7 lines confirmed positive for genomic PCR were positive. Conclusion Four transgenic mouse lines stably expressing hMan2c1 were established, which laid the foundation for the further study on the biological significance of MAN2C1.