人Man2c1转基因小鼠模型的建立

Preparation of hMan2c1 transgenic mice

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。

Objective To develop hMan2cl transgenic mice for studying the biological significance of MAN2C1 in vivo. Methods Recombinant expression plasmid plRKS2-EGFP-hMan2c1 was constructed. The expression of pIRKS2-EGFP-hMan2c1 was indicated in COS-7 cells and then injected into the zygotes of ICR mice to prepare transgenic mice. Genomic PCR was performed to analyze integration of the target gene into the genomic DNA of the host. RT-PCR and Western blot were performed to detect expression of hMan2c1 in the transgenic mice. Results Out of the 116 first generation mice, 7 were genomic PCR positive for hMan2c1. Out of the 20 F1 mice examined, 9 were genomic PCR positive for hMan2cl. Out of the 21 F2 mice detected, 16 were genomic PCR positive for hMan2c1. RT-PCR and Western blot for hMan2c1 expression in mouse tail tissue showed that 4 were positive in the 7 transgenic mouse hnes. Conclusion Four hMan2c1 transgenic mouse lines have been developed, which can be used for studying the biological significance of MAN2C1 in vivo.