大鼠Mettl9基因的克隆及其慢病毒表达载体的构建

Cloning of Rat Mettl9 Gene and Construction of Its Lentiviral Expression Vector

目的:克隆大鼠Mettl9基因,并构建慢病毒表达载体,为研究Mettl9基因功能奠定基础。方法:提取原代培养大鼠星形胶质细胞的总RNA,应用RT-PCR方法,扩增出Mettl9基因的cDNA,克隆至pGEM-T载体上并测序;再将该基因亚克隆至慢病毒载体pLenti6.3-MCS-IRES-EGFP,测序鉴定,利用脂质体lipofectamine 2000转染至293T细胞进行包装,感染U251细胞,荧光显微镜下观察其表达。结果:Mettl9 cDNA的RT-PCR扩增产物为954 bp的基因片段,连接到pGEM-T载体后测序结果向GenBank递交获得收录号HQ898855;构建的pLen-ti6.3-Mettl9-IRES-EGFP慢病毒载体在293T细胞完成包装,测得慢病毒滴度达1.825×108TU/mL;经感染U251细胞,荧光显微镜下可见绿色荧光。结论:成功克隆了SD大鼠Mettl9基因,构建了该基因的慢病毒表达载体pLenti6.3-Mettl9-IRES-EGFP。

Objective ： The Mettl9 gene of rat was cloned and its lentiviral expression vector was constructed in order to investigate the function of Mettl9 gene. Methods： Total RNA from the primatcultured rat astrocytes was extracted. Then the Mettl9 eDNA was amplified by RT-PCR and cloned into the pGEM-T vector. After confirmed by sequencing, Mettl9 gene was subcloned into lentiviral vector pLenti6.3-MCS-IRES-EGFP, sequenced and transfected into 293T cells for virus wraps using lipofectamine 2000. To verify the efficiency of the vector, U251 cells were infected with it and Met- tl9 gene expression was observed under the fluorescence microscope. Results：Mettl9 eDNA amplified product by RT- PCR is 954 bp, and its pGEM-T vector sequencing was numbered HQ898855 from GenBank. Lentiviral vector pLen- ti6.3-Mettl9-IRES-EGFP was packaged in 293T ceils, and the lentiviral virus titer was measured up to 1. 825 x l0s TU/mL. Green fluorescence can be found in U251 cells infected with this Lentiviral vector under fluorescence microscope.Conclusion ： We successfully cloned Mettl9 gene from SD rats and constructed the lentiviral expression vector pLenti6.3- Mettl9-IRES-EGFP.