小鼠单核巨噬细胞Ufm1基因shRNA慢病毒载体构建及鉴定

目的 构建小鼠Ufm1基因RNA干扰慢病毒载体,并实现该基因在小鼠单核巨噬细胞系（RAW264.7）中的稳定表达抑制.方法 根据小鼠Ufm1基因设计两条RNA干扰序列,合成靶序列双链DNA,与pFU-GW-RNAi载体连接,PCR筛选阳性克隆,测序鉴定.pFU-GW-RNAi载体、pHelper 1.0载体及pHelper 2.0载体共转染293T细胞包装产生慢病毒颗粒,并测定其滴度,随后感染RAW264.7细胞,建立稳定转染细胞株.将RAW264.7细胞分为空白组、阴性对照组及RNA干扰慢病毒感染组,用Real-time PCR检测Ufm1基因mRNA的表达量.结果 PCR扩增检测阳性菌落和测序证实,shRNA片段完全正确插入慢病毒载体pFU-GW-RNAi中.在荧光显微镜下观察可见,293T细胞呈绿色荧光,并测得病毒滴度为9×105 TU/μl.重组慢病毒感染RAW264.7后,用Real-time PCR检测到Ufm1基因mRNA的表达受到抑制.结论 成功构建了小鼠RNA干扰慢病毒载体,并能够在RAW264.7细胞中有效沉默靶基因.

Construction of lentiviral shRNA expression vector targeting mouse Ufm1 gene and identification of RNA interference efficiency

Objective To construct lentiviral shRNA expression vector targeting the Ufm1 gene and stably knockdown its expression in mouse monocyte macrophages cell line （RAW264.7）.Methods According to mouse Ufm1 gene sequences,two shRNA sequences were designed,synthesized and cloned into pFU-GW-RNAi plasmid to construct a recombinant plasmid.The recombinant plasmid was identified by PCR and DNA sequencing.293FT cells were co-transfected with pFU-GW-RNAi,pHelper 1.0,pHelper 2.0 to package into lentivirus particles and the titer of virus was measured according to the number of GFP＋ cells.Virus solution was collected to infect RAW264.7 cells,and the stably transfected cell line was established.The RAW264.7 cells were divided into blank group,negative control group and experimental group.The mRNA of Ufm1 gene was detected by real-time PCR.Results The PCR identification and DNA sequencing showed that the fragment and nucleotides were in accordance with the target sequence and the shRNA sequence was inserted into the pFU-GW-RNAi vector correctly.293T cells were observed in green fluorescence,and virus liter reached to 9 × 105 TU/μl.In infected RAW264.7 cells,the inhibition expressions of Ufm1 mRNA was detected by real-time PCR.Conclusion Lentiviral shRNA expression vector targeting the Ufm1 gene has been successfully constructed and it can knockdown the expression of Ufm1 in RAW264.7 cells.