Abstract：Objective To select the effective human nuclear distribution protein C(hNUDC)-specific shRNA using flow cytometry through constructing vectors pDs-NUDC-RFP-TK-U6-shNUDC-A,B,C,D,E,F and H.Methods NUDC-RFP fused gene was cloned into pDs vector,resulting in pDs-NUDC-RFP.Human U6 promoter and hNUDC-specific shRNA were cloned into pDs-NUDC-RFP vector,resulting in pDs-NUDC-RFP-TK-U6-shNUDC-A,B,C,D,E,F and H.Then 293T cells were transfected with high-quality plasmid by liposome complex method.The fluorescence intensity and the number of fluorescent cells were detected by flow cytometry.NUDC-RFP mRNA was analyzed by quantitative real-time PCR and NUDC-RFP protein was detected by Western Blot.Results The 293T cells in blank control group did not show RFP fluorescence.However,a large number of cells in negative control group showed strong expression of RFP,but the fluorescence intensity and the number of fluorescent cells were significantly reduced in 293T cells transfected with hNUDC-specific shRNAs compared with negative control group.The levels of NUDC-RFP fusion protein expression were decreased by 90% and 85% in cells transfected with shNUDC-F and cells transfected with shNUDC-A,respectively.In addition,NUDC-RFP expression was down-regulated by 62%-75% by transfection with pDs-NUDC-RFP-TK-U6-shNUDC-B,C,D,E and H,respectively.NUDC-RFP gene expression was not disrupted in negative control group.There results were consistent with real-time RT-PCR and Western Blot analysis and showed that shNUDC-F was the best effective interference fragment.Conclusion Flow cytometry analysis is an effective method for selecting shRNA vector with fluorescent marker.
廖霞；邹宇光；庞实锋；郑克勤；周汝滨；曹定国；梁朋；胡传银. 用流式细胞仪进行人核迁移蛋白shRNA有效干扰片段的分析和筛选[J]. 南昌大学学报（医学版）, 2012, 52(08): 1-.
LIAO Xia,ZOU Yu-guang,PANG Shi-feng,ZHENG Ke-qin,ZHOU Ru-bin, CAO Ding-guo,LIANG Peng,HU Chuan-yin. Analysis and Selection of Human Nuclear Distribution Protein C-specific shRNA by Flow Cytometry. , 2012, 52(08): 1-.
2012, Vol. 52 
