Specificity of screening of short hairpin RNA targeting Mcl-1 gene in Raw264.7 cells
10.3969/j.issn.1000-484X.2015.02.002
- VernacularTitle:Mcl-1短发夹RNA在Raw264.7细胞内特异性的筛选
- Author:
Chan WANG
;
Xinmin WANG
;
Feiyu WANG
;
Yuqing ZHANG
;
Xudong CAO
;
Jiangdong WU
;
Fang WU
;
Wanjiang ZHANG
;
Le ZHANG
- Publication Type:Journal Article
- Keywords:
Mcl-1;
RNA interference;
Raw264.7
- From:
Chinese Journal of Immunology
2015;(2):151-155
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To transfect Mcl-1shRNA into Raw264.7 cells,and screen out specific shRNA eukaryotic expression plasmids with the most significant effect of silent Mcl-1 gene to figure out the effect of shRNA on Mcl-1 expression in murine macrophage cell line Raw 264.7.Methods: Specific shRNA was transfected into murine macrophage cell line Raw 264.7 via lipofectamine.Semi-quantitative RT-PCR and Western blot were respectively employed to test the changes in Mcl-1 mRNA level and Mcl-1 protein expressions 24 h and 48 h after the transfection ,and the silencing effects of the three pairs of specific shRNA fragments corresponding to different sites were analyzed.Results: Specific shRNA fragments at 24 h and 48 h could effectively reduce Mcl-1 mRNA and protein level ,with higher silencing effects than those of the normal group ,the lipofectamine group and the negative control group.There were statistically significant differences among them ( P<0.05 ).Among the three pairs of specific shRNA fragments corre-sponding to different sites ,Mcl-1 shRNA3 showed the most significant inhibiting effect on Mcl-1 mRNA and proteins.Conclusion:RNA interference can downregulate the level of Mcl-1 mRNA in murine macrophage cell line Raw 264.7 and greatly downregulate the expression of Mcl-1protein.Specific shRNA eukaryotic expression plasmids with the most significant effect of silent Mcl-1 gene have been screened out successfully.
