[RNA interference in three ES cell lines from different mouse strains].

Abstract

RNA interference phenomenon in three different murine ES cell lines (MESPU13, B3, and R1) is reported. A vector(pdsGFP) was used that transcribed hairpin double-stranded RNA of GFP gene to transfect ES cells by using lipofectin. The transient transcription of dsRNA induced RNAi (RNA interference) in the ES cells. That is, the double-stranded RNA of GFP gene potently turned down the expression of the GFP gene. On the hand, the linearized plasmid pdsGFP-puro was electroporated into MESPU13 ES cells, and the expression level of GFP after puromycin screening was turned down obviously in about 30% ES cell clones; and in a few clones, the expression level of GFP was not observed under the fluorescence microscope and GFP mRNA was not detectable by RT-PCR. Further more, another vector (pdsOCT4) was constructed that transcribed double-stranded RNA of OCT-4 gene which is specifically expressed in ES cells. ES cell clones that stably integrated the vector were screened after the electrotransfection of the cells with the above construct. 51 random-selected clones were amplified and 48 of them were checked by semi-quantitative RT-PCR. In 11 of them the mRNA of OCT-4 was undetectable by RT-PCR. This means that RNAi can be used to study mammal and human gene's function in ES cell lines from different strain mice.