[RNA interference in three ES cell lines from different mouse strains].
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.
Meng
Guo-Liang
GL
College of Life Sciences, Peking University, Beijing 100871, China.
Tang
Fu-Chou
FC
Zhang
Jun-Zheng
JZ
Zhao
Wen-Ning
WN
Shang
Ke-Gang
KG
Ding
Ming-Xiao
MX
Xue
You-Fang
YF
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008815:Mice, Inbred Strains; D008856:Microscopy, Fluorescence; D050814:Octamer Transcription Factor-3; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D012330:RNA, Double-Stranded; D014157:Transcription Factors; D049452:Green Fluorescent Proteins