特异性双链RNA对小鼠干细胞和转基因荧光蛋白的抑制作用

Specific RNA Interference in Mouse ES Cells and EGFP-transgenic Mouse

目的研究特异性dsRNA对小鼠ES细胞中对核外基因和核内染色体基因的干扰作用,并对RNA干扰(RNAi)技术在皮肤病基因治疗领域中应用的可能性进行初步探讨。方法选择荧光蛋白基因为靶基因,通过质粒或RNA直接转染技术,将荧光蛋白双链RNA(dsRNA-EGFP)转染小鼠胚胎干细胞(ES细胞),从而观察该细胞中特异性RNAi作用。并通过皮肤局部注射dsRNA-EGFP和局部外用脂质体包裹的dsRNA-EGFP,观察EGFP转基因鼠皮肤EGFP的表达。结果无论是转染质粒产生的dsRNA-EGFP或是直接转染的dsRNA-EGFP,均明显抑制ES细胞核外或染色体中EGFP基因的特异性表达。局部皮肤注射或皮肤外用脂质体-dsRNA-EGFP均特异性抑制EGFP转基因鼠皮肤EGFP的表达。结论本研究证实了小鼠ES细胞中特异性RNAi的作用。

Objective To investigate the feasibility of RNA interference(RNAi)strategy in mouse(embryonic stem)ES cells and study the possibility of RNAi in dermatological research.Methods Specific RNAi was introduced with EGFP gene as a target,either by in situ production of dsRNA from transient trans-fection of a plasmid harboring a547bp inverted repeat,or by direct transfection of dsRNA made by in vitro transcription.Gene silence of EGFP in EGFP-transgenic mouse was investigated by topical delivery of dsRNA.Results This longer dsRNA was capable of inducing a sequence-specific RNAi for the target gene in epi-some and in chromosome of ES cells.It was also shown that this sequence-specific RNAi was observed in EGFP-transgenic mouse by topical delivery of dsRNA.Conclusion These results suggest that ES cell possess RNAi activity by longer dsRNA.We first report the study on RNAi activity by topical delivery of specific dsRNA.Above findings offer a possibility to use dsRNAi for inhibition of gene expression in dermatological re-search.