中文摘要：

【目的】分离用于禽源细胞中表达短干扰RNA（short interference RNA，SiRNA）的禽U6启动子。【方法】以鸡基因组为模板进行PCR扩增，将扩增产物进行序列测定和生物信息学分析，并将其插入报告基因载体pEGFP-N1中，获得siRNA表达载体pGFP—U6，将由软件预测针对GFP基因的SiRNA所对应的shRNA（shorthairpin RNA）插入其中，获得pGFP—U6-shRNA；以含人H1启动子的siRNA表达载体pGFP—H1—shRNA为对照，分别转染COS-1和DF-1细胞，用荧光显微镜和流式细胞仪测定转染细胞培养中GFP阳性细胞数和荧光总量。【结果】克隆的U6启动子位于鸡28号染色体，与发表的鸡U6—3启动子同源性为97．2％，含多个0ct-1序列，不含CACCC框、SPH和PSE等聚合酶Ⅲ启动子序列，TA框不典型；在pGFP-U6-shRNh转染的哺乳动物源COS-1细胞培养中，GFP阳性细胞数和荧光总量均有所下降，但不如pGFP-H1-shRNA转染细胞显著；在pGFP—U6-shRNA转染的禽源DF-1细胞培养中，不仅GFP阳性细胞数和荧光总量显著下降，而且基因沉默效果显著优于pGFP—H1-shRNA转染细胞。【结论】成功克隆的禽U6启动子不仅能有效转录SiRNA，而且转录活性具有相对的细胞种属特异性。

英文摘要：

[ Objective ] To cloning avian U6 promoter for construction of short interference RNA （siRNA） expression vectors in avian cells. [Method] A putative chicken U6 promoter from chicken genomic DNA was amplified by PCR. After sequencing and bioinformatics analysis, the putative promoter was inserted into the reporter plasmid pEGFP-N1 together with GFP-specific short hairpin RNA （shRNA） and resulted in a siRNA expression vector pGFP-U6-shRNA. Then, using human H1 promoter-driven siRNA expression vector pGFP-H 1-shRNA as the control, the simian-originated COS-1 cells or chicken embryonic fibroblast （DF-1） cells were cotransfected with the siRNA expression vector pGFP-U6-shRNA, and the GFP-positive cell numbers and total fluorescence were detected by fluorescence microscopy and flow cytometry. [ Result ] The PCR product was 560bp long and located on chicken chromosome 28 with a percentage identity of 97.2% to the published chicken U6-3 promoter. Bioinformatics analysis showed that the putative chicken U6 promoter contained several Oct-1 motifs and a weak TATA box without other Pol Ⅲ promoter elements. In COS-1 cells, transfection with pGFP-U6-siRNA resulted in significant decreases in both the number of GFP-positive cells and total fluorescence, but significance was lower than that of transfection with pEGFP-H1-shRNA. While in DF-1 cells, transfection with pGFP-U6-shRNA resulted in significantly higher gene silencing effect than cotransfection with pEGFP-HI-shRNA. [ Conclusion] These data suggest that a chicken U6 promoter was successfully cloned, which can efficiently transcribe gene-specific siRNA expression in avian cells.