两种真核启动子体内外转录活性比较研究

Transcriptional Activity Assessment of Two Different Eukaryotic Promoters In Vitro and In Vivo

启动子作为基因工程表达载体的核心组成部分,在很大程度上决定了目的基因的转录活性。为筛选出一个转录活性较高的真核启动子,以目的蛋白与增强型绿色荧光蛋白融合表达的形式,在体内外检测CMV和CAG两种真核启动子的转录活性。首先,通过常规分子生物学技术将神经钙黏着蛋白(N-cad)克隆到p CAG-MCS-EGFP质粒中(携带CAG启动子),菌落PCR、双酶切及测序验证;与p EGFP-N-cad质粒(携带CMV启动子)一起通过磷酸钙法分别转染HEK293T细胞,比较两种质粒在体外细胞水平转染目的基因效率;随后,通过已建立的鸡胚脊髓活体电转方法比较两种质粒在活体鸡胚脊髓内转录目的基因效率,冷冻切片后进行荧光强度观察;而体内外实验均应用常规Western blot和RT-PCR技术验证目的基因N-cad在蛋白及基因水平上的表达。荧光显微镜观察结果表明,CMV启动子仅能在体外细胞水平上行使其转录活性,而CAG启动子可在体内体外高效行使其转录活性,进一步的Western blot和RT-PCR结果与荧光观察结果一致。该研究结果可为体内研究目的基因功能时的载体构建提供借鉴。

As a key element of a plasmid, the promoter determines the transcriptional activity of the exogenous genes. In order to screen for the effective promoter inside plasmid used in studying gene function, the transcriptional activity of two commonly used promoters, CMV and CAG were compared in vitro and in vivo by the fluorescence intensity of enhanced green fluorescent protein （EGFP）. Firstly, the target gene N-cadherin （N-cad） was cloned into the plasmid ofpCAG-MCS-EGFP, and confirmed by colony PCR, double digestion and sequence. Subsequently, these two plasmids （pEGFP-N-cad and pCAG-N-cad-EGFP） were transfected into HEK293T in vitro by calcium phosphate coprecipitation technique, and imaged under a fluorescence microscope, then the expression of N-cad was confirmed by Western blot and RT-PCR. Thirdly, the two plasmids were transfected into chicken embryonic spinal cord by electroporation, and imaged under a stereo fluorescence microscope. Finally, the EGFP positive spinal cords were selected and confirmed by frozen section under a fluorescence microscope, Western blotand RT-PCR. The results showed that these two promoters had equivalent transcriptional activity in vitro, but the artificially designed CAG promoter could effectively drive target gene expression in vivo when compared to the CMV promoter that originated from cytomegalovirus.