摘要
目的构建一种带有DsRed红色荧光蛋白报告基因并可经RU486诱导的真核表达载体,并在体外验证其调控表达作用。方法利用分子生物学技术,将DsRed基因和启动子,以及RU486系统构建成真核可调控载体pRS17-RUDsRed。在转染MFC细胞后,运用荧光显微镜和流式细胞技术检测该载体的调控表达。结果PCR和限制性酶切及测序均证实了载体的正确性。没有RU486时,几乎没有红色荧光蛋白的表达,加入诱导剂RU486后,可以实现红色荧光蛋白的高效表达。结论成功构建了带有红色荧光蛋白报告基因并可经RU486诱导的真核表达载体,实现了对目的基因表达的有效调控,为进一步的基因调控研究和和基因治疗奠定了基础。
Objective To construct an inducible eukaryotic vector carrying red fluorescent protein （DsRed） and evaluate the regulation ofDsRed gene expression in vitro. Methods The vector pRS17-RUDsRed containing DsRed gene, promoter and RU486-inducible system was constructed using molecular biological methods. To minimize potential interference, the two transcriptional elements were spaced with a 1.6 kb insulator. Fluorescence microscopy and flow cytometry were used to observe the activation of this regulatable vector after transfection in MFC cells. Results The vector was identified by digestion with different restriction enzymes, sequencing and PCR. In the absence of RU486, the ceils transfected with the vector exhibited very low DsRed protein expression, and the addition of RU486 induced efficient DsRed expression in the cells. Conclusion The RU486-inducible eukaryotic vector carrying DsRed protein allows effective regulation of the target gene expression in vitro, which provides a useful tool for gene regulation and gene therapy studies.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2008年第12期2113-2116,共4页
Journal of Southern Medical University
基金
国家自然科学基金(30571830)
