Regular Plasmid 現月Promoter Testing V吧一ector (for In Vitro Pr校靜omoter Testing)木自

This vector system is designed for e近路fficient analysis of mammalian p飛們romoters in vitro.&n跳行bsp;Typically, a putativ化生e promoter of interest is 老了cloned into this vect就著or, and the resulti那秒ng construct is used t們歌o transfect mamma秒喝lian cell lines of interest. Expression得筆 of a downstream fluoresc得報ent or chemiluminescent report數村er can then be used a理他s a readout of enhancer a服相ctivity.

This vector system is useful for ide什光ntifying promoter elements, 綠花determining tissue-specificity of prom作草oters, comparing promo們樂ter variants, and many other議國 applications.

This vector can be int公務roduced into mammalian ce相兵lls by conventional transfection. D山兒elivering plasmid vec件爸tors into mammal喝還ian cells by conventional transf他日ection is one of the most widel熱說y used procedures in biomedical researc校土h. While several sophisticated gene de草票livery vector systems have be農志en developed over the years such a歌拿s lentiviral vectors, adenovirus vecto坐匠rs, AAV vectors and piggyBac,服計 conventional plasmid transfection re路生mains the workhorse of gene deli聽知very in many labs. This is largely due場線 to its technical simplicity房通 as well as good e年是fficiency in a wide ra司離nge of cell types. A key feature of tr年我ansfection with regular plas問吃mid vectors is that it is transien男低t, with only a very low fra議和ction of cells st醫民ably integrating the plasmid in t個店he genome (typically less than 1%).

For further informatio錯習n about this vector system, please refe作物r to the papers舊光 below.

Our vector is based on a r務跳egular plasmid syste討資m. The putative promoter to be teste路術d is placed immediately ups知自tream of a reporter gene. Wh行聽ile an active promoter wo麗秒uld drive the expression音知 of the downstream reporter 謝國gene, in the absence of prom唱暗oter activity there姐見 will little or no re間去porter gene expression. A visually d店小etectable bright照醫 fluorescent protein (such as Tu窗劇rboGFP) or a chemi算區luminescent protein (such 雪老as luciferase) is&問村nbsp;used as the reporter, which all舊嗎ows highly sensitive detection o車知f promoter activity in vitro計事.

Technical simplicity: Delivering plasmid vectors into為身 cells by conventional transf金算ection is technic市明ally straightforward, an拿校d far easier than virus-b人弟ased vectors which requ有工ire the packaging of live著如 virus.

Very large cargo space: Our vecto的校r can accommodate ~30 kb of total DNA厭慢. This allows testing of工懂 large putative promo黑拍ter sequences.

Simple and sensi動們tive readout: A visually detec道熱table bright fluorescent p業書rotein (such as Turbo慢信GFP) or a chemilumi離離nescent protein (such as 科那luciferase) is used as the reporter, r電書esulting in highly sensitive rea女山dout of promoter activity in vitro.

Limited cell type 來靜range: The efficiency of plasmid 草放transfection can vary 文微greatly from cell爸民 type to cell type. Non錯道-dividing cells are often mo城少re difficult to transfect than dividing樹服 cells, and primary 習靜cells are often hard妹謝er to transfect than immortalize醫照d cell lines. Some important cell typ醫長es, such as neurons an的拿d pancreatic β cells, are麗裡 notoriously difficult 答說to transfect. Addition秒開ally, plasmid transfection is largely l樂場imited to in vitro applica謝們tions and rarel草男y used in vivo.