Regular Plasmid Enhancer Testing 師北Vector (for In Vitro Enhancer Testing)

This vector system 拿機is designed for efficien木農t analysis of mammalian ci草美s-regulatory elements in vi信機tro. Typically, a putative e土我nhancer of interest is cl我場oned into this vec刀亮tor, and the resulting constr請線uct is used to transfect mammalian ce現關ll lines of interest. Expression of a看日 downstream fluorescent or少數 chemiluminescent report我子er can then be used as a rea匠話dout of enhancer activ些可ity.

This vector system is 報劇useful for identif跳空ying enhancer eleme那熱nts, determining tissue- and spatia在物l-specificity of enhancers, c站呢omparing enhancer variants鐵機, and many other application樹銀s.

This vector can be introduced into事師 mammalian cell習刀s by conventional transfectio我動n. Delivering plasmid vectors in唱紅to mammalian cells物微 by conventional呢通 transfection is one of th錯制e most widely used procedure文厭s in biomedical research. While唱這 several sophistica去快ted gene delivery vector syste通海ms have been develope謝外d over the years such as lentivira文上l vectors, adenovirus大學 vectors, AAV vectors and piggyBac, con師聽ventional plasmid transfection remai場我ns the workhorse of 西線gene delivery in many的相 labs. This is largely due to its techn務又ical simplicity as w生土ell as good efficiency in a wide ran森兒ge of cell types. A k在見ey feature of transfection with regular讀知 plasmid vectors is that it is transien外下t, with only a very low fraction of c些作ells stably integrating the plasmid 樂道in the genome (typically less than 1風暗%).

For further information about this vect笑山or system, please refer森綠 to the papers below.

Technical simplicity: Delivering plasmid vectors into cells b中答y conventional transfectio數門n is technically straig報喝htforward, and far easi個街er than virus-based vectors w秒視hich require the p家票ackaging of live virus.

Very large cargo space:&n他請bsp;Our vector can accommodate ~30 kb 身學of total DNA. This all媽哥ows testing of large putative e家輛nhancer sequences.

Simple and sensitive readout: A visually detectable bright裡新 fluorescent protei照道n (such as TurboG區話FP) or a chemiluminescen公去t protein (such as luciferase)鐵民 is used as the reporte樹那r, resulting in highly s作秒ensitive readout of enhancer 下銀activity in vitro訊西.

Limited cell type技西 range: The efficiency of plasmid transfect花公ion can vary greatly from cell type to 男少cell type. Non-di又地viding cells are often more diffi熱作cult to transfect than dividing cells, 市坐and primary cel你算ls are often harder學為 to transfect than im務視mortalized cell 有但lines. Some important cell type拿舞s, such as neurons and pancrea花路tic β cells, ar作理e notoriously difficult to transf時都ect. Additionally體大, plasmid transfection is larg對分ely limited to in銀服 vitro applications 工花and rarely used in vivo.

Enhancer: Your enhance媽腦r of interest is placed h大上ere.

Minimal promoter: A user-selected minimal 靜銀promoter sequence is placed here年船. This will drive trans頻討cription of the d年章ownstream reporter if an enhancer el視開ement is present to acti綠唱vate it. In the absence of such enhance又票r activity, the minimal promoter妹飛 will be almost comp那但letely inactive.

Kozak: Kozak consensus sequen女年ce. It is placed in front of t弟她he start codon of the ORF of interest b花友ecause it is believed to facili笑女tate translation initiation in euk呢空aryotes.

Reporter: A visually detectab工錯le bright fluores低不cent protein gene (such a身吧s TurboGFP) or a chemiluminescent pro影她tein gene (such as lucifer筆資ase). This allows highly sensitive d視暗etection of enhancer activity i做器n vitro.

SV40 late pA: Simian virus 40 late pol技頻yadenylation signal. I高金t facilitates transcriptional t務錯ermination of the upstream 歌他ORF.

Ampicillin: Ampicillin resi器那stance gene. It 文得allows the plasmid to be 低熱maintained by ampicillin selec技雪tion in E. coli.

pUC ori: pUC origin of replication. 草睡Plasmids carrying this origin exist in 謝黑high copy numbers in 如頻E. coli.