Introduction to Reporter Gene Assays

reporter gene assays are used to study signaling pathways gene regulation and the structure of regulatory elements in its simplest form a reporter essay consists of a regulatory element of interest cloned into a vector with a reporter gene the reporter gene encodes a protein with an easily measurable activity we will be using the bioluminescent activity of luciferase for our reporter upstream regulatory elements affecting expression of the reporter gene can include potential promoters known promoters partial promoters and enhancers reporter assays can also be used to study elements in the three prime untranslated region like micro RNA recognition elements or MREs now that you've created a construct with your regulatory element and a reporter gene the next step is transfecting cells when studying upstream elements the messenger RNA is transcribed from active promoters experimental conditions can modulate the activity of the promoter weak promoters make les messenger RNA while strong promoters make more messenger RNA messenger RNA is translated into active protein so the amount of luciferase is dependent on the activity of the regulatory element in the case of micro RNA recognition elements the signal depends on the binding of micro RNA to the three prime untranslated region blocking translation in animal cells or leading to messenger RNA degradation in plants how do you correct for variables in your reporter essay like cell number and transfection efficiency dual luciferase reporter assays for a dual luciferase assay transfect with a control plasmid constitutively expressing a second luciferase the ratio of experimental luciferase activity to control luciferase activity corrects for variables in your reporter assay per mega supplies three different lucifer aces for reporter assays firefly lucifer ace is great for routine genetic reporter assays Ranil elusive phrase is used as a control and nano luck Lucifer ace a smaller brighter Lucifer ace best suited for research and challenging biological systems can be used as a single reporter or as a primary or secondary reporter in the nano dlr assay

Loading