Presentation on theme: "Microinjection of mRNA and DNA"— Presentation transcript:
1 Microinjection of mRNA and DNA Mary Mullins, UPennWhy inject mRNA?Gain-of-function experiments (today): over & ectopic expressionDominant negative (pseudo loss of function) exptsMutant (today) or morpholino (tomorrow) rescueEpistasis: what genes can and cannot rescue your mutant?Where does your mutant gene lie in a pathway?Why inject DNA?Gain-of-function experiments: over & ectopic expressionDominant negative exptsPromoter analysisTransgenics--in vivo labelling of subsets of cells or subcellular domains,promoters, ectopic expression (UAS/Gal4, heat shock, Cre-lox).Why? A few reasons include:…..When mRNA and when DNA?1. mRNA gives reliable widespread expression. HOWEVER, it’s short-lived:8-14h.2. DNA persists much longer, can be tissue-specific, BUT gives mosaic exp.
2 A BMP Activity Gradient in D-V Pattern Formation BMP antagonistsBMPWild typeDVDorsalizedmutantsVD
3 Dorsalization and Zebrafish Fate Map Class 4 and 5 dorsalizationstailVDyolk
15 Inject through the chorion --Advantage:embryos remain protected by chorion after injection, good survival--Disadvantage: Need a good needle to penetrate the chorion and not break needleAgar troughsNOTE orientation of wells relative to needle: straight side to left, needle to right.Important--very little media in trough, so chorion exposed above media.The surface tension keeps embryo in well, when pull out needle.Otherwise when try to withdraw needle, embryo comes with it.Keep agar plate for tomorrow. Seal with parafilm and put into refrigerator.
16 Ventralized phenotypes Hammerschmidt and Mullins, 2002Overexpression phenotypes of bmp4