Name: quantitative comparison of cis-regulatory element (cre) activities in transgenic drosophila melanogaster
Uploaded: 2011-12-19T12:00:00
Description: Watch this Scientific Journal Video about Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster at JoVE.com

Vi takker: Nicolas Gompel og Benjamin Prud&#39;homme for deres bidrag til udviklingen af ​​denne protokol Melissa Williams og fire anonyme anmeldere for kommentarer til manuskriptet, University of Dayton Graduate School for forskning, stipendier til krig, og University of Dayton Biologi Institut og Research Institute (UDRI) for forskning støtte til TMW. Dette arbejde blev støttet af American Heart Association Grant 11BGIA7280000 til TMW.

Oversigt: Denne video viser en protokol i stand til kvantitativt at måle genet regulerende aktiviteter for cis-regulerende element (CRE) sekvenser i Drosophila (D.) melanogaster. Denne protokol kan bruges til at sammenligne de lovgivningsmæssige aktiviteter i besiddelse af: vild type og mutant CRE former, naturligt forekommende CRE alleler findes inden for en art, eller orthologous Cres mellem afveg arter. 1. Site-specifik integration af journalist transgen...

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cis-regulerende elementer encode det genomiske program, der angiver genekspression mønstre og dermed processen med udvikling 1, og er prominente steder for både mutationer underliggende morfologiske udvikling 2-4 og fænotypiske variation for menneskelige træk 5,6,19. På trods af denne betydning forbliver den lovgivningsmæssige logik for Cres dårligt forstået. En fremtrædende årsag til denne forståelse underskud har været manglen på egnede metoder til kvantitativt at...

quantitative comparison of cis-regulatory element (cre) activities in transgenic drosophila melanogaster

Genekspression mønstre er specificeret af cis-regulerende element (CRE) sekvenser, som også kaldes smagsforstærkere eller cis-regulerende moduler. En typisk CRE besidder et arrangement af bindingssteder for flere transskription faktor, proteiner, der giver en regulerende logik, der angiver, hvornår, hvor og på hvilket niveau de regulerede gen (s) er udtrykt. Det fulde sæt af Cres inden for et dyr genom koder organismens program for udvikling 1, og empiriske og teoretiske undersøgelser viser, at mutationer i Cres spillet en fremtrædende rolle i morfologiske udvikling 2-4. Desuden menneskelige genom bred forening undersøgelser viser, at den genetiske variation i Cres bidrage væsentligt til fænotypisk variation 5,6. Således, forståelse lovgivningsmæssige logik og hvordan mutationer påvirker en sådan logik er et centralt mål for genetik. Reporter transgener give en kraftfuld metode til at studere in vivo-funktionertion af Cres. Her en kendt eller mistænkt CRE sekvens er koblet til heterologe promotor og kodningssekvenser for en reporter gen, der koder en let observerbar protein produkt. Når en journalist transgen er indsat i en værtsorganisme, at CRE aktivitet bliver synlig i form af den indkodede reporter protein. P-element medieret transgenese i bananfluen arten Drosophila (D.) melanogaster 7 er blevet brugt i årtier til at indføre reporter transgener i denne model organisme, selvom det genomiske placeringen af transgener er tilfældig. Derfor er reporter geners aktivitet stærkt påvirket af den lokale kromatin og gen-miljø, der begrænser CRE sammenligninger til at være kvalitative. I de senere år var phiC31 baseret integration, som er tilpasset til brug i D. melanogaster at indsætte transgener i specifikke genom landingssteder 8-10. Denne mulighed har gjort kvantitativ måling af gen-og, relevant her, CRE aktivitet 11-13 feasible. Produktionen af ​​transgene bananfluer kan outsources, herunder phiC31-baseret integration, hvilket eliminerer behovet for at købe dyrt udstyr og / eller have samme færdigheder på specialiserede transgenet injektion protokoller. Her præsenterer vi en generel protokol til kvantitativt evaluere en CRE aktivitet, og vise, hvordan denne tilgang kan bruges til at måle virkningerne af en introduceret mutation på en CRE aktivitet og til at sammenligne aktiviteter orthologous Cres. Selv om de anførte eksempler er for en CRE aktive under frugtflue metamorfose, kan den fremgangsmåde anvendes på andre udviklingsstadier, frugt flyve arter, eller model organismer. I sidste ende bør en mere udbredt anvendelse af denne metode til at studere Cres fremme en forståelse af lovgivningsmæssige logik og hvordan logik kan variere og udvikle sig.

Watch this Scientific Journal Video about Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster at JoVE.com

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Fænotypiske variation for egenskaber kan skyldes mutationer i cis-regulerende element (CRE) sekvenser, der kontrollerer genekspression mønstre. Metoder afledt til brug i Drosophila melanogaster kan kvantitativt sammenligne niveauet af rumlige og tidsmæssige mønstre af genekspression medieret ved ændret eller naturligt forekommende CRE varianter.

Kvantitativ sammenligning af Cis-Regulatory Element (CRE) Aktiviteter i transgene Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

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Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

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