Name: dna vector-based rna interference to study gene function in cancer
Uploaded: 2012-06-04T13:00:00
Description: Watch this Scientific Journal Video about DNA Vector-based RNA Interference to Study Gene Function in Cancer at JoVE.com

Dette arbejde blev delvist støttet af de forskningsstipendiat Tilskud (116.403-RSG-09-082-01-MGO) fra American Cancer Society og Egne fonde for Wake Forest University Health Sciences til GS. DBS blev støttet af NCI træning tilskud 5T32CA079448.

1. Generering af shRNA konstruktioner <ol><li> Identificer et siRNA-målsekvens (20-23 nukleotider) baseret på tidligere offentliggjorte kriterier 6 eller bruge en web-baseret algoritme server, såsom &quot;siRNA Target Finder&quot; fra Ambion og GenScript. </li><li> Syntetisere oligonukleotider baseret på udformning i figur 1 og eksempel sekvenserne i tabel 1. Udføre PCR-opformering ved anvendelse af oligonukleotider P1 og P2 (30 cyklusser af denaturering ved 94 ° C i ...

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Denne protokol beskrives en metode til at vælte et gen under anvendelse shRNA og visualisere den biologiske virkning ved hjælp af bioluminescerende billeddannelse af tumorcellevækst in vivo. Målstedet for en shRNA bør ikke indeholde nogen af de tre restriktionssteder (BamHI, HindIII og EcoRI), der anvendes til subkloning. En sjælden scenario, når som helst af disse sites er til stede, kan en yderligere restriktionsenzym anvendes til at erstatte det i generering af konstruktionen. <p class="jove_conte...

Ud over almindelige laboratorieudstyr anvendt til RNA-og protein analyser og cellekultur, er de følgende materialer og reagenser kræves til denne protokol. <table border="1"><tbody><tr><td> Navn på reagenset </td></tr><tr><td> Biosikkerhed Kabinet egnet til biosikkerhedsniveau 2 inddæmning </td></tr><tr><td> PCR termocyklusapparat </td></tr><tr><td> Ultracentrifuge </td></tr><tr><td> Anæstesi-induktion kammer med isofluran scavengers </td></tr><tr><td> Digital skydelære </td></tr><tr><td> IVIS billeddannelsessystem </td></tr><tr><td> Meget kompetente DH5a E. coli </td></tr><tr><td> EcoRI, BamHI, og HindIII-restriktionsenzymer </td></tr><tr><td> T4 DNA-ligase </td></tr><tr><td> Tredje generation lentivirusinfektioner emballage plasmider VSV-G, pRSV-Rev og pMDLg / pRRE </td></tr></tbody></table> Materialer List

dna vector-based rna interference to study gene function in cancer

RNA-interferens (RNAi) hæmmer genekspression ved specifikt at nedbryde target mRNA. Siden opdagelsen af dobbeltstrenget lille interferens RNA (siRNA) i gen-nedregulering 1 har RNAi blevet et stærkt forskningsværktøj i genfunktion undersøgelser. Sammenlignet med genetisk deletion har RNAi-medieret gen-nedregulering mange fordele, såsom lethed, hvormed den er udført og dets egnethed til de fleste cellelinjer. Flere undersøgelser har påvist anvendelser af RNAi teknologi i kræftforskning. Navnlig har udviklingen af DNA-vektor-baseret teknologi til at producere små hårnålen RNA (shRNA) drevet af U6 eller H1 promotor fremstillet langvarig og inducerbare gen-nedregulering muligt 2,3. Dets anvendelse i kombination med gensplejsede virusvektorer, såsom lentivirus, letter høje virkningsgrader af shRNA levering og / eller integration i genomisk DNA for stabil shRNA ekspression. Beskriver vi en detailed under anvendelse af DNA-vektoren er baseret RNAi teknologi til at bestemme genfunktion, herunder konstruktion af lentivirale vektorer, der udtrykker shRNA, lentivirus produktion og celleinfektion, og funktionelle undersøgelser under anvendelse af en muse-xenotransplantat-model. Forskellige strategier er blevet rapporteret at generere shRNA konstruktioner. Protokollen beskrevet her anvender PCR-amplifikation og en 3-fragment-ligering kan anvendes til direkte og effektivt at generere shRNA indeholdende lentivirale konstruktioner uden at efterlade nogen ekstra nukleotid støder op til en shRNA kodende sekvens. Idet shRNA-ekspressionskassetterne er skabt af denne strategi kan skæres ud af restriktionsenzymer, kan de let flyttes til andre vektorer med forskellige fluorescerende eller antibiotiske markører. De fleste kommercielle transfektionsreagenser kan anvendes i lentivirus produktionen. Men i denne rapport, giver vi en økonomisk metode ved hjælp af calciumphosphat nedbør, der kan opnå over 90% transfektionseffektivitet i293T-celler. Sammenlignet med konstitutive shRNA ekspressionsvektorer, en inducerbar shRNA system er især egnet til at vælte et gen afgørende for celleproliferation. Vi viser genet undertrykkelse af Yin Yang 1 (YY1), en potentiel oncogen i brystkræft 4,5, med en Tet-On inducerbare shRNA systemet og dets virkninger på tumordannelse. Forskning ved hjælp af lentivirus kræver gennemgang og godkendelse af en protokol om biosikkerhed ved biosikkerhed udvalget en forskers institution. Forskning ved hjælp af dyremodeller kræver gennemgang og godkendelse af et dyr protokol fra Animal Care og Use Committee (ACUC) af en forskers institution.

Watch this Scientific Journal Video about DNA Vector-based RNA Interference to Study Gene Function in Cancer at JoVE.com

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA-interferens (RNAi) har mange fordele i forhold til gen-knockout og er bredt anvendt som et redskab i gen funktionelle undersøgelser. Opfindelsen af ​​DNA-vektor baseret RNAi teknologi har gjort lang sigt og inducerbare gen knockdown muligt, og også øget muligheden for gen-nedregulering<em&gt; In vivo</em&gt;.

DNA Vector-baseret RNA-interferens til Studieinformationen genfunktion i Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

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