Name: an ecdysone receptor-based singular gene switch for deliberate expression of transgene with robustness, reversibility, and negligible leakiness
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Description: Watch this Scientific Journal Video about An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness at JoVE.com

Forfatterne takke S-Y Choi (Chonnam National University) for værdifulde kommentarer og grundig læsning af vores manuskript. Dette arbejde blev støttet af en forskningsfond i Chungnam National University.

1. transgen Subcloning
<ol><li>Vælg en passende begrænsning enzym anerkendelse websted (eller websteder) i MCS af pEUI(+), og subclone gen af interesse i den ønskede retning (figur 1). Bemærk: Den EGFP eller FLAG epitop-tagged ankyrin gentage domæne 13A (ANKRD13A) transgen var subcloned i EcoRI site af pEUI(+) vektor ved hjælp af T4 DNA polymerase i en sekvens - og ligatur-uafhængig kloning metode19.</li>
	<li>Linearize pEUI(+) med EcoRI ...

<ol>
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Den største ulempe ved at bruge en binær gen udtrykket switch må samtidig levere to separate plasmider (driver og effektor) i målcellen. Dette kan føre til en ulige fordeling af plasmider og resultere i en inkonsekvent svar af overgangen til induktorer1,2,3. En anden mangel ved to-vektor-system er, at mindst to runder af antibiotika valg forpligtes til at identificere lovende cellelinjer. En gen-inducerbar kassette beståen...

Flere forskellige gen afbrydere har været undersøgt for deres evne til at regulere netop transgen udtryk i cellekultur og dyremodeller, med varierende grader af succes1,2,3. Potentielle gen switch systemer skal opfylde flere strenge kriterier, herunder: præcise retningsbestemt udtryk for transgen, afhængig af dosis og tidspunkt lydhørhed over for induktorer, ubetydelig leakiness af initiativtageren, reversibilitet af transgenet aktivering via inducer fjernelse og inducer toksicitet niveauer tolerabel at cellelinjer og laboratorium dyr1,2,3. Flere små molekyle induktorer er blevet udnyttet, herunder tetracyklin4,5, rapamycin6,7,8, mifepriston9,10, og ecdysone11,12,13,14. Generelt er disse systemer kræver mindst to plasmider, der indeholder to eller tre enheder, diskret udtryk, en eller to som komponere en regulerende element (inducer plasmid), der binder en lille molekyle inducer at få transcriptional aktivitet; og en anden plasmid (effektor plasmid) indeholdende transgen under kontrol af en regulerende DNA-regionen, der giver mulighed for adgang af den inducer-bundet regulerende element. Den største ulempe ved det binære system er, at det kræver den samtidige indførelse af to vektorer (inducer og effektor) i målcellen. I forbigående transgen udtryk eksperimenter producerer den samtidige Transfektion af to plasmider uundgåeligt en befolkning af celler, der enkeltvis er transfekteret med enten Induceren eller effektor eller co transfekteret ulige. Derudover kræver det binære system mindst to runder af antibiotika valg at etablere stabile cellelinjer, som er tidskrævende og besværlige. Således, ved at kombinere alle komponenterne, der kræves for transgen udtryk og forordning i en enkelt vektor ville være ideel til at garantere de samtidig udtryk for alle de obligatoriske elementer i en enkelt celle og mulighed for regulering af transgenet udtryk gennem behandling med lille molekyle induktorer.
For at overvinde manglerne i binære gen switche, udviklet vi for nylig en enestående gen switch, ved hjælp af en Drosophila melanogaster Sml-baserede gen inducerbar system15. Ecdysone medierer genekspression, når det binder sig til Sml. / ultraspiracle (USP) heterodimer, som igen inducerer binding af EcR DNA regulerende elementer3,11. Hvirveldyr retinoid X receptor (RXR), en ortholog af insekt USP, rekrutter Sml for at danne en aktiv transcriptional aktivator16. Således, for den målrettede udtryk for en transgen i hvirveldyr celler eller væv, EcR og RXR skal være co udtrykte samtidig før bliver stimuleret af ecdysone eller dens agonister. Da funktionen heterodimerisk af parameteren Sml-baserede gen kan være påvirket af den endogene RXR niveau, Padidam mfl. erstattet de Sml. DNA-bindende og aktivering domæner med heterolog GAL4 DNA-bindende, herpes simplex virus protein vmw65 (VP16) aktivering domæner smeltet til Sml ligand-bindende domæne, som derefter blev ikke reagerer på endogene RXR og dannede en homodimer at få transcriptional aktivitet17. Parameteren Sml-baserede gen blev yderligere forbedret ved at konstruere en kimære protein består af minimal VP16 aktivering domæne kombineret med GvEcR, som dannede en homodimer og lokaliseret i cytosol i mangel af ecdysone agonist, Teb16, 18. Ved binding til Teb, GvEcR ændret sin subcellulært lokalisering fra cytosol til kernen til at genkende en hybrid promotor består af zebrafisk E1b minimal promotor kombineret med en ti tandem gentages opstrøms aktivering sekvenser (UASs) at indlede den transskription af target gen16.
For at forenkle den tidligere rapporteret Sml-baserede gen skifter16,18, vi kombineret de binære funktioner i systemet til en enkelt vektor udstyret med alle de obligatoriske elementer til stimulerende transgen udtryk, og derefter udpeget den nyligt oprettede vector, pEUI(+) (GenBank stammesamlingsnummer: KP123436, figur 1A)15. Effektor svarer til GvEcR-driveren (herefter driver) består af ti tandem UAS gentager ved siden af en E1b minimal promotor efterfulgt af en flere kloning websted (MCS) med EcoRI, PmlI, NheI, BmtI, AfeI og AflII anerkendelse sekvenser (figur 1B). Desuden at lette udvælgelsen af transfekteret celler, en SV40 promotor-drevet puromycin N-acetyl-transferase (PAC)-genet blev placeret mellem regionerne driver og effektor at opretholde stabile cellelinjer (figur 1).
Vi beskriver en detaljeret protokol for den forbigående og stabil graduering af transgenet udtryk ved hjælp af pEUI(+). Derudover giver vi detaljeret vejledning til vellykket brug af Teb som en ecdysone agonist.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pEUI(+)</td>
			<td style="width:71px;">TransLab</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">The patent license was transferred to TransLab Inc.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gene-Fect Transfection Reagent</td>
			<td style="width:71px;">TransLab</td>
			<td style="width:119px;">TLC-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEK293</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tebufenozide</td>
			<td style="width:71px;">Fluka</td>
			<td style="width:119px;">31652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cloning cylinder</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">CLS31668</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puromycin</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">58-58-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibiotics</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">15240-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin-EDTA</td>
			<td style="width:71px;">Welgene</td>
			<td style="width:119px;">LS015-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:71px;">Welgene</td>
			<td style="width:119px;">LM001-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Welgene</td>
			<td style="width:119px;">S001-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell culture dish</td>
			<td style="width:71px;">SPL life science</td>
			<td style="width:119px;">11090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mouse FLAG M2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F3165</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-alpha tubulin antibody</td>
			<td style="width:71px;">Calbiochem</td>
			<td style="width:119px;">CP06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cloning cylinder</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">CLS31668</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NucleoBond Xtra Midi</td>
			<td style="width:71px;">Macherey-Nagel</td>
			<td style="width:119px;">740410.1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">M-PER Mammalian Protein 
			Extraction Reagent</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">78505</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100X Protease inhibitor Cock. III</td>
			<td style="width:71px;">T&#38;I</td>
			<td style="width:119px;">BPI-9200</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ecdysone receptor-based singular gene switch for deliberate expression of transgene with robustness, reversibility, and negligible leakiness

Præcis styring af transgenet udtryk er ønskeligt i biologiske og kliniske undersøgelser. Men fordi den binære funktion i øjeblikket ansat gen skifter kræver overførsel af to terapeutiske udtryk enheder samtidigt til en enkelt celle, den praktiske anvendelse af ordningen for genterapi er begrænset. For at forenkle ordningen transgen udtryk, skabt vi et gen switch udpeget som pEUI(+) omfatter et komplet sæt af transgenet udtryk moduler i en enkelt vektor. Bestående af GAL4 DNA-bindende domæne og modificerede Sml (GvEcR), en minimal VP16 aktivering domæne sammensmeltet med en GAL4 DNA-bindende domæne, samt en modificeret Drosophila ecdysone receptor (EcR), den nyudviklede ental gen switch er meget lydhør administration af en kemisk inducer på en måde, afhængig af tid og dosering. PEUI(+) vektor er et potentielt kraftfuldt værktøj til at forbedre kontrol af transgenet udtryk i både biologisk forskning og prækliniske undersøgelser. Vi præsenterer her, en detaljeret protokol for graduering af en forbigående og stabil transgen udtryk ved hjælp af pEUI(+) vektor ved behandling af tebufenozide (Teb). Derudover deler vi vigtige retningslinjer for brug af Teb som en kemisk inducer.

Parameteren GvEcR-baserede ental genet er afbildet i figur 1A. PEUI(+) vektor var optimeret til regulerede transgen udtryk ved behandling med Teb. Regionen effektor af pEUI(+) består af en 10xUAS og en E1b minimal promotor efterfulgt af en MCS, der indeholder EcoRI, PmlI, NheI, BmtI, AfeI og AflII begrænsning enzym genkendelsessekvenser. En SV40 poly(A) signal blev tilføjet bag MCS (figur 1B). En PAC gen var indsat me...

Watch this Scientific Journal Video about An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness at JoVE.com

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

En Ecdysone Receptor-baserede ental gen Switch til bevidst udtryk for transgen med robusthed, reversibilitet og ubetydelig Leakiness

Vi præsenterer her, en protokol om en graduering af transgenet udtryk ved hjælp af pEUI(+) ental gen switch ved tebufenozide behandling.

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

Research

JoVE Journal

Genetics

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