Name: methods to discover alternative promoter usage and transcriptional regulation of murine bcrp1
Uploaded: 2016-05-27T14:00:00
Description: Watch this Scientific Journal Video about Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1 at JoVE.com

Dette arbejde blev støttet af Merit anmeldelse Awards til Douglas D. Ross og til Arif Hussain fra Institut for Veterans &#39;Affairs.

1. in silico Forudsigelse af Alternative Første exoner Bcrp1 Brug af BLAST Analyse af Mouse EST-database Bemærk: Denne protokol beskriver, hvordan man søger musen udtrykt sekvens tag (EST) database for EST&#39;eme med sekvens lighed med exon 2 af Bcrp1 (som indeholder det translationelle startsted), og derefter hvordan at tilpasse de matchende EST-sekvenser til genomiske sekvenser at fastslå deres placering i musen Bcrp1 gen i forhold til 5&#39;-enden af ​​Bcrp1 exon 2. <o...

<ol>
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Hovedparten af metoderne og repræsentative resultater, der præsenteres, er beskrevet i tidligere arbejde med titlen &quot;B crp1 transkription i muse testis styres af en promotor opstrøms for en hidtil ukendt første exon (E1U) reguleret af steroidogene faktor-1&quot;, som blev offentliggjort i 2013 5 . Foruden de repræsentative resultater afbildet her, den tidligere papir billede estimater af alternativ første exon udnyttelse under anvendelse 5&#39;-RACE PCR og RT-PCR-metodik. Endvidere in-...

Mere end halvdelen af menneskelige gener er reguleret af alternative initiativtagere 1. Hver alternativ promotor kan indeholde regulatoriske elementer, der kan være forskellige fra dem i andre alternative promotorer. Promotoren (r) anvendes i en væv kan afvige fra dem, der anvendes i en anden væv. For eksempel er det muligt, at aktivering af en given signalvej kan udløse den alternative promotor til et gen anvendes i en væv, men har ingen virkning på eller undertrykke en separat alternativ promotor til det samme gen, der er udnyttet i et andet væv. Ekspression af Bcrp1 genet styres af alternative initiativtagere. Bcrp1 er den murine ortholog af human brystcancer Resistance Protein (BCRP) genet. BCRP er en ATP-bindende kassette (ABC) transportør formelt udpeget ABCG2 2, 3. Som en apikal plasmamembranprotein, at BCRP / Bcrp1 funktioner udstrømning til en lang række naturlige og miljøfremmede substrater <sup&gt; 3, 4. Hos mennesker og mus, er BCRP / Bcrp1 stærkt udtrykt i farmakologisk relevante organer, såsom leveren (galde-canaliculi), tarm og nyre, samt organer med blod-vævsbarrierer såsom placenta, hjerne og testis 2, 5 - 12. Angivelse af BCRP / Bcrp1 i hæmatopoietiske og andre stamceller, herunder kræft stamceller, kan give resistens af disse celler til miljøfremmede stoffer og cancer kemoterapeutiske lægemidler 3. I begyndelsen af arbejde at forstå reguleringen af BCRP udtryk i normale og neoplastiske celler, blev 5 &#39;hurtig opformering af cDNA ender (5&#39;-RACE) analyse af BCRP mRNA udført for at bestemme dens nøjagtige transkriptionelle startsted 13. Ikke alene var flere transkriptionel start- sites fundet; også stødte var tre alternative former af det første exon, som i BCRP er utranslateret. Disse alternative første exoner - udpegede E1a, E1b, E1c - blev udtrykt forskelligt i en række humane væv. To yderligere første exon varianter blev opdaget i en BLAST-søgning af den humane EST database ved hjælp det andet exon af BCRP 13. Fire kampe afslørede en første exon&gt; 70 kb opstrøms fra exon 2, som blev betegnet E1u; fire andre kampe afslørede BCRP mRNA, manglede en første exon helt, som blev betegnet E1- 13. Tilstedeværelsen af alternative leader exons anses for at være en manifestation af alternativ promotoranvendelse 14. Hos mus er fire alternative første exoner af Bcrp1 beskrevet, der kan svare til alternative promotere, der styrer Bcrp en transskription i forskellige mus væv; disse exoner / promotorer betegnes E1U, E1A, E1B og E1C, og er placeret ca. 70, 58, 15, og 5 kb opstrøms fra Bcrp1 exon 2 5, 15. E1A mRNA isoform viste sig at være fremherskende i Murine hæmatopoietiske stamceller, hjerte, lunge, milt og hjerne, mens E1B isoform blev udtrykt i musetarm, føtale leverceller, og erythroide precursorceller i knoglemarv 5, 15. Promotoren opstrøms fra E1B blev vist at være den væsentligste alternativ promotor regulerer Bcrp1 transkription i musetarm, reguleret i det mindste delvis ved phospho-cyklisk-AMP-responselement bindingsprotein (p-CREB) og et CREB responselement unik for denne alternative Bcrp1 promotor 16. Den E1C mRNA isoform udtrykkes overvejende i voksen murin lever og nyre 5. Den E1U isoform kan ikke påvises i de fleste testede bortset murine testis, hvor det er den fremherskende isoform udtrykt 5 væv. Bcrp1 udtrykt i rotte testikler findes i både somatiske (endotelceller tætte sammenføjninger, peritubulære myoid celler og Sertoli-celler) og kimceller (i seminifere endotel, hvor det kan beskytte sene spermatider 4). Den region opstrøms fra E1U indeholder et funktionelt respons element for steroidogene faktor-1 (SF-1) 5. Bcrp1 mRNA og protein er markant reduceret i testiklerne af Sertoli cellespecifikke SF-1 knockout-mus, hvilket antyder, at Bcrp1 ekspression i murine Sertoli-celler styres af SF-1 5. De protokoller præsenteret detalje metoder til at opdage alternative første exoner af Bcrp1 i-silico, og at etablere luciferase-baserede reporter analyser for formodede initiativtagere opstrøms fra de alternative første exoner identificeret.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>COMMERCIAL BIOLOGIC MATERIALS AND KITS:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>First Choice RLM-RACE kit</td>
			<td>Ambion Inc., Austin, TX, currently available through Life Technologies</td>
			<td>AM1700</td>
			<td>5&#39;-RACE PCR kit</td>
		</tr>
		<tr>
			<td>TOPO TA cloning kit&#160;</td>
			<td>Life Technologies</td>
			<td>K450001</td>
			<td>This kit contains the TOP10 chemically competent E. coli bacteria.</td>
		</tr>
		<tr>
			<td>T4 DNA ligase quick ligation kit</td>
			<td>New England Biolabs</td>
			<td>M2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Faststart high-fidelity Taq- DNA polymerase</td>
			<td>Sigma-Aldrich/Roche</td>
			<td>3553400001/RMB-4738284001&#160;</td>
			<td>Contains a high-fidelity Taq polymerase enzyme blend</td>
		</tr>
		<tr>
			<td>XtremeGENE HP DNA transfection reagent</td>
			<td>Roche</td>
			<td>6366236001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-luciferase reporter assay kit</td>
			<td>Promega</td>
			<td>E1910</td>
			<td>Includes the pGL3-basic empty reporter vector (firefly luciferase), pRLTK renilla luciferase expressing vector, and other control vectors.</td>
		</tr>
		<tr>
			<td>QIAprep</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>For plasmid miniprep - isolation and purification of plasmid DNA from bacterial colonies</td>
		</tr>
		<tr>
			<td>pCR2.1 vector</td>
			<td>part of the TOPO TA cloning kit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pGL3-basic luciferase containing vector</td>
			<td>Promega</td>
			<td>E1751</td>
			<td></td>
		</tr>
		<tr>
			<td>pRL-TK Renilla luciferase expressing vector</td>
			<td>Promega</td>
			<td>E2241</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial artificial chromosome</td>
			<td>BACPAC Resources Center, Children&#8217;s Hospital Oakland Research Institute, Oakland, CA</td>
			<td>RP23-285A12 and RP24-314E24</td>
			<td>http://bacpac.chori.org/clones.htm</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>REAGENTS/CHEMICALS/MEDIA/SUPPLIES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Life Technologies</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard&#174; SV Gel and PCR Clean-Up System&#160;</td>
			<td>Promega</td>
			<td>A9281</td>
			<td>Useful for purifying PCR products following digestion with restriction endonucleases</td>
		</tr>
		<tr>
			<td>CRYOVIALS</td>
			<td>Denville Scientific</td>
			<td>V9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>SOFTWARE:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ensembl software</td>
			<td>Ensembl project</td>
			<td>http://Ensembl.org&#160;</td>
			<td>The Ensembl project produces genome databases for vertebrates and other eukaryotic species, and makes this information freely available online.</td>
		</tr>
		<tr>
			<td>Blast software</td>
			<td>NCBI</td>
			<td>http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD= 
			Web&#38;PAGE_TYPE=BlastHome</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer 3 software</td>
			<td>Simgene</td>
			<td>http://simgene.com/Primer3</td>
			<td>Useful for designing primers for 5&#39;-RACE PCR</td>
		</tr>
		<tr>
			<td>NCBI Nucleotide database</td>
			<td>NCBI</td>
			<td>http://www.ncbi.nlm.nih.gov/nucleotide/</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>CELL LINES:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TM4 murine Sertoli cells</td>
			<td>ATCC, Manassas, Virginia</td>
			<td>CRL-1715&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>INSTRUMENTS:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Turner Biosystems</td>
			<td>TD-20/20</td>
			<td>This is a relatively inexpensive, manually operated luminometer. Automated systems are also available from the same manufacturer that utilize 96 well plates.</td>
		</tr>
		<tr>
			<td>NanoDrop spectrophotometers</td>
			<td>Thermo Scientific, Inc.</td>
			<td>NanoDrop 2000</td>
			<td>Spectrophotometer can use very small quantities of sample</td>
		</tr>
		<tr>
			<td>DU 800 UV/VIS spectrophotometer and Nucleic Acid Analysis II software</td>
			<td>BeckmanCoulter Inc, Fullerton, CA</td>
			<td>DU 800</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods to discover alternative promoter usage and transcriptional regulation of murine bcrp1

Genekspression i forskellige væv er ofte styret af alternative promotorer, der resulterer i syntesen af ​​mRNA med unikke - sædvanligvis utranslaterede - første exoner. Bcrp1 (ABCG2), det murine ortholog af ABC transportør Breast Cancer Resistance Protein (BCRP, ABCG2), har mindst fire alternative promotere, der er udpeget af de tilsvarende fire alternative første exoner produceres: E1U, E1A, E1B, og E1C. Heri in-silico protokoller præsenteres til at forudsige alternativ promotoranvendelse for Bcrp1. Desuden er reporter analysemetoder beskrevet at producere reporterkonstruktioner til alternative initiativtagere og bestemme funktionaliteten af ​​formodede initiativtagere opstrøms for de alternative første exoner, der er identificeret.

Identifikation af Bcrp en alternativ promotor udnyttelse i mus testikel ved analyse af leder exons Når databasen EST hos NCBI blev probet (april 2015) ved at følge trinene skitseret i protokol 1, fandt de EST&#39;er, der var sammenhængende med 5&#39;-enden af exon 2 i Bcrp 1 mRNA er vist i tabel 1, sammen med deres position i genomisk DNA ...

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Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Metoder til at opdage Alternativ Promoter Usage og Transkriptionel forordning af murint Bcrp1

Med det murine ABC transportør Bcrp1 (ABCG2) som et eksempel, i-silico protokoller præsenteres for at detektere alternativ promotoranvendelse i gener udtrykt i musevæv, og vurdere funktionaliteten af de alternative promotorer identificeret ved anvendelse reporter assays.

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

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