Name: using a gfp-tagged tmem184a construct for confirmation of heparin receptor identity
Uploaded: 2017-02-17T13:00:00
Description: Watch this Scientific Journal Video about Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity at JoVE.com

Research in the Lowe-Krentz lab is supported by research grant HL54269 from the National Institutes of Health to LLK.

1. Design av en GFP-protein Construct <ol><li> Kjøp, eller design og bygge, en GFP-merket konstruksjon basert på protein i spørsmålet. MERK: For en kjøpt konstruksjon, vanlige vektorer er tilgjengelige fra kommersielle laboratorier som inkluderer noen eller alle av følgende forslag: For en membran protein, velge en C-terminal plasseringen av GFP fordi det er mindre sannsynlighet for å forstyrre membran protein menneskehandel. Betrakt en forlengelse mellom genet av interesse og den GFP hvis det er grunn t...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmembrane+Protein+184A+Is+a+Receptor+Required+for+Vascular+Smooth+Muscle+Cell+Responses+to+Heparin.">Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin.</a> J Biol Chem. 291, 5326-5341 (2016).</li><li>Daher, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Toxoplasma+TgPH1,+a+pleckstrin+homology+domain-containing+protein+that+binds+to+the+phosphoinositide+PI(3,5)P.">Identification of Toxoplasma TgPH1, a pleckstrin homology domain-containing protein that binds to the phosphoinositide PI(3,5)P.</a> Mol Biochem Parasitol. , (2016).</li><li>Vit, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+identification+of+membrane+proteins+based+on+analysis+of+trypsin-protected+transmembrane+segments.">Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments.</a> J Proteomics. , (2016).</li><li>Attwood, M. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin+responses+in+vascular+smooth+muscle+cells+involve+cGMP-dependent+protein+kinase+(PKG).">Heparin responses in vascular smooth muscle cells involve cGMP-dependent protein kinase (PKG).</a> J Cell Physiol. 229, 2142-2152 (2014).</li><li>Farwell, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin+Decreases+in+Tumor+Necrosis+Factor+alpha+(TNFalpha)-induced+Endothelial+Stress+Responses+Require+Transmembrane+Protein+184A+and+Induction+of+Dual+Specificity+Phosphatase+1.">Heparin Decreases in Tumor Necrosis Factor alpha (TNFalpha)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1.</a> J Biol Chem. 291, 5342-5354 (2016).</li><li>Xu, D., Esko, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demystifying+heparan+sulfate-protein+interactions.">Demystifying heparan sulfate-protein interactions.</a> Annu Rev Biochem. 83, 129-157 (2014).</li><li>Chiodelli, P., Bugatti, A., Urbinati, C., Rusnati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin/Heparan+sulfate+proteoglycans+glycomic+interactome+in+angiogenesis:+biological+implications+and+therapeutical+use.">Heparin/Heparan sulfate proteoglycans glycomic interactome in angiogenesis: biological implications and therapeutical use.</a> Molecules. 20, 6342-6388 (2015).</li><li>Slee, J. B., Lowe-Krentz, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+realignment+and+cofilin+regulation+are+essential+for+barrier+integrity+during+shear+stress.">Actin realignment and cofilin regulation are essential for barrier integrity during shear stress.</a> J Cell Biochem. 114, 782-795 (2013).</li><li>Patton, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+heparin-binding+protein+using+monoclonal+antibodies+that+block+heparin+binding+to+porcine+aortic+endothelial+cells.">Identification of a heparin-binding protein using monoclonal antibodies that block heparin binding to porcine aortic endothelial cells.</a> The Biochemical journal. 311, 461-469 (1995).</li><li>Doggett, T. M., Breslin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+actin+cytoskeleton+in+live+endothelial+cells+expressing+GFP-actin.">Study of the actin cytoskeleton in live endothelial cells expressing GFP-actin.</a> J Vis Exp. , (2011).</li><li>Skalamera, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+genome+scale+lentiviral+vector+library+for+EF1alpha+promoter-driven+expression+of+human+ORFs+and+identification+of+human+genes+affecting+viral+titer.">Generation of a genome scale lentiviral vector library for EF1alpha promoter-driven expression of human ORFs and identification of human genes affecting viral titer.</a> PloS one. 7, 51733 (2012).</li><li>Castro, M., Nikolaev, V. O., Palm, D., Lohse, M. J., Vilardaga, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turn-on+switch+in+parathyroid+hormone+receptor+by+a+two-step+parathyroid+hormone+binding+mechanism.">Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism.</a> Proc Natl Acad Sci U S A. 102, 16084-16089 (2005).</li><li>Albertazzi, L., Arosio, D., Marchetti, L., Ricci, F., Beltram, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+FRET+analysis+with+the+EGFP-mCherry+fluorescent+protein+pair.">Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair.</a> Photochem Photobiol. 85, 287-297 (2009).</li><li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+organization+of+the+ATP-sensitive+potassium+channel+complex+examined+by+fluorescence+resonance+energy+transfer.">Domain organization of the ATP-sensitive potassium channel complex examined by fluorescence resonance energy transfer.</a> J Biol Chem. 288, 4378-4388 (2013).</li><li>Christiansen, E., Hudson, B. D., Hansen, A. H., Milligan, G., Ulven, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Characterization+of+a+Potent+Free+Fatty+Acid+Receptor+1+(FFA1)+Fluorescent+Tracer.">Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer.</a> J Med Chem. 59, 4849-4858 (2016).</li><li>Chiang, C. F., Okou, D. T., Griffin, T. B., Verret, C. R., Green Williams, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein+rendered+susceptible+to+proteolysis:+positions+for+protease-sensitive+insertions.">fluorescent protein rendered susceptible to proteolysis: positions for protease-sensitive insertions.</a> Arch Biochem Biophys. 394, 229-235 (2001).</li></ol>

Protokollene som er rapportert her ble utformet for å gi bekreftende bevis for identifisering av TMEM184A som en heparin-reseptor i vaskulære celler 1. Knockdown-teknikker rutinemessig anvendes som en mekanisme for å bekrefte identifisering av nye proteiner. Men noen funksjonell tap etter knockdown er vanligvis ikke tilstrekkelig bevis for at et kandidatprotein er den samme som reseptoren (eller annen funksjonelt protein). Det er også viktig å ha bevis for at kandidaten proteinet faktisk vis...

Identifisering av kandidat proteiner for nye funksjoner avhenger ofte på affinitet-basert isolasjon protokoller fulgt av delvis sekvensbestemmelse. Nyere eksempler på nylig identifiserte proteiner inkluderer trans protein 184A (TMEM184A), en heparin reseptor identifisert etter heparin affinitetsinteraksjoner 1, og TgPH1, en pleckstrin homologi domene protein som binder phosphoinositide PI (3,5) P 2 2. Annet nytt protein identifikasjon innebærer direkte sekvensanalyse av peptider slik som den som ved Vit, et al. som brukte trans peptider å identifisere proteinprodukter fra tidligere uncharacterized gener 3. Tilsvarende kan identifisering av nye proteinsekvenser oppnås ved hjelp av bioinformatikk som søker av tidligere karakteriserte protein familier som identifisering av nye 4TM proteiner 4. Undersøkelse av aquaporin familie gensekvenser har alså ga identifisering av nye medlemmer med nye funksjoner 5. Etter identifikasjon og analyse av protein funksjon er typisk et neste trinn som noen ganger kan bli undersøkt ved hjelp av en spesifikk analyse av protein funksjon slik som i det tilfelle aquaporin. Når det er mulig, kan funksjonen til en nylig identifiserte protein undersøkes med spesifikk enzymatisk eller lignende in vitro funksjonsanalyser. Fordi mange funksjoner av nye proteiner er avhengig av komplekse interaksjoner som forekommer bare i intakte celler eller organismer, in vitro analyser er ikke alltid effektive. Imidlertid må de in vivo, være utformet på en slik måte at de er avhengige av gensekvensen. I cellekultur, og / eller enkle modellorganismer, kan knockdown gi støtte bevis for protein / funksjonsidentifikasjon 6. Med nye proteiner som er identifisert som nevnt ovenfor, er det ofte tilstrekkelig å bare slå ned et protein for å bekrefte funksjon, end utforming av in vivo funksjonelle analyser som er avhengige av gensekvens blir viktig for karakterisering av nye proteiner. Den senere identifisering av TMEM184A som en heparin-reseptor (som modulerer proliferasjon i vaskulær glatt muskulatur og inflammatoriske responser i endotelceller) ved hjelp av affinitetskromatografi og MALDI MS 1, 7 gitt en mulighet til å utvikle en samling av analyser etter knockdown ga resultater i samsvar med identifikasjons . En nylig gjennomgang bekreftet at heparin interagerer spesifikt med flere vekstfaktorer og deres reseptorer, ekstracellulære matriks-komponenter, celle adhesjonsreseptorer, og andre proteiner 8. I det vaskulære system, heparin og heparansulfat proteoglykaner (inneholdende heparansulfat kjeder tilsvarende i struktur til heparin) kommunisere med flere hundre proteiner 9. Å funksjonelt bekrefte that TMEM184A var involvert med heparin opptak og binding, ble teknikker som benyttes genkonstruksjonen for TMEM184A utviklet. Denne rapporten inneholder en samling av analyser basert på en GFP-TMEM184A konstruere for bruk i å bekrefte identiteten til TMEM184A som heparin reseptoren.

<table border="0" cellpadding="0" cellspacing="0" width="767">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GFP-TMEM184A construct</td>
			<td style="width:71px;">OriGene</td>
			<td style="width:119px;">RG213192</td>
			<td style="width:361px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Rhodamine-Heparin</td>
			<td style="width:71px;">Creative PEGWorks</td>
			<td style="width:119px;">HP-204</td>
			<td style="width:361px;">Light Sensitive</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fluorescein-Heparin</td>
			<td style="width:71px;">Creative PEGWorks</td>
			<td style="width:119px;">HP-201</td>
			<td style="width:361px;">Light Sensitive</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Mowiol</td>
			<td style="width:71px;">EMD Millipore</td>
			<td style="width:119px;">475904-100GM</td>
			<td style="width:361px;"></td>
		</tr>
		<tr height="141">
			<td height="141" style="height:141px;width:216px;">Paraformaldehyde (methanol free)</td>
			<td style="width:71px;">Thermo Sci Pierce Biotech, available through Fisher Scientific</td>
			<td style="width:119px;">PI28908 at Fisher</td>
			<td style="width:361px;">Use in Fume Hood</td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:216px;">Reacti-bind neutravidin plates (Avidin coated black 96 well dishes)</td>
			<td style="width:71px;">Thermo Sci Pierce Biotech, through Fisher Scientific</td>
			<td style="width:119px;">PI15510 at Fisher</td>
			<td style="width:361px;">Pay attention to shelf-life</td>
		</tr>
		<tr height="161">
			<td height="161" style="height:161px;width:216px;">Black 96 well plates</td>
			<td style="width:71px;">Corning Life Sciences Plastic, purchased through Fisher Scientific</td>
			<td style="width:119px;">064432 at Fisher</td>
			<td style="width:361px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">A7r5 vascular smooth muscle cell line</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">CRL 1444</td>
			<td style="width:361px;">Can be exchanged into MEM medium1</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">BAOEC bovine aortic endothelial cells</td>
			<td style="width:71px;">Cell Applications, Inc.</td>
			<td style="width:119px;">B304-05</td>
			<td style="width:361px;">Culture as recommended initially, can be exchanged into MEM medium for continuing culture1,7</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">BAOSMC bovine aortic smooth muscle cells</td>
			<td style="width:71px;">Cell Applications, Inc.</td>
			<td style="width:119px;">B354-05</td>
			<td style="width:361px;">Culture as recommended initially, can be exchanged into MEM medium for continuing culture1</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">RAOEC rat aortic endothelial cells</td>
			<td style="width:71px;">Cell Applications, Inc.</td>
			<td style="width:119px;">R304-05a</td>
			<td style="width:361px;">Culture as recommended initially, can be exchanged into MEM medium for continuing culture7</td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:216px;">Biotinylated anti-GFP</td>
			<td style="width:71px;">Thermo Sci Pierce Biotech, through Fisher Scientific</td>
			<td style="width:119px;">MA5-15256-BTIN</td>
			<td style="width:361px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptavidin-coated beads</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S1638</td>
			<td style="width:361px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">HeBS</td>
			<td style="width:71px;">Available from Bio-Rad</td>
			<td style="width:119px;"></td>
			<td style="width:361px;">Can be prepared in the lab.&#160; The pH is 6.8</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">TMEM184A antibody to the N-terminus</td>
			<td style="width:71px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc292006</td>
			<td style="width:361px;">Only known TMEM184A antibody to N-terminal region.</td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:216px;">TMEM184A antibody to the C-terminus</td>
			<td style="width:71px;">Obtained from ProSci Inc, Poway, CA&#160;</td>
			<td style="width:119px;">Pro Sci 5681</td>
			<td style="width:361px;">ProSci used in figure 1</td>
		</tr>
		<tr height="20">
			<td height="41" style="height:41px;width:216px;">GFP antibodies</td>
			<td style="width:71px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc9996</td>
			<td style="width:361px;">Used in figures 5</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Secondary antibodies, labeled with TRITC or Cy3</td>
			<td style="width:71px;">Jackson ImmunoResearch Laboratories, Inc, West Grove, PA</td>
			<td style="width:119px;">711 025 152 (donkey anti-rabbit, TRITC) 
			715 165 150 (donkey anti-mouse, Cy3)</td>
			<td style="width:361px;">Minimal cross-reactivity to minimize any non-specific staining.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">CHAPS</td>
			<td style="width:71px;">Purchased from Sigma</td>
			<td style="width:119px;">C5849</td>
			<td style="width:361px;">Note that this specific catalog number has been discontinued.&#160; Supplier will provide information regarding replacement.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Live imaging 35 mm dishes</td>
			<td style="width:71px;">MatTek (Ashland MA)</td>
			<td style="width:119px;">P35G-1.0 &#8211; 20 mm - C</td>
			<td style="width:361px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Confocal Microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">LSM 510 Meta with a 63X oil-immersion lens</td>
			<td style="width:361px;">Used for images and live-imaging in Figures 1, 2 and 3</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Confocal Microscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">C2+ confocal with a 60X oil-immersion lens</td>
			<td style="width:361px;">Used for images in Figure 5</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Confocal Microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Zeiss LSM 880 with a 63X oil-immersion lens</td>
			<td style="width:361px;">Used for images in Figure 2C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Electroporation equipment</td>
			<td style="width:71px;">Bio-Rad</td>
			<td style="width:119px;">Gene Pulser X-Cell System</td>
			<td style="width:361px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Electroporation cuvettes</td>
			<td style="width:71px;">Available from MidSci</td>
			<td style="width:119px;">EC2L</td>
			<td style="width:361px;">Can also be obtained from equipment supplier</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Plate reader</td>
			<td style="width:71px;">TECAN</td>
			<td style="width:119px;">TECAN Infinite&#174; m200 Pro plate reader</td>
			<td style="width:361px;">Readings in the middle of the wells rather than at the surface.</td>
		</tr>
		<tr height="40">
			<td height="101" style="height:101px;width:216px;">Computer program for measuring staining intensity</td>
			<td style="width:71px;">Image J</td>
			<td style="width:119px;">https://imagej.nih.gov/ij/ 
			Program and information available on-line</td>
			<td style="width:361px;">Any appropriate program can be used. See https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html for additional detail&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell Culture trypsin solution</td>
			<td style="width:71px;">Sigma</td>
			<td>T4174</td>
			<td style="width:361px;">purchased as a 10X solution</td>
		</tr>
	</tbody>
</table>

using a gfp-tagged tmem184a construct for confirmation of heparin receptor identity

Når nye proteiner er identifisert ved affinitets-baserte isolasjon og bioinformatikk analyse, er de ofte er ukarakterisert i stor grad. Antistoffer mot spesifikke peptider innenfor den antatte sekvensen tillate noen lokaliserings eksperimenter. Men andre mulige interaksjoner med antistoffene ofte kan ikke utelukkes. Denne situasjonen gitt en mulighet til å utvikle et sett av analyser avhengig av proteinsekvensen. Nærmere bestemt ble en konstruksjon inneholdende gensekvensen koplet til GFP-kodende sekvensen ved den C-terminale ende av proteinet erholdt og anvendt for disse formål. Forsøk å karakterisere lokalisering, ligandaffinitet, og gevinst på funksjon ble opprinnelig designet og utført for å bekrefte identifiseringen av TMEM184A som heparin reseptor 1. I tillegg kan konstruksjonen anvendes for undersøkelser rettet mot membran topologi spørsmål og detaljerte protein-ligand interaksjoner. Denne rapporten presenterer arange av eksperimentelle protokoller basert på den GFP-TMEM184A konstruksjonen uttrykt i vaskulære celler som lett kan tilpasses for andre nye proteiner.

Selv om i teorien transfeksjon av en hvilken som helst DNA-konstruksjon inn i cellene kan oppnås med lipofile transfeksjon reagenser, tidligere rapporter indikerer mer effektiv transfeksjon av GFP-konstruksjonene til endoteliale celler ved hjelp av elektroporering 12. Protokollen gitt her oppnås typisk GFP-konstruere ekspresjon i mer enn 80% av de primære avledet endotelceller og glatte muskelceller som brukes. Utformingen av den anvendte konstruksjonen benytte...

Watch this Scientific Journal Video about Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity at JoVE.com

Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

Et konstrukt som koder for TMEM184A med en GFP-tag ved karboksy-terminus konstruert for eukaryot ekspresjon, ble anvendt i analyser konstruert for å bekrefte identifisering av TMEM184A som en heparin-reseptor i vaskulære celler.

Ved hjelp av en GFP-merket TMEM184A Construct for bekreftelse av Heparin Receptor Identity

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies ...

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JoVE Journal

Genetics

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Prosedyre for Adaptive Laboratory Utviklingen av mikroorganismer ved hjelp av en kjemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Påvisning av Kopier nummer Endringer Bruke enkelt celle Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Bestemmelse av den optimale Chromosomal plassering(er) gir et DNA i Escherichia coli bruker en ny Transposon-mediert tilnærming

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Høyoppløselig smeltende PCR for komplementreceptor 1 Lengdepolymorfisme Genotyping: Et innovativt verktøy for Alzheimers sykdomssesmerksvurdering

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

En metode for å studere C924T polymorfisme av Thromboxane A2 reseptor genet

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

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

En isoinokosterone reseptor-baserte entall Gene bryter for bevisst uttrykk for Transgene robusthet, Reversibilitet og ubetydelig Leakiness

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

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

Høy gjennomstrømming identifikasjon av regulatoriske gensekvenser med neste generasjons sekvensering av runde kromosom konformasjon fange (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

Endogene Protein merking i menneskelig indusert Pluripotent stilk celler med CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

qKAT: kvantitative Semi-automated å skrive Killer-cellen immunglobulin-lignende reseptor gener

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Masse massespektrometri-baserte Proteomikk analyser bruker OpenProt databasen å avsløre romanen proteiner oversatt fra ikke-kanoniske åpent lesing rammer

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...