Name: use of alu element containing minigenes to analyze circular rnas
Uploaded: 2020-03-10T12:00:00
Description: Watch this Scientific Journal Video about Use of Alu Element Containing Minigenes to Analyze Circular RNAs at JoVE.com

Dit werk werd ondersteund door het ministerie van Defensie DoD subsidie AZ180075. Stefan Stamm bedankt Jacqueline Noonan Endowment. Anna Pawluchin werd ondersteund door de DAAD, Duitse academische uitwisseling programma, Justin R. Welden was een ontvanger van de Universiteit van Kentucky Max Steckler Award.

1. Ontwerp van de constructies
<ol><li>Gebruik de UCSC genoombrowser24 om repetitieve elementen te identificeren die nodig zijn voor cirkelvormige RNA-vorming en deze op te nemen in de constructies. Belangrijk is dat primers voor versterking buiten de repetitieve elementen moeten zijn.</li>
	<li>Plak de cirkelvormige RNA-sequentie(Supplemental Figuur 1 is een testsequentie) in <a href="https://genome.ucsc.edu/cgi-bin/hgBlat?command=start" target="_blank">https://genome.ucsc.edu/...

<ol>
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Y., Eisenberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+Alu+editability.">Genome-wide analysis of Alu editability.</a> Nucleic Acids Research. 42 (11), 6876-6884 (2014).</li><li>Levanon, E. Y., 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=Systematic+identification+of+abundant+A-to-I+editing+sites+in+the+human+transcriptome.">Systematic identification of abundant A-to-I editing sites in the human transcriptome.</a> Nature Biotechnology. 22 (8), 1001-1005 (2004).</li><li>Hansen, T. B., 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=Natural+RNA+circles+function+as+efficient+microRNA+sponges.">Natural RNA circles function as efficient microRNA sponges.</a> Nature. 495 (7441), 384-388 (2013).</li><li>Kelly, S., Greenman, C., Cook, P. R., Papantonis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exon+Skipping+Is+Correlated+with+Exon+Circularization.">Exon Skipping Is Correlated with Exon Circularization.</a> Journal of Molecular Biology. 427 (15), 2414-2417 (2015).</li><li>Li, Z., 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=Exon-intron+circular+RNAs+regulate+transcription+in+the+nucleus.">Exon-intron circular RNAs regulate transcription in the nucleus.</a> Nature Structural &amp; Molecular Biology. 22 (3), 256-264 (2015).</li><li>Yang, Y., 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=Extensive+translation+of+circular+RNAs+driven+by+N(6)-methyladenosine.">Extensive translation of circular RNAs driven by N(6)-methyladenosine.</a> Cell Research. 27 (6), 626-641 (2017).</li><li>Abe, N., 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=Rolling+Circle+Translation+of+Circular+RNA+in+Living+Human+Cells.">Rolling Circle Translation of Circular RNA in Living Human Cells.</a> Scientific Reports. 5, 16435 (2015).</li><li>Salzman, J., Chen, R. E., Olsen, M. N., Wang, P. L., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type+specific+features+of+circular+RNA+expression.">Cell-type specific features of circular RNA expression.</a> PLOS Genetics. 9 (9), 1003777 (2013).</li><li>Ottesen, E. W., Luo, D., Seo, J., Singh, N. N., Singh, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Survival+Motor+Neuron+genes+generate+a+vast+repertoire+of+circular+RNAs.">Human Survival Motor Neuron genes generate a vast repertoire of circular RNAs.</a> Nucleic Acids Research. 47 (6), 2884-2905 (2019).</li><li>Stoss, O., Stoilov, P., Hartmann, A. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+minigene+systems+to+dissect+alternative+splicing+elements.">Use of minigene systems to dissect alternative splicing elements.</a> Methods. 37 (4), 331-340 (2005).</li><li>Baralle, D., Baralle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+in+action:+assessing+disease+causing+sequence+changes.">Splicing in action: assessing disease causing sequence changes.</a> Journal of Medical Genetics. 42 (10), 737-748 (2005).</li><li>Percifield, R., Murphy, D., Stoilov, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medium+throughput+analysis+of+alternative+splicing+by+fluorescently+labeled+RT-PCR.">Medium throughput analysis of alternative splicing by fluorescently labeled RT-PCR.</a> Methods in Molecular Biology. 1126, 299-313 (2014).</li><li>Stoilov, P., Lin, C. H., Damoiseaux, R., Nikolic, J., Black, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+screening+strategy+identifies+cardiotonic+steroids+as+alternative+splicing+modulators.">A high-throughput screening strategy identifies cardiotonic steroids as alternative splicing modulators.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (32), 11218-11223 (2008).</li><li>Shen, M., 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=Pyrvinium+pamoate+changes+alternative+splicing+of+the+serotonin+receptor+2C+by+influencing+its+RNA+structure.">Pyrvinium pamoate changes alternative splicing of the serotonin receptor 2C by influencing its RNA structure.</a> Nucleic Acids Research. 41 (6), 3819-3832 (2013).</li><li>Noto, J. J., Schmidt, C. A., Matera, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+and+expressing+circular+RNAs+via+tRNA+splicing.">Engineering and expressing circular RNAs via tRNA splicing.</a> RNA Biology. , 1-7 (2017).</li><li>Schmidt, C. A., Noto, J. J., Filonov, G. S., Matera, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+Expressing+and+Imaging+Abundant,+Stable,+Circular+RNAs+In+Vivo+Using+tRNA+Splicing.">A Method for Expressing and Imaging Abundant, Stable, Circular RNAs In Vivo Using tRNA Splicing.</a> Methods in Enzymology. 572, 215-236 (2016).</li><li>Welden, J. R., van Doorn, J., Nelson, P. T., Stamm, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+MAPT+locus+generates+circular+RNAs.">The human MAPT locus generates circular RNAs.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. , 2753-2760 (2018).</li><li>Casper, 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=The+UCSC+Genome+Browser+database:+2018+update.">The UCSC Genome Browser database: 2018 update.</a> Nucleic Acids Research. 46, 762-769 (2018).</li><li>Grozdanov, P. N., MacDonald, C. C. <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+plasmid+vectors+expressing+FLAG-tagged+proteins+under+the+regulation+of+human+elongation+factor-1alpha+promoter+using+Gibson+assembly.">Generation of plasmid vectors expressing FLAG-tagged proteins under the regulation of human elongation factor-1alpha promoter using Gibson assembly.</a> Journal of Visualized Experiments. (96), (2015).</li><li>Wang, Y., 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=A+novel+strategy+to+engineer+DNA+polymerases+for+enhanced+processivity+and+improved+performance+in+vitro.">A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro.</a> Nucleic Acids Research. 32 (3), 1197-1207 (2004).</li><li>Gibson, D. G., 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=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nature Methods. 6 (5), 343-345 (2009).</li><li>Conn, S. 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=The+RNA+binding+protein+quaking+regulates+formation+of+circRNAs.">The RNA binding protein quaking regulates formation of circRNAs.</a> Cell. 160 (6), 1125-1134 (2015).</li><li>Jeck, W. R., Sharpless, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+and+characterizing+circular+RNAs.">Detecting and characterizing circular RNAs.</a> Nature Biotechnology. 32 (5), 453-461 (2014).</li><li>Pamudurti, N. R., 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=Translation+of+CircRNAs.">Translation of CircRNAs.</a> Molecular Cell. 66 (1), 9-21 (2017).</li><li>Kramer, M. 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=Combinatorial+control+of+Drosophila+circular+RNA+expression+by+intronic+repeats,+hnRNPs,+and+SR+proteins.">Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins.</a> Genes &amp; Development. 29 (20), 2168-2182 (2015).</li><li>Starke, 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=Exon+circularization+requires+canonical+splice+signals.">Exon circularization requires canonical splice signals.</a> Cell Reports. 10 (1), 103-111 (2015).</li><li>Suzuki, H., Tsukahara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+view+of+pre-mRNA+splicing+from+RNase+R+resistant+RNAs.">A view of pre-mRNA splicing from RNase R resistant RNAs.</a> International Journal of Molecular Sciences. 15 (6), 9331-9342 (2014).</li><li>Zhang, X. 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=Diverse+alternative+back-splicing+and+alternative+splicing+landscape+of+circular+RNAs.">Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.</a> Genome Research. 26 (9), 1277-1287 (2016).</li><li>Liang, D., Wilusz, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+intronic+repeat+sequences+facilitate+circular+RNA+production.">Short intronic repeat sequences facilitate circular RNA production.</a> Genes &amp; Development. 28 (20), 2233-2247 (2014).</li><li>Wang, Y., Mandelkow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+in+physiology+and+pathology.">Tau in physiology and pathology.</a> Nature Reviews Neuroscience. 17 (1), 5-21 (2016).</li><li>Fejes-Toth, K., 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=Post-transcriptional+processing+generates+a+diversity+of+5'-modified+long+and+short+RNAs.">Post-transcriptional processing generates a diversity of 5'-modified long and short RNAs.</a> Nature. 457 (7232), 1028-1032 (2009).</li><li>Gao, Q. 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=Complex+regulation+of+tau+exon+10,+whose+missplicing+causes+frontotemporal+dementia.">Complex regulation of tau exon 10, whose missplicing causes frontotemporal dementia.</a> Journal of Neurochemistry. 74 (2), 490-500 (2000).</li><li>Hartmann, A. M., 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=Regulation+of+alternative+splicing+of+human+tau+exon+10+by+phosphorylation+of+splicing+factors.">Regulation of alternative splicing of human tau exon 10 by phosphorylation of splicing factors.</a> Molecular and Cellular Neuroscience. 18 (1), 80-90 (2001).</li><li>Wang, Y., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+backsplicing+produces+translatable+circular+mRNAs.">Efficient backsplicing produces translatable circular mRNAs.</a> RNA. 21 (2), 172-179 (2015).</li><li>Yang, Y., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constructing+GFP-Based+Reporter+to+Study+Back+Splicing+and+Translation+of+Circular+RNA.">Constructing GFP-Based Reporter to Study Back Splicing and Translation of Circular RNA.</a> Methods in Molecular Biology. 1724, 107-118 (2018).</li><li>Zheng, Q., 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=Circular+RNA+profiling+reveals+an+abundant+circHIPK3+that+regulates+cell+growth+by+sponging+multiple+miRNAs.">Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs.</a> Nature Communications. 7, 11215 (2016).</li><li>Jia, W., Xu, B., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNA+expression+profiles+of+mouse+ovaries+during+postnatal+development+and+the+function+of+circular+RNA+epidermal+growth+factor+receptor+in+granulosa+cells.">Circular RNA expression profiles of mouse ovaries during postnatal development and the function of circular RNA epidermal growth factor receptor in granulosa cells.</a> Metabolism. 85, 192-204 (2018).</li><li>Liang, 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=The+Output+of+Protein-Coding+Genes+Shifts+to+Circular+RNAs+When+the+Pre-mRNA+Processing+Machinery+Is+Limiting.">The Output of Protein-Coding Genes Shifts to Circular RNAs When the Pre-mRNA Processing Machinery Is Limiting.</a> Molecular Cell. 68 (5), 940-954 (2017).</li><li>Legnini, I., 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=Circ-ZNF609+Is+a+Circular+RNA+that+Can+Be+Translated+and+Functions+in+Myogenesis.">Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis.</a> Molecular Cell. 66 (1), 22-37 (2017).</li><li>Li, X., 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=Coordinated+circRNA+Biogenesis+and+Function+with+NF90/NF110+in+Viral+Infection.">Coordinated circRNA Biogenesis and Function with NF90/NF110 in Viral Infection.</a> Molecular Cell. 67 (2), 214-227 (2017).</li><li>Zhang, Y., 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=The+Biogenesis+of+Nascent+Circular+RNAs.">The Biogenesis of Nascent Circular RNAs.</a> Cell Reports. 15 (3), 611-624 (2016).</li></ol>

In het algemeen, cirkelvormige RNA's zijn laag overvloedig1, die de studie van hun functie en vorming compliceert. Net als bij lineaire RNAs13,maakt het gebruik van reporter minigenes de identificatie van cis en trans-acting factoren die de vorming van cirkelvormige RNAs reguleren. Deze aanpak genereert dus hypothesen die verder kunnen worden getest met behulp van de endogene genen.
De meest kritische stap is het ontwerp van het reporter gen. De ...

Circulaire RNA's Circulaire RNAs (circRNAs) zijn covalently gesloten enkele gestrande RNAs die worden uitgedrukt in de meeste organismen. Ze worden gegenereerd door zich aan te sluiten bij een downstream 5' splice site naar een upstream 3 ' splice site, een proces genaamd back-splicing (Figuur 1A)1. Sequenties in het pre-mRNA die basis complementair vertonen zo kort als 30-40 nt brengen back-splice sites in de juiste uitlijning voor circRNA vorming2. Bij de mens vormen Alu-elementen 1, die ongeveer 11% van het genoom3vertegenwoordigen , uitgebreide dubbelstrengs RNA-structuren in pre-mRNA vanwege hun zelfcomplementariteit4,5 en bevorderen zo de vorming van circRNAs1.
Momenteel zijn drie belangrijke functies van circRNAs beschreven. Sommige circRNAs binden microRNAs (miRNAs) en door sekwestratie fungeren als miRNA sponzen6. CircRNAs zijn betrokken bij transcriptie- en posttranscriptieregeling, door concurrentie met lineaire splicing7 of modulatie van transcriptiefactoractiviteit8. Ten slotte bevatten circRNAs korte open leeskaders en bewijzen van principestudies tonen aan dat ze kunnen worden vertaald9,10. De functie van de meeste circRNAs blijft echter raadselachtig. De meeste circulaire RNA's zijn gedetecteerd met behulp van volgende generatie sequencing methoden11. Gedetailleerde analyses van individuele genen met behulp van gerichte RT-PCR benaderingen blijkt dat een groot aantal circulaire RNAs nog moet worden ontdekt12.
Gebruik van reporter genen om pre-mRNA verwerking te analyseren De analyse van mRNA afgeleid van DNA reporter construeert transfected in cellen is een gevestigde methode om alternatieve pre-mRNA splicing te bestuderen, die kan worden toegepast op cirkelvormige RNAs. In het algemeen worden de alternatieve exon, de omringende intronen en constitutieve exonen versterkt en gekloond tot een eukaryotische expressievector. Vaak worden de introns ingekort. De constructies worden omgezet in eukaryotische cellen en meestal geanalyseerd door RT-PCR13,14. Deze aanpak is op grote schaal gebruikt om regelgevende splice-sites en trans-acting factoren in co-transfection experimenten13,15,16,17,18in kaart te brengen . Bovendien kon de productie van eiwituitdrukkende minigenen worden gescreend op stoffen die alternatieve splicing19,20veranderen .
De methode is toegepast op circulaire RNA's. Momenteel zijn ten minste 12 minigene backbones beschreven in de literatuur en samengevat in tabel 1. Met uitzondering van het op tRNA gebaseerde expressiesysteem21,22zijn ze allemaal afhankelijk van polymerase II-promotors. Hier beschrijven we een methode om menselijke reporter minigenen te genereren om cis en trans-acting factoren die betrokken zijn bij de generatie van circulaire RNAs te bepalen. Een overzicht van de methode met behulp van sequenties van een gepubliceerd reporter gen23 is te zien in figuur 1.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(PEI) Hydrochloride</td>
			<td>Polysciences</td>
			<td>24765-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Builder tool</td>
			<td>NEB</td>
			<td></td>
			<td><a href="https://nebuilder.neb.com/#!/" target="_blank">https://nebuilder.neb.com/#!/</a></td>
		</tr>
		<tr>
			<td>Dark Reader Transilluminator.</td>
			<td>Clare Chemical Research</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic DNA assembly kit</td>
			<td>NEB</td>
			<td>E2621S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel and PCR cleanup kit</td>
			<td>Promega</td>
			<td>A9282</td>
			<td></td>
		</tr>
		<tr>
			<td>Glyco Blue</td>
			<td>Thermo Fisher</td>
			<td>AM9516</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.1 cloning site</td>
			<td>Polycloning site</td>
			<td></td>
			<td><a href="https://assets.thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_1_man.pdf" target="_blank">https://assets.thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_1_man.pdf</a></td>
		</tr>
		<tr>
			<td>Polymerase 1</td>
			<td>NEB</td>
			<td>M0491L</td>
			<td>Q5 DNA polymerase</td>
		</tr>
		<tr>
			<td>Polymerase 2</td>
			<td>Biorad</td>
			<td>1725310</td>
			<td>Long range polymerase (NEB), iproof (BioRad)</td>
		</tr>
		<tr>
			<td>Polymerase 2</td>
			<td>Qiagen</td>
			<td>206402</td>
			<td>Qiagen long range polymerase kit</td>
		</tr>
		<tr>
			<td>Reverse Transcriptase</td>
			<td>Thermo Fisher</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA isolation kit</td>
			<td>Life Technologies</td>
			<td>12183025</td>
			<td>Ambion by Life Technologies</td>
		</tr>
		<tr>
			<td>RNAse R</td>
			<td>Lucigen</td>
			<td>RNR07250</td>
			<td>Epicenter/Lucigen</td>
		</tr>
		<tr>
			<td>Stable competent cells</td>
			<td>NEB</td>
			<td>C3040H</td>
			<td>NEB stable cells</td>
		</tr>
		<tr>
			<td>Standard cloning bacteria</td>
			<td>NEB</td>
			<td>C2988J</td>
			<td>NEB5-alpha competent</td>
		</tr>
		<tr>
			<td>Web tool to design primers</td>
			<td>NEB</td>
			<td></td>
			<td><a href="https://nebuilder.neb.com/#!/" target="_blank">https://nebuilder.neb.com/#!/</a></td>
		</tr>
		<tr>
			<td>Web-based temperature calculations</td>
			<td>NEB</td>
			<td></td>
			<td><a href="https://tmcalculator.neb.com/#!/main" target="_blank">https://tmcalculator.neb.com/#!/main</a></td>
		</tr>
	</tbody>
</table>

use of alu element containing minigenes to analyze circular rnas

Naast lineaire mRNAs genereren veel eukaryotische genen circulaire RNA's. De meeste circulaire RNAs worden gegenereerd door lid te worden van een 5 'splice site met een upstream 3 ' splice site binnen een pre-mRNA, een proces genaamd back-splicing. Deze circularisatie wordt waarschijnlijk geholpen door secundaire structuren in het pre-mRNA die de splice sites in de nabijheid brengen. In menselijke genen wordt gedacht dat Alu-elementen deze secundaire RNA-structuren bevorderen, omdat Alu-elementen overvloedig aanwezig zijn en basiscomplementariteiten met elkaar vertonen wanneer ze in tegengestelde richtingen aanwezig zijn in het pre-mRNA. Hier beschrijven we de generatie en analyse van grote, Alu-element met reportergenen die cirkelvormige RNA's vormen. Door optimalisatie van kloonprotocollen kunnen reporter genen met een afstandslengte tot 20 kb worden gegenereerd. Hun analyse in co-transfection experimenten maakt het mogelijk de identificatie van regelgevende factoren. Zo kan deze methode identificeren RNA sequenties en cellulaire componenten die betrokken zijn bij cirkelvormige RNA vorming.

Reporter genen maken bepaling van regelgevende factoren die van invloed zijn op circulaire RNA vorming. Echter, deze reporter genen zijn groot en bevatten repetitieve elementen die vaak DNA-constructies instabiel. Vanwege hun grote omvang is het vaak noodzakelijk om delen van de introns te verwijderen, wat wordt bereikt door het versterken van genomische stukken met de exonen en kleinere flankerende intronic-delen. Deze DNA-stukken zijn enzymatisch geassembleerd, waardoor de bouw zonder beperking enzymen.
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Watch this Scientific Journal Video about Use of Alu Element Containing Minigenes to Analyze Circular RNAs at JoVE.com

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We klonen en analyseren reporter genen die circulaire RNA's genereren. Deze reporter genen zijn groter dan constructies om lineaire splicing te analyseren en Alu-elementen bevatten. Om de cirkelvormige RNAs te onderzoeken, worden de constructies omgezet in cellen en wordt resulterend RNA geanalyseerd met behulp van RT-PCR na verwijdering van lineair RNA.

Gebruik van Alu Element met minigenen om circulaire RNA's te analyseren

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Dit protocol is belangrijk omdat het de gebruiker in staat stelt om een genomisch expressiesysteem te genereren dat alternatieve splicing kan ondergaan en in dit geval circulaire RNAs kan vormen die kunnen worden getest op hun functie en ziekteassociatie. Het belangrijkste voordeel van deze techniek is dat het de weg zal effenen voor anderen om dit protocol te gebruiken in de moleculaire biologie en klonen bij het bestuderen van alternatieve splicing in circulaire RNA formatie en functie. Mijn advies aan iemand proberen deze techniek voor de eerste keer zou zijn om de PCR voorwaarden te optimaliseren.Voer verlopen uit of voer touchdown PCR uit met verschillende temperaturen en verlengingstijden. Meestal langere fragmenten meer dan zes KB zal een KB per cyclus uit te breiden en verschillende annealing temperaturen zou moeten worden getest op de beste versterking. Demonstratie van deze methode is van cruciaal belang omdat het de gebruikers een betere visuele weergave van de moeilijkere aspecten van de procedure met name de generatie van primers zal geven.Om deze procedure te beginnen, laadt u de UCSC Genome Browser en gebruikt u deze om repetitieve elementen te identificeren die nodig zijn voor de vorming van cirkelrna en deze in de constructies op te nemen. Belangrijk is dat primers voor versterking buiten de repetitieve elementen moeten staan. Plak de cirkelvormige RNA-sequentie in het menselijke BLAT-zoeken en selecteer het juiste organisme.Verzend de volgorde, ga naar de browserweergave en zoom 1,5 timers of naar gelang van het geval uit. Vervolgens muis over de repetitieve elementen om hun subtype te identificeren in een zwevend venster. Alu elementen zijn in de sinuslijn.Gebruik de standaardtrackingknop onder het venster om de browser opnieuw in te stellen als er een onjuiste afbeelding wordt verkregen. Ga eerst naar het bekijken, DNA op de bovenste lijn van de USCS Genome Browser om de DNA-sequentie in het venster te downloaden. Selecteer in de optie opmaak voor sequencing de optie Uitgebreide aanvraag-/kleuropties.Selecteer de standaardcase lager en selecteer de schakelcase voor NCBI RefSeq. Selecteer onderstrepen en vet en cursisch voor RepeatMasker. Klik op verzenden.Er zullen exons als hoofdletters en intronen als kleine letters. Controleer de exon/intron borders. Als de browser de omgekeerde aanvulling weergeeft, gaat u terug en selecteert u het omgekeerde complementvak totdat de juiste exon/intronranden worden weergegeven.Kopieer vervolgens het bestand met de juiste afdrukstand naar een tekstverwerkingsdocument en markeer de exonen. Selecteer de te versterken fragmenten, zodat de intron niet begint of eindigt in een repetitief gebied, omdat primers in deze regio's specifieke sequenties niet versterken. Gebruik om te beginnen een webtool om de primers voor klonen te ontwerpen.Voeg voor de vectorsequentie de invoegplaats toe als laatste nucleotide en voeg vervolgens de fragmenten toe. Aangezien de vectornummering niet begint met een bepaalde invoegplaats, bevindt de plaats van invoegpositie in de vector zich en wordt het downstream-gedeelte voor de upstream-sequentie geplaatst. Pas primers aan als hun smeltpunten meer dan vier graden Celsius uit elkaar liggen en niet werken in versterking.In deze studie worden reportergenen die circulaire RNAs genereren gekloond en geanalyseerd. Geoptimaliseerde PCR producten worden gescheiden op een 1%agarose gel met 1X gel groen. De afzonderlijke banden vertegenwoordigen de PCR-producten die zullen worden gebruikt in enzymatische DNA-assemblage.Deze banden worden vervolgens uit de gel gesneden en gezuiverd. Het gezuiverde PCR-product voldoet niet aan de verwachte grootte met gelgroen, zodat de producten ook gescheiden zijn op een 1%agarose gel die vervolgens wordt bevlekt met ethidium bromide om ervoor te zorgen dat de producten de juiste grootte hebben. Om te beginnen, zet een enzymatische DNA assemblage kit.Combineer de vector en inserts in een kiesverhouding van één tot twee. Voeg vervolgens 10 microliter dna-assemblagemastermix toe. Incubeermonsters gedurende 60 minuten bij 50 graden.Ontdooi bevoegde cellen op ijs tijdens incubatie. Cellen moeten in een volume van 50 microliter. Transformeer vervolgens competente cellen met de totale montagereactie.Voeg twee microliter van het gekoelde geassembleerde product toe aan de bevoegde cellen. Meng door zachtjes flicking de buis vier tot vijf keer. Niet vortex.Zet het mengsel 30 minuten op ijs. Verwarm schok het mengsel op 42 graden Celsius gedurende 30 seconden. Zet het dan twee minuten terug op het ijs.Voeg hierna 950 microliters van soc-media bij kamertemperatuur toe aan de buis. Incubeer op 37 graden Celsius gedurende 60 minuten tijdens het schudden op 300 RPM. Tijdens deze incubatie, warme twee selectieplaten die het juiste antibioticum bevatten.Na de incubatiecentrifugeren de reactiebuis gedurende 30 seconden op 10,000 g om de cellen te pelleteren en 25% van de cellen op de ene selectieplaat en 75% op de andere plaat uit te voeren. Broed deze platen 's nachts bij 37 graden Celsius. Voordat u begint met transfectie, controleer uw DNA door beperking verteren.Hier wordt een representatieve tau minigene die exons negen tot en met 12 bevat gesneden met beperkingsenzymen die zijn aangegeven om grote recombinaties uit te sluiten. Los voor transfectie eerst lineair polyethyleenhydrchloride op in water bij een concentratie van één milligram per milliliter bij pH twee. Gebruik natriumhydroxide om de pH tot zeven te brengen en steriel filter de oplossing met een filter van 0,22 micrometer.Bewaar de oplossing op vier graden Celsius tot het gebruiksklaar is. Splits vervolgens de cellen in de putten van een zes-put plaat en laat ze groeien 's nachts op DMEM media met 10%FBS. De volgende dag, aliquot een microgram van het gerapporteerde gen in een steriele buis en voeg 200 microliter van steriele gefilterd 150 millimolar natriumchloride.Meng door vortexing. Voeg vervolgens de polyethyleenoplossing toe aan dit mengsel met een verhouding van drie microliters polyethyleen voor elk microgram DNA. Centrifuge kort om monsters te verzamelen aan de onderkant van de buis.Incubeer de monsters op kamertemperatuur gedurende 10 minuten. Voeg het vervolgens direct toe aan HEK293-cellen. Incubeer de cellen 's nachts bij 37 graden Celsius met 5%kooldioxide.De volgende dag gebruikt u een RNA-isolatiekit om het RNA voor RT-PCR te isoleren. cDNA uit twee monsters afkomstig van menselijke hersenweefsels worden versterkt met cirkelvormige RNA primers circ tau exon 1210 reverse en circ tau exon 1211 naar voren. Terwijl de verwachte band die overeenkomt met tau cirkel RNA wordt gezien, de andere sterke banden zijn artefacten die niet overeenkomen met het menselijk genoom.Dit experiment wordt herhaald met identieke PCR voorwaarden, maar de omgekeerde transcriptie werd alleen uitgevoerd met de circ tau exon 1210 reverse primer. Alleen de verwachte band wordt versterkt en gevalideerd door middel van sequencing. Het RNA wordt vervolgens behandeld met RNase R die lineair RNA verwijdert.Het cirkelvormige RNA is detecteerbaar na de behandeling, terwijl lineair RNA niet langer een detecteerbaar signaal geeft. RNA is geïsoleerd 24 uur na de transfectie en geanalyseerd door RT-PCR. Versterking van het lineaire tau mRNA toont twee banden als gevolg van alternatieve splicing van exon 10.Hun verhouding verandert in de over expressie van splicing factoren. Versterking van de circulaire 1210 tau RNA toont de afhankelijkheid van tau circ RNA expressie op de expressie van een aantal splicing factoren met name de Cdc2-achtige kinase CLK2 en het SR-eiwit 9G8. Het belangrijkste om te onthouden bij een poging deze procedure is het ontwerp en de locatie van de primers bij de bouw van een minigene.De primer mag niet in repetitieve elementen liggen en moet onder verschillende omstandigheden worden geoptimaliseerd, afhankelijk van het fragment dat wordt versterkt. Na deze procedure zullen andere methoden die kunnen worden uitgevoerd, het testen van de functie van de circulaire RNAs zijn. De gebruikers kunnen vertaling of sekwestratie van eiwitten testen om een functie te identificeren voor het specifieke circulaire RNA waarin de gebruiker geïnteresseerd is.Deze techniek maakte de weg vrij om genomische fragmenten te gebruiken in een minigeen systeem dat de circulaire RNAs kan uitdrukken, waardoor de gebruiker zijn functie kan testen en identificeren en in sommige aspecten hun associatie met ziekte kan. De implicatie van deze techniek strekt zich uit tot potentiële therapieën en diagnostiek van een bepaalde ziekte, bijvoorbeeld tauopathieën, neurodegeneratieve ziekten geassocieerd met tau pathologie. We hebben ontdekt dat het microtubuli-geassocieerde eiwit tau, bekend als MAPT, circulaire RNAs genereert waarvan wij geloven dat ze bijdragen aan de ziektepathologie en deze methode stelt ons in staat om deze circulaire RNAs volledig te bestuderen en hun functie en relatie met ziekten te identificeren.Deze methode zou inzicht kunnen geven in een beter begrip van circulaire RNAs, wat hun functies zijn en de rol die zij kunnen spelen bij bepaalde ziekten. Deze methode kan worden toegepast bij het klonen van andere minigenen die circulaire RNAs uitdrukken over soorten waar het inzicht kan geven in hun functie. Versterking van de PCR-producten blijkt het moeilijkst te zijn met lange fragmenten versterkt uit genomisch DNA, samen met het hebben van meerdere repetitieve elementen in het fragment.

use-of-alu-element-containing-minigenes-to-analyze-circular-rnas

Research

JoVE Journal

Biochemistry

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

De combinatie van natte en droge Lab Technieken om de kristallisatie van grote opgerolde-coil bevattende eiwitten Gids

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Methode om te visualiseren en te analyseren Membrane interagerende eiwitten door Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

Kwantificering van de overvloed en het opladen van de niveaus van overdracht RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

Gelijktijdige meting van Superoxide/waterstofperoxide en NADH productie door Flavin-bevattende mitochondriale Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

Gebruik van twee dimensionale semi-denatureren wasmiddel Agarose gelelektroforese te bevestigen grootte heterogeniteit van amyloïde of Amyloid-achtige vezels

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

De grootschalige productie van recombinante RNAs op een circulaire steiger met behulp van een systeem van Viroïde afkomstige in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Expressie, zuivering, kristallisatie, en enzym assays van Fumarylacetoacetate hydrolase domein-bevattende eiwitten

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

Gebruik van thermoforese op microschaal om eiwit-lipide-interacties te meten

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a multitude of samples. It has been extensively used in many research areas and achieved industrial application in fields such as human diagnostics and trait selection in crops of genetically modified organisms (GMO) crops. However, qPCR is not an error-proof technique. Mixing all reagents into a single master mix subsequently distributed onto 96 wells of a regular qPCR plate might lead to operator mistakes such as incorrect mixing of reagents or inaccurate dispensing into the wells. Here, a technique called gelification is presented, whereby most of the water present in the master mix is substituted by reagents that form a sol-gel mixture when submitted to a vacuum. As a result, qPCR reagents are effectively preserved for a few weeks at room temperature or a few months at 2-8 oC. Details of preparing each solution are shown here along with the expected aspect of a gelified reaction designed to detect&#160;T. cruzi satellite DNA (satDNA). A similar procedure can be applied to detect other organisms. Starting a gelified qPCR run is as simple as removing the plate from the refrigerator, adding the samples to their respective wells, and starting the run, thus decreasing the setup time of a full-plate reaction to the time it takes to load the samples. Additionally, gelified PCR reactions can be produced and controlled for quality in batches, saving time and avoiding common operator mistakes while running routine PCR reactions.

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

The present work describes the steps for producing ready-to-use qPCR for T. cruzi DNA detection that can be pre-loaded on the reaction vessel and stored in the refrigerator for several months.

Kant-en-klare qPCR voor detectie van DNA van Trypanosoma cruzi of andere pathogene organismen

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a ...

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

Formulering en karakterisering van bioactieve stof die nanoschijven bevat

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...