We thank Anne Wochele for her assistance in the laboratory and Vera Augsburger for help with the automated gel sequencer. We thank the Deutsche Forschungsgemeinschaft for funding by grants BE4038/2 and BE4038/5 within the &#8220;priority programmes&#8221; SPP1316 and SPP1617.

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescently+labeled+oligonucleotide+extension:+a+rapid+and+quantitative+protocol+for+primer+extension.">Fluorescently labeled oligonucleotide extension: a rapid and quantitative protocol for primer extension.</a> Biotechniques. 35, 90-94 (2003).</li><li>Schuster, C. F., 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=Characterization+of+a+mazEF+Toxin-Antitoxin+Homologue+from+Staphylococcus+equorum.">Characterization of a mazEF Toxin-Antitoxin Homologue from Staphylococcus equorum.</a> J. Bacteriol. 195, 115-125 (2013).</li><li>Nolle, N., Schuster, C. F., Bertram, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+paralogous+yefM-yoeB+loci+from+Staphylococcus+equorum+encode+functional+toxin-antitoxin+systems.">Two paralogous yefM-yoeB loci from Staphylococcus equorum encode functional toxin-antitoxin systems.</a> Microbiology. 159, 1575-1585 (2013).</li><li>Yamaguchi, Y., Park, J. H., Inouye, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxin-antitoxin+systems+in+bacteria+and+archaea.">Toxin-antitoxin systems in bacteria and archaea.</a> Annu. Rev. Genet. 45, 61-79 (2011).</li><li>Schuster, C. F., Bertram, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxin-antitoxin+systems+are+ubiquitous+and+versatile+modulators+of+prokaryotic+cell+fate.">Toxin-antitoxin systems are ubiquitous and versatile modulators of prokaryotic cell fate.</a> FEMS Microbiol. Lett. 340, 73-85 (2013).</li><li>Goeders, N., Van Melderen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxin-antitoxin+systems+as+multilevel+interaction+systems.">Toxin-antitoxin systems as multilevel interaction systems.</a> Toxins. 6, 304-324 (2014).</li><li>Yamaguchi, Y., Inouye, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+interferases,+sequence-specific+endoribonucleases+from+the+toxin-antitoxin+systems.">mRNA interferases, sequence-specific endoribonucleases from the toxin-antitoxin systems.</a> Progress in molecular biology and translational science. 85, 467-500 (2009).</li><li>Park, J. H., Yamaguchi, Y., Inouye, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+subtilis+MazF-bs+(EndoA)+is+a+UACAU-specific+mRNA+interferase.">Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase.</a> FEBS Lett. 585, 2526-2532 (2011).</li><li>Zhu, 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=et+al.Staphylococcus+aureus+MazF+specifically+cleaves+a+pentad+sequence,+UACAU,+which+is+unusually+abundant+in+the+mRNA+for+pathogenic+adhesive+factor+SraP.">et al.Staphylococcus aureus MazF specifically cleaves a pentad sequence, UACAU, which is unusually abundant in the mRNA for pathogenic adhesive factor SraP.</a> J. Bacteriol. 191, 3248-3255 (2009).</li><li>Zhu, 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=The+mRNA+interferases,+MazF-mt3+and+MazF-mt7+from+Mycobacterium+tuberculosis+target+unique+pentad+sequences+in+single-stranded+RNA.">The mRNA interferases, MazF-mt3 and MazF-mt7 from Mycobacterium tuberculosis target unique pentad sequences in single-stranded RNA.</a> Mol. Microbiol. 69, 559-569 (2008).</li><li>Fu, Z., Donegan, N. P., Memmi, G., Cheung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+MazFSa,+an+endoribonuclease+from+Staphylococcus+aureus.">Characterization of MazFSa, an endoribonuclease from Staphylococcus aureus.</a> J. 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Microbiol. 5, 501-509 .</li><li>Yu, H., Goodman, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+HIV-1+and+avian+myeloblastosis+virus+reverse+transcriptase+fidelity+on+RNA+and+DNA+templates.">Comparison of HIV-1 and avian myeloblastosis virus reverse transcriptase fidelity on RNA and DNA templates.</a> J. Biol. Chem. 267, 10888-10896 (1992).</li><li>Ying, B. W., Fourmy, D., Yoshizawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+of+the+use+of+radioactivity+by+fluorescence+for+biochemical+studies+of+RNA.">Substitution of the use of radioactivity by fluorescence for biochemical studies of RNA.</a> RNA. 13, 2042-2050 (2007).</li></ol>

The authors have nothing to disclose that would present a conflict of interest.

Primer extension fluorescente è un metodo semplice e rapido per determinare l&#39;estremità 5 &#39;di RNA, sia per TSP- o l&#39;identificazione di trasformazione secondaria dell&#39;RNA. Grazie all&#39;uso di primer fluorescenti, le reazioni possono essere impostati e gestiti senza misure di sicurezza supplementari (a differenza in caso di primer marcati radioattivamente). Poiché i campioni vengono rilevati mediante fluorescenza, possono essere esposte mentre l&#39;elettroforesi è in corso che consente l&#39;analisi...

Estensione Primer 1 è un metodo molecolare per determinare l&#39;estremità 5 &#39;di molecole di RNA specifici fino ad una risoluzione di una base. Il vantaggio di altri metodi come 5&#39;-RACE (amplificazione rapida delle estremità del cDNA) è il tempo di ritorno veloce e la capacità di analizzare facilmente una miscela di diverse lunghezze di molecole di RNA. Questo metodo funziona sottoponendo molecole di RNA per invertire le reazioni di trascrizione utilizzando primer fluorescenti specifiche, generando frammenti di cDNA di alcune lunghezze. Queste molecole di cDNA vengono eseguiti insieme reazioni di sequenziamento tradizionali Sanger 2 su gel di poliacrilammide denaturante e possono essere rilevate da loro fluorescenza a causa dell&#39;uso di primer fluorescente. Le lunghezze dei frammenti di cDNA vengono quindi valutati rispetto alla scala sequenziamento, consentendo la mappatura delle estremità 5 &#39;RNA. Tradizionalmente, le reazioni di estensione dei primer sono utilizzati in combinazionecon isotopi radioattivi per rilevare molecole di cDNA su film X-ray. A causa di rischi per la salute, problemi di smaltimento e maneggevolezza, protocolli più recenti utilizzano fluorescenza per la rilevazione del primer extension con sequenziatori automatici, benché la loro sensibilità è leggermente inferiore. Utilizzando primer fluorescente, la procedura di radio-marcatura ricorrenti può essere omesso, come primer fluorescenti sono stabili per un lungo periodo di tempo (più di un anno nelle nostre mani). Il metodo che descriviamo qui utilizza un sequenziatore automatizzato gel, ma con lievi modifiche, sequenziatori capillari può essere utilizzato anche per la separazione cDNA e rilevamento 3. Il parallelismo tra analisi del gel permette di rilevare anche una piccola quantità di RNA scissione o trasformazione. Un altro vantaggio è l&#39;alta risoluzione di questo metodo, come scissione o la trasformazione anche di una sola base del terminale può essere rilevato. Per quanto riguarda la rilevazione di RNA scissione o trasformazione, typically due diversi tipi di estensioni di primer si distinguono. In un caso, il trattamento enzimatico è fatto in vitro utilizzando RNA purificato e l&#39;enzima purificato, mentre nell&#39;altro caso, la lavorazione avviene in vivo e l&#39;RNA risultante viene purificato. In entrambi i casi l&#39;RNA viene sottoposto ad un primer extension effettuata in vitro, tuttavia, a seconda della fonte di RNA, il metodo o è chiamato un primer extension vivo o in vitro. Nel protocollo presentiamo, ci concentriamo esclusivamente sulla vivo primer extension in, a causa della facilità d&#39;uso (non proteine ​​purificate necessario) e la possibilità di determinare punti di partenza trascrizionali e trasformazione in una sola volta. Tuttavia, in vitro estensioni di primer sono in linea di principio configurare allo stesso modo e questo protocollo può servire come punto di partenza. Il metodo qui illustrato può essere applicato a molte specie batteriche purché siano suscettibili di altapurezza e alto rendimento preparazione di acidi nucleici. La ricerca nel nostro laboratorio si concentra sulla portata normativa di tossina-antitossina (TA) Sistemi di 4,5, un campo in cui è ampiamente utilizzato il metodo primer extension. TA-sistemi sono piccoli elementi genetici presenti nei genomi procariotici che consistono di una proteina tossica stabile e endogenamente attiva e una proteina o RNA antitossina lo più instabile che contrasta la tossicità 6,7. Attività di tossina è a volte esercitata da inibizione della replicazione, sintesi della parete cellulare o altri meccanismi, ma il più delle volte per attività RNasi 8,9. Tipicamente, RNasi specificità è determinata effettuando diverse prove, una delle quali è il metodo di primer extension. Reazioni primer extension sono adatti per questa applicazione, come miscela di frammenti di lunghezza clivati ​​e completi possono essere analizzati simultaneamente per determinare la loro 5 &#39;estremità. Utilizzando un mix di in vitro e in vivo estensioni di primer, laspecifica RNasi clivaggio tossina, ad esempio, specificità di sequenza può essere determinata 10-13. <img alt="Figura 1" fo:content-width="6in" src="/files/ftp_upload/52134/52134fig1highres.jpg" width="600" /> Figura 1. Panoramica della procedura di primer extension. Le colture batteriche sono incubate e trattati in base alle esigenze sperimentali. L&#39;RNA totale viene estratto dalle cellule, trattate con DNasi I per rimuovere tracce di DNA e sottoposti ad una reazione di trascrizione inversa utilizzando bersaglio specifici primer di DNA fluorescenti rendimento cDNA. Il DNA genomico o plasmidi vengono estratti e successivamente utilizzati per fluorescenti reazioni di sequenziamento Sanger per il confronto dimensioni con i frammenti di cDNA. Prodotti primer extension vengono eseguiti insieme a prodotti di sequenziamento Sanger su un gel denaturante di poliacrilammide di urea e analizzati con un laser automatico e microscopio. La base sequenziamento che si allinea con la band cDNA è il lav base 5 &#39;del cDNA (freccia blu). Maggiori informazioni in Fekete, et al. 3 <a href="https://www.jove.com/files/ftp_upload/52134/52134fig1highres.jpg" target="_blank">Fate clic qui per visualizzare una versione più grande di questa figura.</a> Una panoramica di tutta la procedura di primer extension può essere trovato in Figura 1. In breve, le cellule batteriche vengono coltivate, raccolte, il pellet cellulare lisato e l&#39;RNA estratto. RNA purificato viene quindi trattato con DNasi I per rimuovere tracce di molecole di DNA che potrebbero agire come modelli per la trascrittasi inversa. Primer fluorescenti specifici sono aggiunti al RNA, ibridato alla regione di interesse e successivamente trascrizione inversa, con conseguente DNA a singolo filamento complementare (cDNA). Una scaletta sequenziamento è stato creato da sequenziamento Sanger tradizionale impiegando primer fluorescenti e separati su un gel di poliacrilammide denaturante a fianco del primer frammenti di estensione di cDNA. La risultantegel viene analizzato confrontando le bande fluorescenti, che permette l&#39;identificazione delle estremità 5 &#39;di interesse. Punti di partenza di trascrizione e siti di lavorazione sono poi valutato individualmente in sequenza comparazioni.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;"><a name="RANGE!A1:D16">Name</a></td>
			<td style="width:196px;">Supplier</td>
			<td style="width:244px;">Catalog Number(s)</td>
			<td style="width:363px;">Comment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">AMV Reverse Transcriptase (20-25 U/&#181;l)</td>
			<td style="width:196px;">NEB / Roche</td>
			<td style="width:244px;">NEB: M0277-T / Roche: 10109118001</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:399px;">DNase I (RNase free)</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td style="width:244px;">AM2222</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">FastPrep-24 Instrument&#160;</td>
			<td style="width:196px;">MPBio</td>
			<td align="right">116004500</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:399px;">Fluorescently labeled primers</td>
			<td style="width:196px;">Biomers</td>
			<td style="width:244px;">n/a</td>
			<td style="width:363px;">5&#8217; DY-681 modification of &#8220;ordinary&#8221; DNA oligonucleotides. Compatible dyes such as the LICOR IRDye 700/800 are also available from other suppliers such as IDTdna.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Li-Cor 4200 Sequencer incl. ImagIR Data collection software</td>
			<td style="width:196px;">Li-Cor</td>
			<td style="width:244px;"></td>
			<td style="width:363px;">Product discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">NanoDrop 2000</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Nuclease free water</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td style="width:244px;">AM9915G</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Plasmid mini preparation kit</td>
			<td style="width:196px;">QIAGEN</td>
			<td align="right">12125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">RapidGel-XL-40% Concentrate</td>
			<td style="width:196px;">USB</td>
			<td style="width:244px;">US75863</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">RNA STORAGE BUFFER</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td>AM7000</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:399px;">Roti-Aqua-P/C/I</td>
			<td style="width:196px;">Carl Roth</td>
			<td style="width:244px;">X985.3</td>
			<td style="width:363px;">Alternative: &#8220;Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)&#8221; from Ambion (AM9720)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">SUPERase&#8226;In RNase Inhibitor</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td style="width:244px;">AM2696</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:399px;">Thermo Sequenase fluorescently labelled primer cycle sequencing kit with 7-deaza-dGTP</td>
			<td style="width:196px;">GE Healthcare</td>
			<td style="width:244px;">RPN2538</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">TRIzol reagent</td>
			<td style="width:196px;">life technologies</td>
			<td style="width:244px;">15596-026</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Zirconia/Silica Beads 0.1 mm</td>
			<td style="width:196px;">BioSpec</td>
			<td style="width:244px;">11079101z</td>
			<td style="width:363px;"></td>
		</tr>
	</tbody>
</table>

fluorescence based primer extension technique to determine transcriptional starting points and cleavage sites of rnases in vivo

Fluorescenza estensione primer a base (FPE) è un metodo molecolare per determinare i punti di partenza di trascrizione o siti di trasformazione di molecole di RNA. Ciò è ottenuto mediante trascrizione inversa di RNA di interesse utilizzando specifici primer fluorescente e successiva analisi dei frammenti di cDNA risultante dalla elettroforesi su gel denaturante di poliacrilammide. Contemporaneamente, un tradizionale reazione di sequenziamento Sanger viene eseguito sul gel per mappare le estremità dei frammenti di cDNA alle loro basi corrispondenti esatti. In contrasto con 5&#39;-RACE (Rapid Amplification di cDNA Ends), in cui il prodotto deve essere clonato e sequenziato più candidati, la maggior parte dei frammenti di cDNA generato dall&#39;estensione del primer può essere simultaneamente rilevata in una corsa di gel. Inoltre, l&#39;intera procedura (da trascrizione inversa di analisi finale dei risultati) può essere completato in un giorno lavorativo. Usando primer fluorescente, l&#39;uso di isotopi radioattivi pericolosi reagenti etichettatopossono essere evitati e tempi di lavorazione sono ridotti come i prodotti possono essere rilevati durante la procedura di elettroforesi. Nella seguente protocollo, si descrive un metodo di estensione in vivo di primer fluorescente per rilevare in modo affidabile e rapido l&#39;estremità 5 &#39;di RNA dedurre trascrizionale punti di partenza e siti di lavorazione RNA (ad esempio, dai componenti di sistema tossina-antitossina) a S. aureus, E. coli e altri batteri.

Come illustrato nella figura 6, una reazione di primer extension può essere utilizzato per determinare i punti di partenza trascrizionali di trascritti di interesse e può aiutare a dedurre regioni promotrici (tipicamente identificati da -10 e -35 elementi). Il più alto (più lunga) frammento di cDNA rappresenta il &#39;estremità del mRNA 5 e quindi può essere facilmente mappato rispetto alla scala sequenziamento. <img alt="Figura 6" src="/files/ftp_upload/52134/52134fig6...

Watch this Scientific Journal Video about Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo at JoVE.com

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

Fluorescenza base tecnica Primer Extension determinare trascrizionali punti di partenza e siti di taglio di RNasi<em&gt; In Vivo</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

fluorescence-based-primer-extension-technique-to-determine

Research

JoVE Journal

Biology

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

26.8K Views.  University of Tübingen. We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

Video: Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Le cellule umane ES: Cultura partire da cellule congelate

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Ricerca

Biologia

Proboscis Extension Response (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth (labellum), there are hair-like structures called sensilla which houses taste neurons. When an attractive substance makes contact to the labellum, the fly extends its proboscis to consume the material. Proboscis Extension Response (PER) assay measures this taste behavior response, and it is a useful method to learn about food preferences in a single fly. Solutions of various sugars, such as sucrose, glucose and fructose, are very attractive to the fly. The effect of aversive substances can also be tested as reduction of PER when mixed in a sweet solution.Despite the simplicity of the basic procedure, there are many things that can prevent it from working. One of the factors that requires attention is the fly's responsive state. The required starvation time to bring the fly to the proper responsive state varies drastically from 36 to 72 hours. We established a series of controls to evaluate the fly's state and which allows screening out of non-responsive or hyper-responsive individual animals. Another important factor is the impact level and the position of the contact to the labellum, which would be difficult to describe by words. This video presentation demonstrates all these together with several other improvements that would increase the reproducibility of this method.

Proboscis Extension Response PER Assay in Drosophila

Proboscis extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the proboscis makes contact with an attractive substance, the fly extends its proboscis to consume the substance. Solutions of various sugars are very attractive to the fly.

Proboscide Estensione di risposta (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Mesoscopici fluorescenza per Tomografia In-vivo Imaging di sviluppo Drosophila</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

Primer Capture Estensione: Recupero sequenza mirata da fonti DNA fortemente degradate

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Singola molecola di imaging del regolamento Gene<em&gt; In vivo</em&gt; Utilizzo di attivazione Cotranslational da Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Un quantitativo test per studiare proteine: DNA interazioni, Discover trascrizionali regolatori dell&#39;espressione genica, e identificare nuovi agenti anti-tumorali

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

A Proteoliposome-Based Efflux Assay per determinare le proprietà singola molecola di Cl<sup&gt; -</sup&gt; Canali e trasportatori

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

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

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

I metodi per scoprire alternative Uso Promotore e regolazione trascrizionale di Murine Bcrp1

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

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.