L&#39;originale GFP-Pem1 era un dono del Dr. Lei Li. Questo lavoro è stato supportato anche da finanziamenti del NIH e la Ellison Medical Foundation di VG e AS

In questo protocollo viene descritto il metodo per l&#39;analisi di riparazione del DNA con DSB giornalista cromosomicamente integrato costruisce 1,2 DSB in cui sono indotti in vivo l&#39;espressione transiente un&#39;endonucleasi taglio raro SCEI I-3. Il test integrato offre il vantaggio di analizzare riparazione DSB nel contesto cromosomico. Tuttavia, questo protocollo richiede prolungata passaging cellula, che può essere problematico quando si lavora con le cellule primarie. Un approccio alternativo è un test extracromosomico dove vengono digeriti costrutti reporter in vitro con I-SCEI o endonucleasi HindIII e poi trasfettate in cellule il DNA lineare. Il protocollo del saggio exctrachromosomal 4 è simile al test integrato descritto qui di seguito con le seguenti modifiche. Per il saggio extracromosomico omettere l&#39;integrazione dei costrutti reporter e, invece di co-trasfezione delle cellule con 0,5 mcg di giornalista NHEJ plasmide linearizzato in vitro con I-SCEI o HindIII e 0,1 mg di pDsRed2-N1 controllo trasfezione. Per l&#39;analisi delle risorse umane co-trasfezione due reporter di HR mcg plasmide linearizzato con I-SCEI o HindIII e 0,1 mcg pDsRed2-N1. Analizzare le cellule FACS tre giorni dopo la trasfezione. Il test permette extracromosomico per l&#39;analisi di riparazione DSB in isolati primari, evitando cellula passaging ampia, e facilitando l&#39;analisi di linee cellulari multiple. 1. Costrutti reporter per NHEJ e HR La cassetta giornalista NHEJ 5 contiene un gene GFP con un ingegnerizzato 3 kb introne del gene Pem1 (GFP-Pem1). L&#39;introne Pem1 contiene un esone adenovirale affiancata da sequenze di riconoscimento per la endonucleasi HindIII e I-SCEI (Figura 1a) per l&#39;induzione di DSB. L&#39;I-SCEI siti sono in orientamento invertito. I-SCEI ha una sequenza nonpalindromic riconoscimento, quindi due siti invertita generare estremità del DNA incompatibili (Figura 1c). DNA incompatibili finisce meglio imitare il DSB naturale. La cassetta è intatta NHEJ GFP negativo come esone adenovirale sconvolge l&#39;ORF GFP. Al momento l&#39;induzione di DSB da I-SCEI, il esone adenovirale viene rimosso e NHEJ ripristina la funzione del gene GFP (Figura 2). La caratteristica unica di questo reporter NHEJ è che si può rilevare un ampio spettro di eventi NHEJ dal introne può tollerare eliminazioni e inserimenti. La cassetta giornalista HR 1 (figura 1b) si basa anche sul GFP-Pem1. Nella cassetta delle risorse umane, il primo esone del GFP-Pem1 contiene un ragazzo di 22 bp delezione combinato con l&#39;inserimento di tre siti di restrizione, I-SceI/HindIII/I-SceI. La cancellazione assicura che GFP non può essere ricostituito da un evento NHEJ. I due I-SCEI siti sono in orientamento invertito, in modo che io-SCEI digestione lascia finisce incompatibili (Figura 1c). La prima copia di GFP-Pem1 è seguita da un promoter-less/ATG-less primo esone e introne di GFP-Pem1. Il costrutto è intatto GFP-negativo. Su induzione di un DSB di I-SCEI digestione del gene GFP funzionale è ricostituito da conversione genica intramolecolare o intermolecolare tra le due copie mutate del primo GFP-Pem1 esone. Dal momento che la seconda copia del gene GFP che manca il primo codone ATG e il secondo esone, attraversando o single-strand annealing non ripristinerà l&#39;attività GFP. Questo design permette la rilevazione esclusiva di conversione genica, che è la via prevalente delle risorse umane in cellule di mammifero (Figura 3). 2. Integrazione del Reporter Costruisce nel genoma <ol><li> Fai una preparazione di alta qualità di giornalista o NHEJ HR plasmide. Per ottenere i migliori risultati, utilizzare Qiagen Endo Kit gratuito. Misurare la concentrazione di plasmide e la purezza di assorbanza a 260/280 nm. </li><li> Linearizzare 10 mcg del reporter plasmide da digerire con 50 U di NheI in 50 microlitri (volume totale della reazione) per 6 ore a 37 ° C incubatore (non utilizzare il blocco secco, per evitare l&#39;evaporazione). Purificare il DNA dalla soluzione digestione con Qiagen QiaexII Kit, eluire il DNA in 20 ml di 10 mM Tris-HCl pH 8.0. Misurare la concentrazione e la purezza del DNA mediante assorbimento a 260/280 nm. Di solito, si perde il 20-30% di DNA durante la purificazione. Confermare la resa e la digestione eseguendo una piccola aliquota (2 mL) su un gel, insieme a un giornalista non digerito costruire. Il costrutto purificato giornalista linearizzato può essere conservato a 20 ° C. </li><li> In preparazione per la trasfezione, le cellule portano a migliori condizioni di crescita. Se partendo da una fiala congelata, dividere le celle due volte prima di trasfezione. Se a partire da un piatto di cellule confluenti, dividerle una volta. </li><li> Crescere le cellule al 70-80% confluenza (di solito due giorni, se placcato 5x10 5 cells/100 piastra mm, per i normali fibroblasti umani). E &#39;fondamentale per la migliore efficienza di trasfezione per portare le cellule in crescita logaritmica. Cultura in crescita attiva contiene cellule in fase M (visto come le cellule arrotondate attaccati alla superficie). </li><li> Trasfect le cellule utilizzando le seguenti raccomandazioni Amaxa produttore di Nucleofector. Per i fibroblasti usare la soluzione NHDF e U20 programma. Per la trasfezione, raccogliere le cellule da due piastre di 100 mm (~ 2x10 6 celle). E &#39;fondamentale non trypsinize sulle celle. Fermare tripsinizzazione appena l&#39;80% delle cellule staccato dal piatto. Risospendere le cellule in soluzione NHDF. Le cellule sono sensibili alla soluzione NHDF, quindi, minimizzare il tempo di incubazione e mescolare delicatamente le cellule. Trasfezione delle cellule con 0,5 mcg di costruire giornalista linearizzato. Queste condizioni di trasfezione, se utilizzato con normali fibroblasti umani risultato nell&#39;integrazione di una singola copia di un reporter costruzione per la maggior parte dei integrants. </li><li> Selezionare le celle con costrutti reporter di cromosomicamente integrato con l&#39;aggiunta di 1 mg / mL geneticin (G418) 24 ore dopo la trasfezione. Continua la selezione per 7-10 giorni. </li><li> Scegli le colonie resistenti G418 (si può scegliere singole colonie o in piscina i cloni resistenti). Espandere la cultura a 100 piatti circa cinque millimetri. Congelare tre piatti per un uso futuro ed espandere restanti due piastre per il numero di cella desiderata (generalmente dieci piastre 100 mm sono sufficienti per cinque trasfezioni). </li><li> Quando le cellule raggiungono il 70-80% confluenza (due giorni dopo la placcatura 5x10 5 celle per 100 mm per piastra fibroblasti), le cellule sono pronte per essere transfettate con I-SCEI esprimendo plasmide. </li></ol> 3. L&#39;induzione di DSB da Espressione transitoria di I-SCEI endonucleasi <ol><li> Trasfezione ~ 2x10 6 cellule (due piatti) della cultura logaritmico in crescita con una miscela di 5 mg I-SCEI esprimendo plasmide e 0,1 mcg pDsRed2-N1 (Clontech) come controllo di efficienza di transfezione con Amaxa Nucleofector (usare soluzione NHDF e U20 programma per fibroblasti ). </li><li> Allo stesso tempo, preparare tre controlli di calibrazione per FACS da trasfecting ~ 2x10 6 celle con (i) 5 mcg EGFP che esprimono plasmide, (ii) 5 mcg pDsRed2-N1, (iii) 5 mcg di un controllo plasmide che non esprime una proteina fluorescente (stiamo usando una codifica plasmide HPRT minigene). </li><li> Crescere le cellule per quattro giorni di tempo sufficiente per l&#39;espressione di GFP e proteine ​​DsRed. </li><li> (Opzionale) Tre giorni dopo la transfezione verificare l&#39;efficienza di trasfezione ed espressione di GFP e proteine ​​fluorescenti DsRed da microscopio invertito con filtri per la rilevazione di tali pratiche e DsRed. </li></ol> 4. Analisi della GFP + + e cellule DsRed mediante citometria di flusso <ol><li> Quattro giorni dopo la transfezione raccolta I-SCEI cellule transfettate e le cellule di controllo. Risospendere le cellule in 500 l di PBS e cellule per il trasferimento dei tubi FACS. In questa fase è fondamentale raccogliere tutte le cellule e le cellule sono di forma rotonda. Per raggiungere questo obiettivo, si consiglia di utilizzare un tempo più lungo o un tripsinizzazione tripsina più forte. Confermano che il 99% delle cellule si staccano dalla piastra e hanno forma rotonda osservando le cellule al microscopio. Mantenere le cellule in ghiaccio al riparo dalla luce (cover secchiello del ghiaccio con un foglio). E &#39;possibile conservare le cellule in ghiaccio per qualche ora. Per ottenere i migliori risultati di analizzare le cellule immediatamente dopo la raccolta. </li><li> FACS calibrare con GFP, DsRed e controlli negativi. Regolare la tensione e la compensazione del colore al fine di includere tutte le cellule fluorescenti per l&#39;analisi (Figura 4). Tenere presente che l&#39;intensità fluorescente dei campioni sperimentali saranno inferiore a quello dei controlli. </li><li> Analizzare I-SCEI trasfettate cellule di FACS. Si contano almeno 20.000 cellule per ogni trattamento. Per evitare possibili interferenze tra GFP e DsRed fluorescenza si raccomanda di mantenere la percentuale di GFP + o + DsRed cellule inferiore al 25%. Se la percentuale di + DsRed o cellule GFP + è superiore a ridurre la quantità di pDsRed2-N1 o I-SCEI plasmide. Nei nostri esperimenti abbiamo solo bisogno di regolare la quantità di pDsRed2-N1 plasmide, ma non del I-SCEI plasmide. </li><li> La percentuale di cellule GFP + corrisponde alla efficienza della riparazione del DNA DSB e la percentuale di cellule + DsRed indica l&#39;efficienza di trasfezione. Calcolare l&#39;efficienza relativa di riparazione del DNA DSB come il rapporto tra cellule GFP + per + le cellule DsRed. </li><li> Le giunzioni fine riparato può essere sequenziato per testare l&#39;accuratezza di riparazione. I costrutti giornalista contengono origine di replicazione e di E.coli geni di resistenza alla kanamicina (Figura 1). Ciò consente salvataggio i costrutti di digestione del DNA genomico con enzimi EcoRI, recircularization, e la trasformazione in cellule competenti di E.coli. I plasmidi salvati vengono poi analizzati da digestione di restrizione e sequenziamento. </li></ol> 5. Rappresentante Risultati Tipico risultati FACS del trasfezioni di controllo e di NHEJ ed esperimenti HR sono mostrati nelle Figure 4 e 5. Intensità fluorescente e il numero di cellule fluorescenti sarà inferiore al I-SCEI cellule transfettate contenenti inteed NHEJ e costruisce HR (Figura 5) rispetto ai controlli (Figura 4) a causa della differenza nel numero di copie di GFP e costrutti DsRed in queste cellule. Ogni cella nell&#39;esperimento contiene solo una copia del costrutto giornalista integrata, eventi riparare così il successo ricostituirà il gene GFP in una frazione delle cellule. Trasfezione con 0,1 mcg pDsRed2-N1 in fibroblasti umani eroga circa 1 copia plasmide per cellula. L&#39;efficienza di NHEJ e HR è calcolato come rapporto tra GFP + / + DsRed cellule. NHEJ è un processo più efficiente delle risorse umane nelle cellule umane 2. In fibroblasti umani l&#39;efficienza NHEJ è tipicamente 0,6-1,3, e l&#39;efficienza delle risorse umane è 0,05-0,3. Poiché l&#39;efficienza DSB è misurato come rapporto GFP + / + DsRed variazioni cellule nella miscela di I-SCEI e DsRed2-N1 plasmidi possono influire sul risultato. La qualità plasmide può anche influenzare i risultati, cambiando l&#39;efficienza di trasfezione. Pertanto, per ottenere misure coerenti, è importante utilizzare lo stesso mix plasmide all&#39;interno di un esperimento. Inoltre, l&#39;efficienza riparazione DSB è influenzata dal tipo di cellula, fase del ciclo cellulare, e la localizzazione cromosomica dei costrutti integrato. <img src="/files/ftp_upload/2002/2002fig1.jpg" alt="Figura 1" /> Figura 1. Reporter costruisce per l&#39;analisi di riparazione del DNA DSB da NHEJ (A) e HR (B). (C) DNA incompatibili finisce generato dalla digestione di due invertita I-SCEI siti SD, splicing donatore; SA, splicing accettore; piazze ombreggiate, i siti di poliadenilazione, Neo / Kana, singolo ORF controllato da due promotori:. SV40 resistenza alla neomicina conferimento di mammiferi cellule, e β-lattamasi che conferisce resistenza kanamicyn in E. coli, Ori, E. Coli origine pUC di replica. <img src="/files/ftp_upload/2002/2002fig2.jpg" alt="Figura 2" /> Figura 2. Reporter costruire per l&#39;analisi di NHEJ In questo costrutto il gene GFP è inattivo;. Ma sulla induzione di una NHEJ DSB e di successo del costrutto diviene GFP +. Qui sotto è un prodotto di riparazione di questo giornalista da NHEJ. <img src="/files/ftp_upload/2002/2002fig3.jpg" alt="Figura 3" /> Figura 3. Reporter costruire per l&#39;analisi delle risorse umane mediante la conversione del gene. Dopo l&#39;induzione del DSB di I-SCEI, HR da gene conversione (GC) ricostituisce un gene attivo GFP. Mostrati di seguito sono prodotti per la riparazione di questo giornalista attraverso le vie principali di riparazione del DNA: GC, crossing-over (X-over), singolo filamento annealing (SSA), e NHEJ. X-over riparazione del prodotto è indicato per la ricombinazione intramolecolare, ricombinazione exramolecular darà lo stesso prodotto, ma trova su un cromosoma. Geni che esprimono una proteina attiva GFP (GFP +) sono indicati con colore verde. <img src="/files/ftp_upload/2002/2002fig4.jpg" alt="Figura 4" /> Figura 4. Calibrazione dei parametri per l&#39;analisi FACS. (A) I fibroblasti transfettate con il plasmide pHPRT, utilizzato come controllo negativo per l&#39;esclusione automatica delle cellule fluorescenti. (B) I fibroblasti transfettate con plasmide pEGFP, utilizzato per impostare il gate di cellule GFP + (area verde). (C) I fibroblasti transfettate con plasmide pDsRed2-N1, utilizzato per impostare il gate per le cellule + DsRed (area rossa). <img src="/files/ftp_upload/2002/2002fig5.jpg" alt="Figura 5" /> Figura 5. Risultati tipici dell&#39;analisi dei NHEJ (A) e HR (B) in fibroblasti umani normali con costrutti reporter di cromosomicamente integrato.

<ol>
	<li>Mao, Z., Seluanov, A., Jiang, Y., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRF2+is+required+for+repair+of+nontelomeric+DNA+double-strand+breaks+by+homologous+recombination.">TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination.</a> Proc Natl Acad Sci U S A. 104, 13068-13073 (2007).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <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+nonhomologous+end+joining+and+homologous+recombination+in+human+cells.">Comparison of nonhomologous end joining and homologous recombination in human cells.</a> DNA Repair (Amst). 7, 1765-1771 (2008).</li><li>Rouet, P., Smih, F., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+site-specific+endonuclease+stimulates+homologous+recombination+in+mammalian+cells.">Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.</a> Proc Natl Acad Sci U S A. 91, 6064-6068 (1994).</li><li>Mao, Z., Jiang, Y., Xiang, L., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+homologous+recombination,+but+not+by+nonhomologous+end+joining,+is+elevated+in+breast+cancer+cells.">DNA repair by homologous recombination, but not by nonhomologous end joining, is elevated in breast cancer cells.</a> Neoplasia. 11, 683-691 (2009).</li><li>Seluanov, A., Mittelman, D., Pereira-Smith, O. M., Wilson, J. H., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+end+joining+becomes+less+efficient+and+more+error-prone+during+cellular+senescence.">DNA end joining becomes less efficient and more error-prone during cellular senescence.</a> Proc Natl Acad Sci U S A. 101, 7624-7629 (2004).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+nonhomologous+end+joining+and+homologous+recombination+during+cell+cycle+in+human+cells.">DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.</a> Cell Cycle. 7, 2902-296 (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

Le fluorescenti NHEJ e saggi giornalista HR forniscono un modo quantitativo per misurare separatamente ciascun percorso riparazione DSB in vivo. I test sono molto sensibili, come FACS di rilevare in modo affidabile 10 cellule GFP + in 20.000 cellule. Il test può essere adattato per la misurazione eventi riparazione in &quot;tempo reale&quot;, rilevando la comparsa di cellule GFP + giro di pochi minuti o ore dopo l&#39;induzione di DSB 2. Inoltre, l&#39;analisi di cellule GFP + non si basa sulla prol...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>EndoFree Plasmid Maxi kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12362</td>
<td>&nbsp;</td>
<tr>
<td>Qiaex II Gel Extraction Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>20021</td>
<td>&nbsp;</td>
<tr>
<td>Amaxa Nucleofector</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>AAD-1001</td>
<td>&nbsp;</td>
<tr>
<td>Geneticin (G418)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11811-031</td>
<td>&nbsp;</td>
<tr>
<td>pDsRed2-N1</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>632406</td>
<td>&nbsp;</td>
<tr>
<td>Round bottom tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352058</td>
<td>FACS tubes</td>
</tr>		</tbody>
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analysis of dna double-strand break (dsb) repair in mammalian cells

DNA a doppio filamento sono le lesioni del DNA più pericolosa che può causare la perdita massiccia di informazioni genetiche e morte cellulare. DSB riparare le cellule utilizzando due percorsi principali: nonhomologous fine unirsi (NHEJ) e ricombinazione omologa (HR). Perturbazioni di NHEJ e HR sono spesso associate a invecchiamento precoce e tumorigenesi, quindi è importante avere un modo quantitativo per misurare ogni percorso riparazione DSB. Il nostro laboratorio ha sviluppato costruisce giornalista fluorescenti che permettono di misurazione sensibile e quantitativa di NHEJ e HR. I costrutti si basano su un gene GFP progettato contenenti siti di riconoscimento per una rara taglio I-endonucleasi SCEI per l&#39;induzione di DSB. I costrutti di partenza sono GFP negativi come il gene della GFP viene inattivato da un esone aggiuntivo, o da mutazioni. Riparazione di successo della I-SCEI indotta break da NHEJ o HR ripristina il gene funzionale GFP. Il numero di cellule GFP positive contati mediante citometria di flusso fornisce una misura quantitativa della NHEJ o efficienza delle risorse umane.

Watch this Scientific Journal Video about Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells at JoVE.com

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

Questo articolo descrive GFP-based fluorescenza<em&gt; In vivo</em&gt; Test che separatamente quantificare ricombinazione omologa e nonhomologous fine unirsi in cellule di mammifero.

Analisi del DNA doppio filamento Break (DSB) di riparazione in cellule di mammifero

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

analysis-of-dna-double-strand-break-dsb-repair-in-mammalian-cells

Research

JoVE Journal

Biology

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

31.5K Views.  University of Rochester. Questo articolo descrive GFP-based fluorescenza In vivo Test che separatamente quantificare ricombinazione omologa e nonhomologous fine unirsi in cellule di mammifero.

Video: Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

Transfection DNA dei mammiferi muscoli scheletrici con In Vivo Elettroporazione

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Ricerca

Biologia

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Microiniezione intranucleari di DNA in dissociato neuroni dei mammiferi adulti

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Pulse-chase Analisi di N-linked catene di zuccheri da glicoproteine ​​in cellule di mammifero

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenesi e Analisi funzionale dei canali ionici Heterologously Espresso in cellule di mammifero

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Analisi di Schwann-astrociti Interazioni utilizzo In Vitro Saggi

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Analisi della cinetica di esportazione mRNA nucleare in cellule di mammifero da Microiniezione

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analisi della posizione ciclo cellulare nelle cellule di mammifero

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

In tempo reale Imaging del singolo Engineered RNA trascritti in cellule viventi Utilizzando Ratiometric bimolecolari Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

La misurazione dei livelli Heme sintesi in cellule di mammifero

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.