Siamo grati allo Stato dell&#39;Ohio Centro Comprehensive Cancer dell&#39;Università per il finanziamento per questo lavoro.

1. Linea cella con un substrato ricombinazione integrato <ol><li> Le cellule HeLa sono state trasformate stabilmente con PDR-GFP (plasmide 7 dono di M. Jasin, Memorial Sloan-Kettering Cancer Center) e mantenuti in puromicina per la selezione dei trasformanti. </li><li> Le cellule sono diversi passaggi in DMEM / FBS, contenente 10% siero fetale bovino, 2 mM GlutaMAX, 1 mM sodio piruvato, penicillina / streptomicina (100 U / mL/0.1 mg / mL), e 1,5 ug / mL puromicina. </li></ol> Il substrato ricombinazione del PDR-GFP plasmide, e in trasformanti stabili, contiene due alleli inattivi di GFP (Figura 1). Un allele è inattivo a causa dell&#39;inclusione nella sua sequenza codificante del sito di riconoscimento degli enzimi di restrizione 18 bp per I-SCEI. Espressione di I-SCEI in queste cellule crea un doppio filamento rottura del DNA (DSB). C&#39;è un secondo allele di GFP inattivi su questo DNA, ma la parte della sua sequenza correlata con la I-SCEI sito sul allele prima wild-type. Questo allele GFP secondo può funzionare come un donatore sequenza per ricombinazione omologa la riparazione del DSB e si tradurrebbe in un allele GFP attivo, che è prontamente rilevata mediante citometria di flusso. In alternativa, il DSB possono essere riparati con nonhomologous fine-adesione, che avrebbe distrutto l&#39;I-SCEI sito di restrizione e si tradurrebbe in negativo GFP-cellule. Il livello di ricombinazione omologa in una cella è determinato dal conteggio della percentuale di cellule GFP-positive. Questa linea cellulare, pHeLa-DR13-9, è stato selezionato tra tutti i cloni per lo zero segnale di fondo GFP e una percentuale relativamente elevata di cellule GFP-positive sulla trasfezione con l&#39;I-SCEI esprimendo plasmide, pCBASce. (Il pCBASce e il suo controllo vettoriale, pCAGGS, sono stati entrambi forniti da M. Jasin del Memorial Sloan-Kettering Cancer Center.) La linea HeLa-DR13-9 celle è disponibile presso gli autori su richiesta. 2. Trasfezione di impoveriscono BRCA1 endogena e Aggiungi Indietro BRCA1 Mutant. <ol><li> Il giorno prima della trasfezione prima (di solito un Martedì): Iniziare con un piatto di 10 cm di HeLa-DR13-9 celle di almeno il 90% confluenti. </li><li> Trypsinize in 1 ml e interrompere la reazione con l&#39;aggiunta di 9 ml DMEM / FBS, mescolare bene nel pipettaggio. </li><li> Aggiungere 40 ml di cellule trypsinized per pozzetto di una piastra ben 24 in 0,5 ml di DME / FBS. </li><li> Giorno 1 trasfezioni (Mercoledì): le cellule devono essere confluenti ~ 40-50%, molto meno se ricominciare da capo. Trasfezione mescolando: 5 siRNA pmol + 0,3 ug BRCA1-mutante che esprime DNA plasmidico in 12,5 microlitri Opti-MEM, e 0,5 microlitri Lipofectamine 2000 a 12,5 microlitri Opti-MEM. Procedere con la transfezione secondo il protocollo Lipofectamine (Invitrogen). </li><li> Sostituire le 4-6 ore dei media dopo. L&#39;siRNA che utilizziamo per esaurire BRCA1 si basa sul GCUCCUCUCACUCUUCAGU sequenza specificando BRCA1 3&#39;-UTR sequenze 80.780 a 80.798 (adesione numero AY273801). </li><li> Giorno 2: Trasferire le cellule trypsinizing dalla piastra 24 bene a un piatto ben 6. </li><li> 3 ° giorno trasfezioni: 25 siRNA pmol + 0,75 ug BRCA1-mutante che esprime DNA plasmidico + 0,75 pCBASce ug (o pCAGGS controllo vettoriale) a 62,5 microlitri Opti-MEM e 2,5 microlitri Lipofectamine 2000 a 62,5 microlitri Opti-MEM. Sostituire le 4-6 ore dei media dopo. </li></ol> 3. Analisi dei trasfettate. <ol><li> Il giorno 6, trypsinize cellule di ogni pozzetto e fermare in 1 ml di DMEM / FBS. Analizzando le cellule per l&#39;espressione GFP può essere effettuata nei giorni 5 o 7, ma a nostro parere giorno 6 funziona meglio. </li><li> Analizzare 10.000 cellule per pozzetto mediante citometria di flusso. Usiamo un Becton Dickinson strumento FACSCalibur in Ohio State University Comprehensive Cancer Center Citometria analitica risorsa condivisa. Singolo, cellule vive sono gated utilizzando i parametri scatter in avanti e laterale. GFP-positive le cellule vengono rilevati tramite il rilevatore GFP fluorescenza (FL1), ed i risultati sono presentati come un istogramma (figura 2, in basso). </li><li> Sinistra su cellule possono essere saltuariamente per la fotografia al microscopio a fluorescenza, se desiderato. </li></ol> 4. Rappresentante dei risultati: BRCA1 regola il percorso per la riparazione delle doppie rotture del DNA bloccati dalla ricombinazione omologa 6,11. Quelle cellule che hanno subito la riparazione per ricombinazione omologa sono prontamente rilevate al microscopio a fluorescenza (Figura 2, in alto). Esaurimento dei risultati BRCA1 in una diminuzione della GFP-positive rilevate cellule (Figura 2, a destra). L&#39;output del FACSCalibur è un istogramma con intensità GFP per cella sul asse X e il numero di celle dell&#39;asse Y (Figura 2, in basso). In un tipico insieme di risultati di un singolo esperimento, il 15,5% delle cellule sono state GFP-positive dopo la I-SCEI espressione e di controllo esaurimento RNAi (Figura 3). In confronto, l&#39;esaurimento di BRCA1 e vettore aggiungere di nuovo ridotto GFP-conversione al 0,7%. Add-back di tipo selvaggio BRCA1 o di BRCA1 (I21V) completamente restaurato ricombinazione omologa, come rilevato da ottenere 15-16% delle cellule GFP-positive. Add-posteriore del (BRCA1 M18T) mutante (Figura 3, corsia 5) ha avuto un effetto simile a quello della reintegrazione del controllo vettoriale. Dato che questa variante espressa a livelli simili a quelli della proteina endogena 9, abbiamo quindi interpretare le (M18T) BRCA1 variante non è funzionale a ricombinazione omologa. Noi in genere hanno il 10-20% delle cellule convertire GFP-positive dopo la trasfezione I-SCEI plasmide di espressione. L&#39;esaurimento delle BRCA1 riduce riproducibile la conversione di cellule GFP-positive di circa 8-10 volte. Anche se le percentuali effettive di GFP-positive cellule possono cambiare da esperimento per esperimento, il rapporto tra le cellule GFP-positive a seguito di esaurimento BRCA1 rispetto al depauperamento controllo all&#39;interno di un esperimento è del tutto coerente. Noi normalizzare i risultati impostando il tasso di conversione disinibita al 100% e risultati esprimono con svuotamenti BRCA1 e aggiungere-backs come percentuali. In questo modo, siamo in grado di combinare molteplici esperimenti, e la variazione sperimentale non è elevato. Aggiungere nuovamente di mutanti BRCA1 hanno finora dato risultati robusti: ogni variante BRCA1 o ha completamente restaurato ricombinazione omologa attività o ha avuto alcun effetto. Una considerazione importante per ottenere questi risultati è che le nostre condizioni di trasfezione produrre livelli di proteina BRCA1 che approssimativamente uguale alla concentrazione della proteina endogena in cellule non trattate. Troppo alto di una sovraespressione di anche un mutante difettoso di BRCA1 può comportare in alcuni positivi attività ricombinazione omologa. Un problema che abbiamo dovuto superare è stata la tossicità di trasfezione. L&#39;esaurimento delle BRCA1 si può rallentare la crescita cellulare, e l&#39;equilibrio del DNA Lipofectamine-2000 e plasmide rispetto al numero di celle nel piatto deve essere tenuto ad un livello costante. DNA troppo Lipofectamine o plasmide sarà ridurre il numero delle cellule, e il numero di cellule non sarà sufficiente per ottenere risultati affidabili citometria a flusso. Il saldo dei reagenti plasmide e Lipofectamine rispetto al numero di cellule trasfettate è molto importante per il mantenimento della vitalità delle cellule. <img src="/files/ftp_upload/2468/2468fig1.jpg" alt="Figura 1" /> Figura 1. Il substrato ricombinazione. La strategia che era stato sviluppato dal gruppo di M. Jasin 7,12 è raffigurato. Il PDR-GFP plasmide contiene due alleli difettosi di GFP, uno dei quali contiene un I-SCEI sito endonucleasi di restrizione. Un derivato linea cellulare HeLa contiene questo substrato di DNA integrato in un unico sito nel genoma 9, e la riparazione attiva della doppia elica del DNA rompere al I-SCEI sito ricombinazione omologa risultati nella conversione di un allele di GFP-positive. <img src="/files/ftp_upload/2468/2468fig2.jpg" alt="Figura 2" /> Figura 2. Rilevamento di GFP-positive le cellule al microscopio a fluorescenza. Le cellule sono state testate di cui al presente protocollo e di controllo impoverito le cellule erano attivi in ​​ricombinazione omologa, come rilevato da un&#39;alta percentuale di cellule con espressione di GFP (a sinistra). L&#39;esaurimento delle BRCA1 dai risultati RNAi in una forte riduzione del numero di cellule GFP-positive (a destra). <img src="/files/ftp_upload/2468/2468fig3.jpg" alt="Figura 3" /> Figura 3. I risultati di un add-back di mutanti missense BRCA1. I risultati di un singolo esperimento sono riportati in questo istogramma. In assenza di transfettate I-SCEI esprimendo plasmide (corsia 1) non ci sono cellule GFP-positive rilevate. I risultati in corsie 2-6 sono stati tutti presi dalle cellule in cui è stato transfettate I-SCEI plasmide che esprime al giorno 3. Nelle cellule che sono state esaurite per un gene irrilevante (luciferasi) e con vettore aggiungere-back, il 15,5% delle cellule sono state GFP-positive (corsia 2). L&#39;esaurimento delle BRCA1 e vettore di add-back rivela l&#39;effetto della perdita di BRCA1 sulla ricombinazione omologa (corsia 3). Gli effetti di impoverimento delle BRCA1 e add-retro di wild-type BRCA1 (corsia 4), BRCA1 (M18T) (corsia 5), ​​e BRCA1 (I21V) (corsia 6) sono mostrati. Questi risultati provengono da un singolo esperimento, e potrebbe essere combinata con almeno due esperimenti ripetere più al fine di ottenere una misura della ricombinazione omologa supportata da una data proteina BRCA1 mutato.

<ol>
	<li>Carvalho, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+cancer+risk+associated+with+germ+line+BRCA1+missense+variants+by+functional+analysis.">Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis.</a> Cancer Res. 67, 1494-1501 (2007).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+epidemiology+of+BRCA1.">Genetic epidemiology of BRCA1.</a> Cancer Biol Ther. 3, 509-514 (2004).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+functional+effects+of+unclassified+genetic+variants.">Assessment of functional effects of unclassified genetic variants.</a> Hum Mutat. 29, 1314-1326 (2008).</li><li>Tomlinson, G. E. <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+breast+cancer+cell+line+derived+from+a+germ-line+BRCA1+mutation+carrier.">Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier.</a> Cancer Res. 58, 3237-3242 (1998).</li><li>Ruffner, H., Joazeiro, C. A., Hemmati, D., Hunter, T., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-predisposing+mutations+within+the+RING+domain+of+BRCA1:+loss+of+ubiquitin+protein+ligase+activity+and+protection+from+radiation+hypersensitivity.">Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity.</a> Proc Natl Acad Sci U S A. 98, 5134-5139 (2001).</li><li>Moynahan, M. E., Chiu, J. W., Koller, B. H., 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=Brca1+controls+homology-directed+DNA+repair.">Brca1 controls homology-directed DNA repair.</a> Mol Cell. 4, 511-518 (1999).</li><li>Nakanishi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Fanconi+anemia+monoubiquitination+pathway+promotes+homologous+DNA+repair.">Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair.</a> Proc Natl Acad Sci U S A. 102, 1110-1115 (2005).</li><li>Pierce, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+breaks+and+tumorigenesis.">Double-strand breaks and tumorigenesis.</a> Trends Cell Biol. 11, 52-59 (2001).</li><li>Ransburgh, D. J., Chiba, N., Ishioka, C., Toland, A. E., Parvin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+breast+tumor+mutations+in+BRCA1+that+abolish+its+function+in+homologous+DNA+recombination.">Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination.</a> Cancer Res. 70, 988-995 (2010).</li><li>Easton, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+genetic+assessment+of+1,433+sequence+variants+of+unknown+clinical+significance+in+the+BRCA1+and+BRCA2+breast+cancer-predisposition+genes.">A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes.</a> Am J Hum Genet. 81, 873-883 (2007).</li><li>Snouwaert, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1+deficient+embryonic+stem+cells+display+a+decreased+homologous+recombination+frequency+and+an+increased+frequency+of+non-homologous+recombination+that+is+corrected+by+expression+of+a+brca1+transgene.">BRCA1 deficient embryonic stem cells display a decreased homologous recombination frequency and an increased frequency of non-homologous recombination that is corrected by expression of a brca1 transgene.</a> Oncogene. 18, 7900-7907 (1999).</li><li>Pierce, A. J., Hu, P., Han, M., Ellis, N., 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=Ku+DNA+end-binding+protein+modulates+homologous+repair+of+double-strand+breaks+in+mammalian+cells.">Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells.</a> Genes Dev. 15, 3237-3242 (2001).</li><li>Spearman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinically+applicable+models+to+characterize+BRCA1+and+BRCA2+variants+of+uncertain+significance.">Clinically applicable models to characterize BRCA1 and BRCA2 variants of uncertain significance.</a> J Clin Oncol. 26, 5393-5400 (2008).</li><li>Sweet, K., Senter, L., Pilarski, R., Wei, L., Toland, A. E. <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+BRCA1+ring+finger+variants+of+uncertain+significance.">Characterization of BRCA1 ring finger variants of uncertain significance.</a> Breast Cancer Res Treat. 119, 737-743 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Il vantaggio di questo metodo è che possiamo studiare la funzione della proteina BRCA1 nella regolazione della riparazione del DNA per ricombinazione omologa utilizzando cellule che hanno wild-type BRCA1 endogenamente espresso. Abbiamo adottato due metodi distinti costituiti di studiare il processo di ricombinazione omologa e di utilizzare l&#39;RNAi mediato esaurimento per ottenere il nuovo metodo per il test per la funzione varianti BRCA1 nella riparazione del DNA. La chiave per il successo di questo metodo è stato quello di stabilire una linea di cellule facilmente transfectable, come ad esempio HeLa, con il substrato ricombinazione del genoma in modo tale che si ottengono livelli molto alti di conversione GFP. Avevamo proiettato più cloni della trasformazione originale al fine di ottenere uno che non aveva segnale rilevabile GFP fondo, ma dopo trasfezione delle I-SCEI plasmide di espressione aveva un livello molto elevato di conversione GFP. Con questo metodo, ora stiamo studiando altri specifici residui di aminoacidi BRCA1 per la funzione di ricombinazione omologa. Insieme con la intuizioni biologico da questo lavoro, queste conclusioni possono essere applicate alla genetica clinica sul cancro. Un&#39;alta percentuale di BRCA1 mutazioni missense sono sconosciute significato clinico poiché ci sono molte varianti rare e non ci sono informazioni sufficienti per molte di queste per determinare l&#39;associazione del cancro attraverso l&#39;analisi di segregazione genetica 3,10,13,14. Utilizzando questo metodo, le conseguenze di una variante specifica per la funzione biologica di BRCA1 nella regolazione della ricombinazione omologa potrebbero essere utilizzati per informare le donne che svolgono una di queste varianti nel loro genoma.

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identifying the effects of brca1 mutations on homologous recombination using cells that express endogenous wild-type brca1

L&#39;analisi funzionale di mutazioni missense può essere complicata dalla presenza nella cellula della proteina endogena. Struttura-funzione di analisi del BRCA1 sono state complicate dalla mancanza di un test affidabile per la proteina BRCA1 pieno lunghezza e le difficoltà insite nel lavoro con linee cellulari che esprimono proteine ​​BRCA1 hypomorphic 1,2,3,4,5. Abbiamo sviluppato un sistema in cui la proteina BRCA1 endogena in una cella è stato acutamente impoverito da RNAi intese a promuovere l&#39;3&#39;-UTR del mRNA BRCA1 e sostituito da co-trasfezione un plasmide che esprime una variante del gene BRCA1. Un vantaggio di questa procedura è che il silenzio acuto di sostituzione BRCA1 e simultanea permettono alle cellule di crescere senza mutazioni secondarie o adattamenti che potrebbero sorgere nel corso del tempo per compensare la perdita della funzione del gene BRCA1. Questo impoverimento e add-back procedura è stata fatta in un HeLa derivate linea cellulare che è stato prontamente analizzati per attività di ricombinazione omologa. La ricombinazione omologa test si basa su un metodo precedentemente pubblicato con il quale è integrato un substrato ricombinazione nel genoma (Figura 1), 6,7,8,9. Questo substrato ricombinazione ha il raro taglio I-SCEI sito enzima di restrizione all&#39;interno di un allele inattivo GFP, ed è a valle un allele secondo GFP inattivo. Trasfezione del plasmide che esprime I-SCEI si traduce in un doppio filamento pausa, che può essere riparato per ricombinazione omologa, e se ricombinazione omologa fa riparare la rottura che crea un allele attivo GFP che è prontamente segnato mediante citometria di flusso per l&#39;espressione della proteina GFP . L&#39;esaurimento delle BRCA1 endogena comportato un 8-10-fold riduzione dell&#39;attività ricombinazione omologa, e aggiungere-back di tipo selvaggio funzione plasmide ricombinazione omologa completamente restaurato. Quando mutanti specifico punto di BRCA1 lunghezza sono stati espressi dal co-trasfettate plasmidi, l&#39;effetto della mutazione missense specifico potrebbe essere segnato. A titolo di esempio, l&#39;espressione della (M18T) proteina BRCA1, una variante di significato clinico sconosciuto 10, è stato espresso in queste cellule, non è riuscito a ripristinare BRCA1-dipendente ricombinazione omologa. Al contrario, espressione di un&#39;altra variante, anche di significato sconosciuto, BRCA1 (I21V) completamente restaurato BRCA1-dipendente funzione di ricombinazione omologa. Questa strategia di test della funzione di BRCA1 mutazioni missense è stato applicato a un altro sistema biologico dosaggio per la funzione centrosoma (Kais et al, osservazioni non pubblicate). Nel complesso, questo approccio è adatto per l&#39;analisi dei mutanti missense in qualsiasi gene che deve essere analizzato recessiva.

Watch this Scientific Journal Video about Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1 at JoVE.com

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Forniamo un metodo per testare le varianti BRCA1 in un test di coltura di tessuti a base di ricombinazione omologa la riparazione del danno al DNA mediante deplezione delle proteine ​​endogene BRCA1 da una cella con RNAi e la sua sostituzione con un mutante punto BRCA1 che contiene un cambio di codifica.

Identificare gli effetti delle mutazioni BRCA1 sulla ricombinazione omologa utilizzando cellule che esprimono endogeno wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

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14.6K Views.  The Ohio State University. Forniamo un metodo per testare le varianti BRCA1 in un test di coltura di tessuti a base di ricombinazione omologa la riparazione del danno al DNA mediante deplezione delle proteine ​​endogene BRCA1 da una cella con RNAi e la sua sostituzione con un mutante punto BRCA1 che contiene un cambio di codifica.

Video: Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Studiare gli effetti di rigidità Matrix sulla funzione cellulare utilizzando acrilamide a base di idrogeli

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Ricerca

Biologia

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Analizzando la funzione di GTPasi Small Microiniezione di plasmidi in cellule epiteliali polarizzate

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

A PCR-based metodo di genotipizzazione di distinguere tra le varietà selvatiche di tipo ornamentale e di Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombineering costrutti omologhi ricombinazione<em&gt; Drosophila</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Misurare gli effetti dei batteri su<em&gt; C. Elegans</em&gt; Comportamento utilizzando un saggio di ritenzione dell&#39;uovo

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

gDNA arricchimento da una tecnologia basata trasposasi-NGS per l&#39;analisi della sequenza intera<em&gt; BRCA1</em&gt;,<em&gt; BRCA2</em&gt;, e 9 geni coinvolti nella riparazione del DNA Damage

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Identificare proteina-proteina siti di interazione Uso Peptide Array

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Caratterizzazione funzionale di Na<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; Scambiatori di intracellulare compartimenti, utilizzando Proton-uccidere selezione di esprimerle livello della membrana plasmatica

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Determinazione degli acidi sialici nei campioni di fegato e latte di tipo selvatico e<em&gt; CMAH</em&gt; Mouse Knock-out.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Metodo per valutare gli effetti del batterioiocinio sul microbiota di gatti dei topi

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...