T.R.R conceived the project, designed and performed experiments and prepared the manuscript. E.J.K and S.E.M performed experiments. All authors contributed intellectually to the final manuscript. This work was funded by a University of Leicester Innovation Fellowship to SEM and by a UK Medical Research Council (MRC) Senior Research Fellowship to S.M.C (MR/J009202/1). DNA sequencing was performed by Protein and Nucleic Acid Laboratories (PNACL), Centre for Core Biotechnology Services, University of Leicester. We wish to thank A. Francis Stewart for providing the Rox and Dre plasmids. pL451 and pL452 plasmids were obtained from National Cancer Institute (NCI), Frederick, USA.

1. Gene Targeting design <ol style="margin-left: 40px;"><li> Ordinare un adeguato clone BAC che copre la regione genomica di interesse. Assicurarsi che questo è isogenico al tipo di cellule ES da modificare ad esempio, cloni RPCI-23 e RPCI-24 BAC per gene targeting con C57BL / 6 ES cellule. </li><li> Applicare gene convenzionale criteri di targeting durante la progettazione del vettore targeting. I parametri chiave includono se la modifica è richiesto di essere costitutiva o condizionale, definendo esoni critiche (CE) per la cancellazione in una strategia gene knockout e la spaziatura e la collocazione delle cassette intronici. NOTA: Ognuno di questi è stato discusso in dettaglio altrove 10. </li><li> Scegli regioni genomiche ciascuno di 5-6 kb che fiancheggia il sito di modifica di destinazione. NOTA: La dimensione dell&#39;inserto subclonato è pertanto tipicamente 10-12 kb, anche se il limite superiore può essere alto come 80 kb con una copia bassa subcloning plasmide come p15A, ecc pBR322 e fino a 200 kb con BACvettoriale. </li></ol> 2. Multiplex recombineering Oligos <ol style="margin-left: 40px;"><li> Oligo di progettazione per la spina dorsale plasmide (subcloning plasmide) e il marcatore (cassette di inserimento). Progettare ogni oligo tal da contenere 180 bp HA fiancheggianti il sito di destinazione genomico e 20 bp di sequenza specifica priming della cassetta di inserimento o il plasmide subcloning (Figura 2). NOTA: braccia omologia non dovrebbero contenere elementi ripetitivi, presenza di elementi ripetitivi comporterà il targeting errata e subcloning. Elementi ripetitivi possono essere rilevati utilizzando strumenti web based, come il &#39;&#39; Repeat Maschera &#39;&#39;. </li><li> Include un enzima di restrizione (RE) sito unico su una delle oligo vettoriali per linearizzare il vettore gene targeting per cellule ES di targeting (Figura 2). </li><li> Controllare i parametri di oligo utilizzando un programma di analizzatore di oligo. Fare attenzione ad evitare strutture secondarie nella regione primings. </li><li> Shorter HA (50 bp) della cassetta di inserimento è tollerato e produce minor numero di ricombinanti, ma assicurarsi che il plasmide subcloning HA è sempre più lungo (180 bp). </li><li> Determinare l&#39;orientamento dell&#39;inserto di DNA genomico del particolare clone BAC utilizzando strumenti web based come &#39;&#39; cloneDB &#39;&#39;. Stabilire la direzione di replica da Oris controllando la mappa della backbone plasmide BAC utilizzati nella costruzione della libreria BAC. NOTA: ORIS è generalmente opposta alla direzione trascrizionale del marcatore Cloramfenicolo (Chl) per tutti i plasmidi BAC comunemente usati. </li><li> Aggiungere due fosforotioato (PTO) legami terminali all&#39;estremità 5 &#39;del oligo che è opposta alla direzione di replica sul clone BAC. Aggiungere una modifica fosfato 5 &#39;alla oligo inversa (Figura 2). NOTA: Il asimmetrica phosphorothioated cassette PCR su digestione Red genera un ssDNA intermedio che può innescare il filo in ritardodella forcella replica. NOTA: massima frequenza di ricombinazione multiplex si osserva con le cassette protette filo in ritardo. Leading filo protetta o cassette protette doppio non può produrre efficienza di ricombinazione equivalenti ad una loci. </li><li> Ordinare oligo con PAGE o HPLC purificazione. </li></ol> 3. BAC Trasformazione Clone con pSC101 BADgbaA recombineering plasmidi. <ol style="margin-left: 40px;"><li> Per rendere la E. coli recante la recombineering BAC abile, trasformarlo con il plasmide pSC101 BADgbaA contenente i geni rossi 16. NOTA: Il plasmide pSC101 BADgbaA contiene i geni rossi e RecA sotto il controllo del Arabinose inducibile araC-P promoter BAD, un marcatore di selezione tetraciclina e la temperatura sensibile pSC101 replicone. <ol><li> Pugnalare un puntale sterile cultura BAC agar e inoculare 5,0 ml di brodo lisogenia (LB) pH 8,0 contenente 12.556; g ml -1 Chl. Grow a 37 ° C per 5 ore in agitazione a 200 rpm. </li><li> Raffreddare 10% (v / v) soluzione di glicerolo, microprovette e cuvette elettroporazione su ghiaccio. Raffreddare una grande centrifuga refrigerata e microcentrifuga a 4 ° C. </li><li> Determinare la densità ottica (OD) della coltura utilizzando uno spettrofotometro e misurare l&#39;assorbanza a 600 nm. Preparare cellule electrocompetent (come descritto sotto) quando viene raggiunto un OD 600 lettura da 0.3 a 0.8. </li><li> Spin le cellule in una provetta da centrifuga da 50 ml in un grande centrifugare a 1.216 xg per 5 minuti a 4 ° C. </li><li> Lavare le cellule con 1 ml di acqua refrigerata 10% glicerolo e spin giù cellule a 17.949 xg per 20 sec a 4 ° C. Eseguire il lavaggio passo per un totale di 3 volte. </li><li> Risospendere le cellule in un volume totale di 50 ml di 10% glicerolo e aggiungere 10-200 ng del plasmide recombineering pSC101 BADgbaA. Ottenere una singola sospensione cellulare pipettandosu e giù parecchie volte e poi trasferire le cellule ad una pre-refrigerati 1 mm di aria elettroporazione cuvetta. </li><li> Elettroporazione le cellule con una cornice di 1,8 kv, 25 uF e 200 Ω. NOTA: Controllare le impostazioni corrette per ogni marca di elettroporatore. Una costante di tempo di elettroporazione inferiore 4 indica la presenza di sale e altre impurità. </li><li> Immediatamente recuperare le cellule in 1 ml di LB e trasferire le cellule in una provetta da centrifuga da 50 ml. </li><li> Far crescere la cultura BAC gbaA a 30 ° C per 2 ore agitazione a 200 rpm. NOTA: Il plasmide pSC101 BADgbaA si perde quando le cellule sono coltivate a 37 ° C a causa di inattivazione del fattore di replica rEPE sensibili alla temperatura. Crescere cellule gbaA a 30 ° C per mantenere le funzioni recombineering. </li><li> Aggiungere 9 ml di LB contenente 12,5 mg ml -1 Chl e 4 mg ml -1 tetraciclina (Tet) alla cultura BAC gbaA recuperato. Far crescere laBAC gbaA cultura O / N a 30 &#160; ° C agitazione a 200 rpm. NOTA: L&#39;efficienza della trasformazione electroporating del gbaA plasmide supercoiled è sufficientemente alta da permettere la crescita saturazione O / N in mezzi liquidi. </li></ol></li></ol> 4. Preparazione di inserimento cassette e Subclonaggio Plasmidi <ol style="margin-left: 40px;"><li> Per incorporare HA nel cassetto di inserimento (s) e il plasmide subcloning, effettuare la reazione a catena della polimerasi (PCR) utilizzando oligonucleotidi lunghi modificati come descritto di seguito. <ol><li> Optional: per evitare plasmide riporto nella reazione recombineering, utilizzare un&#39;origine R6K o simile modello ristretta gamma di ospiti plasmide per amplificare la cassetta di inserimento. </li><li> In alternativa, linearizzare il modello plasmide con un RE digest (Tabella 2). Scegliere un RE che taglia fuori la regione di amplificazione PCR ed è il calore inattivato. Il calore inattivare la RE come raccomandato dal manifacturer. </li><li> Impostare la reazione a catena della polimerasi (PCR) utilizzando un sistema di polimerasi hotstart DNA ad alta fedeltà. Preparare una master mix PCR come indicato (tabella 3). Eseguire cicli termici come mostrato (Tabella 3). </li></ol></li><li> Analizzare PCR prodotti di gel elettroforesi. Carico 1-5 microlitri di ogni PCR su un 1% (w / v) gel di agarosio contenente 0,5 mg ml -1 etidio bromuro (EtBr). NOTA: La presenza di prodotti non specifici amplificazione non interferisce nella reazione recombineering. In alcuni casi, tuttavia, di primer-dimeri possono diminuire l&#39;efficienza recombineering. </li><li> Purificare prodotti PCR utilizzando un kit di purificazione PCR. <ol><li> Optional: rimuovere il modello plasmide da PCR per trattamento con DpnI seguita da PCR clean-up. DNA Eluire in un volume minimo di acqua deionizzata sterile (come raccomandato dal costruttore). NOTA: L&#39;aggiunta di DpnI per non purificata PCR reazioni risultati in ridotta efficienza di scissione di the template metilato DNA plasmide. </li></ol></li><li> Quantificare PCR DNA amplificato mediante analisi gel contro una serie nota di standard di DNA ad esempio, λ HindIII digest o utilizzando uno spettrofotometro NanoDrop. </li></ol> 5. Subclonaggio più Inserimento <ol style="margin-left: 40px;"><li> Diluire la O / N BAC gbaA cultura 50 volte con l&#39;aggiunta di 200 ml in 10 ml LB + Chl + Tet. Coltivare a 30 &#160; ° C agitazione a 200 rpm per 1 ora 50 min. Include un campione da utilizzare come controllo negativo. </li><li> Raffreddare tutti i materiali e le attrezzature recombineering a 4 ° C, come descritto al punto 3.1.2. </li><li> Versare LB agar pH 8 piastre contenenti la corretta concentrazione di antibiotico selettiva appropriata (s) che permetterà la selezione del frammento di DNA che viene inserito e per la selezione del vettore subcloning (Tabella 4). NOTA: Alcuni antibiotici sono pH sensibili. Usa LBagar pH 8 come una regola. </li><li> Preparare 10% (w / v) di L-arabinosio. Filtrare sterilizzare attraverso un filtro a siringa da 0,2 micron. NOTA: arabinosio induce l&#39;espressione delle proteine ​​recombineering dal plasmide gbaA. </li><li> Controllare il diametro esterno della cultura BAC gbaA utilizzando uno spettrofotometro. Una volta che un OD 600 di 0,25-0,3 viene raggiunto, indurre le proteine ​​recombineering come descritto nel passaggio seguente. </li><li> Aggiungere 200 microlitri della soluzione di arabinosio 10% a 10 ml della coltura BAC per ottenere una concentrazione finale di arabinosio del 0,2%. Includere una cultura non indotte (senza Arabinose) da utilizzare come controllo negativo. </li><li> Trasferire la cultura BAC a 37 &#160; ° C incubatore agitazione e indurre l&#39;espressione rosso per 45 minuti agitando a 230 rpm. NOTA: L&#39;espressione di proteine ​​rosse è inefficiente a 30 ° C. </li><li> Spin le cellule e lavare con il 10% glicerolo 3 volte come descritto al punto 3.1.5. </li><li> Aggiungi 600-1,000 ng del plasmide ciascuna subcloning e la cassetta di inserimento (s) per la reazione recombineering. Includere un vettore solo e vettoriale, più controlli singoli inserti di verificare la competenza ricombinazione e l&#39;integrità del vettore e le cassette. </li><li> Ottenere una sospensione di cellule singole pipettando su e giù. Eseguire elettroporazione come descritto al punto 3.1.7 e successivo recupero in 1 ml LB a 37 ° C per 1 ora per plasmidi multi-copia o in 10 ml di LB a 37 ° C per 3 ore per i vettori BAC. </li><li> Piatto diverse diluizioni della coltura recuperato esempio, 90%, 10%, 1% sulle piastre doppi selezione agar e crescere a 37 &#160; ° C per 16 ore. </li></ol> 6. Analisi dei ricombinanti <ol style="margin-left: 40px;"><li> Optional: Scegliere da 6 a 12 colonie ed eseguire colonia PCR con un accompagnamento di fondo HA e un primer specifico inserto. Includere il plasmide subcloning e la cas inserimentosette plasmide così come il clone BAC genitore come controlli negativi. <ol><li> Eseguire l&#39;analisi gel delle PCR colonia. Identificare cloni positivi per la presenza di una banda luminosa alla dimensione attesa. NOTA: L&#39;alta efficienza del processo di clonazione SPI genera ricombinanti principalmente corrette. </li></ol></li><li> Selezionare colonie in 5 ml LB pH 8 contenente l&#39;antibiotico selettiva e crescere O / N a 37 ° C. </li><li> Preparare minipreps DNA utilizzando un kit di purificazione colonna come da istruzioni del produttore. </li><li> Preparare BAC miniprep DNA utilizzando standard di isolamento fenolo-cloroformio o un protocollo simile. </li><li> Eseguire digerisce RE sul DNA miniprep. DNA separato mediante elettroforesi su gel di agarosio. Analizzare i modelli RE per identificare i cloni contenenti i frammenti formati attesi di targeting vettore di corretta. Scegliere un RE che discrimina chiaramente tra il vettore carente inserto (s) e il vettore contenente l&#39;inserto (s). </li><li> Optionale: per ottenere cellule che sono privi della BAC, che è ancora presente nella E. coli dopo recombineering, utilizzare il DNA miniprep plasmide trasformare DH5alpha o DH10B E. coli. </li><li> Eseguire il sequenziamento del DNA attraverso l&#39;HA e cassette di inserimento per verificare gli errori di sintesi oligo. </li></ol>

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Biotechnol. 29, 255-265 (2011).</li><li>Mulryan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+vas+deferens+contraction+and+male+infertility+in+mice+lacking+P2X1+receptors.">Reduced vas deferens contraction and male infertility in mice lacking P2X1 receptors.</a> Nature. 403, 86-89 (2000).</li><li>Subramanian, K., Rutvisuttinunt, W., Scott, W., Myers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enzymatic+basis+of+processivity+in+lambda+exonuclease.">The enzymatic basis of processivity in lambda exonuclease.</a> Nucleic Acids Res. 31, 1585-1596 (2003).</li><li>Antoch, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+identification+of+the+mouse+circadian+Clock+gene+by+transgenic+BAC+rescue.">Functional identification of the mouse circadian Clock gene by transgenic BAC rescue.</a> Cell. 89, 655-667 (1997).</li><li>Rostovskaya, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-mediated+BAC+transgenesis+in+human+ES+cells.">Transposon-mediated BAC transgenesis in human ES cells.</a> Nucleic Acids Res. 40, e150 (2012).</li><li>Giraldo, P., Montoliu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+matters:+use+of+YACs,+BACs+and+PACs+in+transgenic+animals.">Size matters: use of YACs, BACs and PACs in transgenic animals.</a> Transgenic Res. 10, 83-103 (2001).</li><li>Hill, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+trimming:+minimizing+clone+overlaps.">BAC trimming: minimizing clone overlaps.</a> Genomics. 64, 111-113 (2000).</li><li>Warming, S., Costantino, N., Court, D. L., Jenkins, N. A., Copeland, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+highly+efficient+BAC+recombineering+using+galK+selection.">Simple and highly efficient BAC recombineering using galK selection.</a> Nucleic Acids Res. 33, e36 (2005).</li><li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Genome+Engineering+Using+CRISPR/Cas+Systems.">Multiplex Genome Engineering Using CRISPR/Cas Systems.</a> Science. 339, 819-823 (2013).</li></ol>

T.R.R is the inventor of a British patent application on multiplex recombineering technology.

Costruzione di linee di cellule ES e modelli di topo è storicamente coinvolto gene targeting utilizzando costrutti plasmide che contenevano l&#39;allele 4 modificato. Tuttavia, la costruzione di questi complessi vettori gene targeting ha dimostrato di essere un collo di bottiglia significativo nella produzione tempestiva di tali modelli. Lo sviluppo di strategie di costruzione vettore recombineering base ha permesso di disegni vettoriali migliorate e assemblaggio vettore più efficiente. Tuttavia, protocolli recombineering attuali coinvolgono ancora più passaggi, richiedono intermedio di purificazione plasmide e utilizzano diversi ceppi batterici. Subcloning oltre inserimento offre un nuovo approccio alla costruzione del vettore che può essere eseguita in un evento elettroporazione nel ceppo ospite BAC residente. L&#39;utilità di SPI in gene targeting stato qui testato in una varietà di applicazioni edili vettoriale. In tutti i casi esaminati qui, SPI ha dimostrato di essere efficiente e il plasmide ricombinante corretto è stato prodotto.Nella maggioranza dei casi, le molteplici cassette diversi stati correttamente inseriti nel vettore targeting. Grandi cassette di DNA e vettori erano facilmente alloggiati nel protocollo SPI e dimostrato la flessibilità del sistema. SPI si basa sull&#39;utilizzo di lunghe sequenze di omologia e fosforotioato (PTO) Protezione delle cassette di DNA lineari. Modifica PTO conferisce protezione da esonucleasi al DNA lineare 20,21 e la lunga HA aumenta l&#39;efficienza di ricombinazione per consentire multiplexing. Tuttavia, la sintesi di sequenze oligo lunghi aumenta le possibilità di accumulare errori soprattutto le eliminazioni. Le mutazioni nel oligo può essere particolarmente dannoso se coprono le regioni codificanti proteine. Sequenziamento del DNA attraverso il HA e copre l&#39;intera lunghezza della cassetta inserita è altamente raccomandato per eliminare cloni con alterazioni sequenza. L&#39;uso di un sistema di DNA polimerasi ad alta fedeltà viene anche suggerito di evitare l&#39;introduzione di unerrori y PCR. La lunghezza e la composizione del HA del plasmide subcloning è più critica rispetto a quella della cassetta di inserimento (dati non mostrati). Per applicazioni particolarmente sensibili come la costruzione di vettori Knockin, dove eventuali mutazioni in regioni esone non sono tollerati, l&#39;HA della cassetta di inserimento può essere ridotto (50-120 bp) per evitare problemi connessi con lunghe oligo. La cassetta di inserimento può anche essere lasciata phosphorothioated non modificato o doppia (dove la conoscenza della direzione di replica non è disponibile). Ma multiplexing in questi casi richiede ancora molto protetto HA plasmidi subcloning. Un avvertimento di questa particolare strategia è l&#39;abbassamento di efficienza multiplexing che possono potenzialmente influire clonazione SPI a diversi loci. La lunghezza della cassetta inserimento e il plasmide subcloning è un altro parametro importante nella clonazione SPI. Molecole di DNA più grandi elettroporazione meno efficiente e l&#39;effetto è cumulativo, dato il requirement introdurre tutte le cassette nella stessa cella (dati non mostrati). Multiplexing è più efficiente con cassette di inserimento più piccoli. DNA frammenti maggiore di 3 kb anche porre un limite elaborazione ssDNA Red mediata, che è più efficace fino a 3 kb 30. Cassette di inserimento superiori a 3 kb sono dual resecato e ricombinano meno efficiente attraverso una beta percorso indipendente 20. Pertanto, lo screening di colonie sufficienti a identificare il clone corretta diventa importante in questi casi. Una durata più lunga di ricombinazione dopo elettroporazione aumenta anche le possibilità di recupero del corretto clone in esercizi SPI difficili. Fino a quattro cassette piccole (&lt;1,5 kb) possono essere inseriti simultaneamente con il processo di SPI, se l&#39;efficienza multiplexing diminuisce con ciascuna cassetta aggiuntiva (dati non mostrati). Tuttavia, questo riflette il risultato di un esperimento ottimale SPI ed è opportuno considerare i limiti di multiplexing utilizzando molte grandi cassette. </p&gt; Lo sviluppo di strumenti di editing del genoma come cluster brevi ripetizioni palindromiche regolarmente intervallati (CRISPR) sistema endonuclease -cas9 ha permesso la creazione di nuove modifiche del genoma e ha portato a più efficiente di ingegneria del genoma 36. Tuttavia, queste nuove tecnologie sono integrate vettori gene targeting piuttosto che soppiantato. Si prevede che il sistema CRISPR-cas potrebbe sostituire la selezione antibiotica e ulteriormente affinare il protocollo recombineering multiplex.

Lo sviluppo di tecnologie di targeting dei geni ha consentito la costruzione di linee cellulari e modelli di topo per studiare diversi sistemi biologici 1-3. Una chiave prima fase nella modifica di una sequenza genomica è il progetto e la costruzione di un gene targeting vettore 4. Vettori targeting sono costrutti plasmide che portano l&#39;allele di interesse contenente le modifiche desiderate (s) affiancato da un marcatore di selezione (ad esempio, la neomicina), e lunghe regioni genomiche necessarie per un efficiente ricombinazione omologa in cellule di mammifero 5. Modificazione genetica precisa si ottiene l&#39;introduzione del vettore targeting in staminali embrionali (ES) cellule o cellule somatiche 6 7, in cui la ricombinazione omologa tra tratti identici di sequenza di DNA sul vettore targeting ei risultati locus genomico nel trasferimento della modificazione previsto al genoma mediante conversione genica 8. Tale modificellule ed ES possono essere iniettati in blastocisti di topo per produrre prole (chimere) che possono poi trasmettere questi alleli modificati tramite la linea germinale 9. Un percorso alternativo per produrre topi transgenici comporta la microiniezione di un vettore di espressione genica in singoli zigoti unicellulari topo, che porta l&#39;integrazione casuale del vettore nel genoma del mouse 10. I metodi tradizionali di costruzione del vettore si sono basati su tradizionali &#39;copia e incolla&#39; clonazione utilizzando enzimi di restrizione e ligasi del DNA per clonare le diverse marcatore di selezione e frammenti genomici in un vettore backbone. Tuttavia, un fattore limitante intrinseca della clonazione tradizionale è il posizionamento e la scelta di siti di restrizione, soprattutto con sequenze di DNA più lunghe. Ciò richiede spesso più passaggi subcloning ed introduce anche sequenze di DNA estraneo nel vettore, che spesso porta ad una minore efficienza di gene targeting. Recombineering (eng recombinogenicineering) è una tecnologia di ingegneria DNA che supera queste limitazioni utilizzando la ricombinazione omologa (HR) mediato da proteine ​​fago ricombinazione a E. coli 11,12. Da una regione di una sequenza omologa può servire come substrato di recombineering, i vincoli di disponibilità di siti di restrizione vengono rimossi. Le grandi sequenze di DNA possono essere facilmente modificati direttamente in vivo, quindi anche preservare la loro integrità strutturale 13. Recombineering è molto efficiente con omologie brevi (50 bp) 14 e, pertanto, le braccia di omologia (HA) possono essere comodamente incorporati in sequenze oligo sintetici. In un esperimento recombineering tipico, un oligo o un DNA a doppia elica (dsDNA) frammento contenente HA è elettroporate in recombineering competente E. coli contenenti il bersaglio sia situato sul cromosoma o su un plasmide 15. Il potenziale di ricombinazione è conferito dalla espressione inducibile della rec Redombination proteine ​​del fago 16,17 o le proteine ​​RecET del rac profago 18. Il exonuclease Red / Rece converte dsDNA lineare ad un singolo filamento di DNA (ssDNA) intermedio, che viene poi legato dal suo partner, Rosso / Rect, un singolo filamento di proteine ​​ricottura (SSAP) 19. La ricottura di un lungo ssDNA o una breve oligo alla sua sequenza bersaglio complementare si verifica sul filamento in ritardo della forcella replica e conduce all&#39;incorporazione della sequenza al sito bersaglio. Fanalino di filo ssDNA ricombinazione è la base della elevata efficienza di recombineering e può essere descritto dal &#39;beta&#39; modello ricombinazione 20,21. Un flusso di lavoro tipico recombineering per costruire un vettore gene targeting coinvolge uno dei due seguenti percorsi. Una via comporta subcloning regione genomica desiderata da un clone BAC topo in un plasmide seguita dall&#39;inserimento sequenziale di siti di ricombinazione LoxP, un marcatore di selezione <sup&gt; 22 ecc, o il percorso alternativo comporta mira il BAC locus genomico con i diversi elementi di targeting vettore di vari cicli di recombineering 10,23 e poi subcloning il locus modificato in un plasmide mediante riparazione gap clonazione 24,25. Variazioni su questo tema sono state utilizzate in diversi gasdotti ad alta produttività recombineering nell&#39;ambito di programmi di produzione di grandi dimensioni del mouse 26,27. Tuttavia, queste procedure comportano fasi complicate e lunghe, richiedono l&#39;uso di vettori specializzati e E. coli ceppi (ad esempio, cellule che esprimono Cre) e utilizzare uno o più stadi intermedi di purificazione del DNA vettoriale e ri-trasformazione (Tabella 1). Subcloning più inserimento (SPI) è una tecnica recombineering romanzo che combina beta ricombinazione e riparazione gap clonazione in un unico processo (Figura 1). Montaggio vettore SPI è semplice, veloce e flessibile e offre un miglioramento significativo recombi serieneering approcci (Tabella 1). Qui, dimostriamo la facilità e l&#39;utilità di usare SPI per diverse applicazioni della costruzione del vettore con una particolare enfasi sulla costruzione di disegni vettoriali non standard e stimolanti. Casi di test inclusi la costruzione di una fluorescenza giornalista knockin vettore, una duplice espressione proteica tag Knockin vettore, un BAC fluorescenza giornalista vettoriale e un ko vettore di condizionale. Questi esaminate variamente il requisito di localizzare un recettore sulla superficie cellulare, purificare un complesso proteico nucleare o condizionatamente ablazione l&#39;espressione di un gene.

<table border="0" cellpadding="0" cellspacing="0" width="1597">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Name of Material/ Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:216px;">Catalog Number</td>
			<td style="width:941px;">Comments/Description</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:315px;">Antibiotics except Zeocin</td>
			<td style="width:127px;">Sigma: http://www.sigmaaldrich.com</td>
			<td style="width:216px;">Ampicillin, A9518-5G; 
			Chloramphenicol , C1919-5G; Blasticidin, 15205-25MG; Gentamicin, G1264-50MG; Hygromycin, H3274-50MG; Kanamycin,: K1876-1G; 
			Tetracycline hydrochloride, T7660-5G; Trimethoprim, 92131-1G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Zeocin</td>
			<td style="width:127px;">Invivogen:&#160;</td>
			<td style="width:216px;">ant-zn-1</td>
			<td>Zeocin is also available from Life technologies/Fisher</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">C57BL/6 BAC clones</td>
			<td style="width:127px;">CHORI: https://bacpac.chori.org/&#160;</td>
			<td style="width:216px;">RPCI-23 or RPCI-24 BAC libraries</td>
			<td>129/Sv BAC clones can be obtained from Invivogen or Life technologies/Fisher</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:315px;">KOD hotstart DNA polymerase system</td>
			<td style="width:127px;">Millipore: https://www.merckmillipore.com</td>
			<td style="width:216px;">71086-3</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:315px;">L-Arabinose</td>
			<td style="width:127px;">Sigma: http://www.sigmaaldrich.com</td>
			<td style="width:216px;">A3256-25G</td>
			<td></td>
		</tr>
		<tr height="189">
			<td height="189" style="height:189px;width:315px;">Long oligos</td>
			<td style="width:127px;">IDT: https://www.idtdna.com; Gene link: https://www.genelink.com</td>
			<td style="width:216px;">IDT Ultramers or Gene Link long oligos</td>
			<td>PTO and Phosphosphate available as custom modifications. PAGE purification strongly recommended for long oligos.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">MinElute PCR purification kit</td>
			<td style="width:127px;">Qiagen: http://www.qiagen.com&#160;</td>
			<td align="right" style="width:216px;">28004</td>
			<td>Minimum elution PCR purifications kits are preferred.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">QIAPrep Spin Mini Prep kit</td>
			<td style="width:127px;">Qiagen: http://www.qiagen.com&#160;</td>
			<td align="right" style="width:216px;">27104</td>
			<td>Silica column or similar matrix kits are preferred.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">Restriction enzymes</td>
			<td style="width:127px;">NEB: https://www.neb.com</td>
			<td style="width:216px;">DpnI: R0176L</td>
			<td>See NEB website for a list of RE.</td>
		</tr>
	</tbody>
</table>

subcloning plus insertion (spi) - a novel recombineering method for the rapid construction of gene targeting vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Watch this Scientific Journal Video about Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors at JoVE.com

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Subcloning più Insertion (SPI) - A Novel Metodo recombineering per la costruzione rapida di gene targeting Vettori

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

subcloning-plus-insertion-spi-novel-recombineering-method-for-rapid

Research

JoVE Journal

Biology

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

16.3K Views.  University of Leicester. Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term ‘subcloning plus insertion’, which can be used to generate custom gene targeting vectors rapidly.

Video: Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Un rapido ad alta velocità Metodo per ribonucleoproteine ​​Mapping (RNP) su Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Ricerca

Biologia

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hi-C: un metodo per studiare l&#39;architettura tridimensionale dei genomi.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

La produzione di C. elegans Transgeni attraverso recombineering con il GalK Marcatori selezionabili

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

Un nuovo metodo per la Cultura e polarizzata stimolazione umani espianti mucosa intestinale

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

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 ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Metodo di Cultura per il mouse embrionale del cuore

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

Zinc-finger nucleasi avanzata gene targeting in cellule staminali embrionali umane

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

L&#39;isolamento di filtrazione di acidi nucleici: DNA metodo di estrazione semplice e veloce

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

A Rapid, metodo scalabile per l&#39;isolamento, studio funzionale, e analisi di Cell-derivati ​​matrice extracellulare

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

Protocolli per testare la tossicità di romanzo chimiche insetticide per le zanzare

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...