Gli autori sono molto grati a RJ Sommer e X Wang per l&#39;assistenza con microiniezione, così come i commenti penetranti dal revisori anonimi. Questo lavoro è supportato dal NIH concedere SC2GM089602.

1. Transgenesi: preparazione del DNA <ol><li> Dominante co-iniezione marcatore: pRL3 [ppa-PRL-1 (tu92)] Il plasmide pRL3 è un dominante co-iniezione marker per individuare visivamente eventi di trasformazione di successo. Questo plasmide codifica per un allele mutante dominante (tu92) della PPA-PRL-1 gene strettamente correlata alla SQT-1 gene del collagene in C. elegans e trasforma il tipo selvatico locomozione sinusoidale movimenti di torsione in senso orario lungo il corpo del worm asse 1,5. L&#39;animale trasformato è molto simile al marcatore di selezione popolare dominante rol-6 (su1006) utilizzato in C. elegans negli ultimi 20 anni. </li><li> Obiettivo giornalista transgene: ppa-EGL-4p:: GFP Un gene di interesse possono essere collegati trascrizionalmente o traduzionali ad una sequenza codificante GFP 9. In questo studio, il PPA-EGL-4promoter:: GFP (EGL-4, proteina chinasi cGMP dipendente) was usato per determinare se un frammento 2 kb a monte dell&#39;inizio della traduzione può conferire espressione GFP in P. pacificus PS312. Questo vettore GFP contiene una regione codificante GFP modificato con introni il multiuso ppa-rpl-23 3 &#39;UTR Terminator 5 gentilmente fornito da Xiaoyue Wang e Ralf J. Sommer (Max-Planck Institute, Tuebingen, Germania, UE). e </li><li> DNA genomico: PS312 L&#39;aggiunta di tipo selvatico P. pacificus DNA genomico PS312 è necessario per ottenere l&#39;eredità transgene stabile, in particolare da animali transgenici F 1 F 2 alla loro progenie 5. Non è ancora certo se il DNA genomico forme complesse array supplementare cromosomica con i due transgeni (la co-iniezione marcatore PRL-3 e la ppa-EGL-4p:: reporter GFP) simili a quelli osservati in stabile F 2 linee transgeniche in C. elegans 3. Tuttavia, l&#39;obbligo di avere il DNA genomico e transgeni di condividere Identicasovrasta l coesa suggerisce fortemente la formazione di complessi extra-array cromosomico della cellula ospite può riconoscere per la trasmissione corretta di DNA (gDNA preparati utilizzando Sigma G1N10 kit). </li><li> Digestione del DNA Enzimi di restrizione sono usati per linearizzare il PPA-EGL-4p:: GFP DNA vettore e di produrre sbalzi appiccicoso compatibile con il marcatore pRL3 co-iniezione e PS312 gDNA. Mescolare agitando, non vortex. Incubare per un&#39;ora a 37 ° C. </li></ol><table border="1"><tbody><tr><td> pRL3 </td><td> 4 microgrammi </td></tr><tr><td> 10xbuffer </td><td> 10 microlitri </td></tr><tr><td> PstI (10 U / mL) </td><td> 4 microlitri </td></tr><tr><td> dH 2 O </td><td> ~ </td></tr><tr><td> Totale: </td><td> 100 ul </td></tr><tr><td> Ppa-EGL-4p:: GFP </td><td> 4 microgrammi </td></tr><tr><td> Tampone 10x </td&gt; <td> 10 microlitri </td></tr><tr><td> Sali (10 U / mL) </td><td> 4 microlitri </td></tr><tr><td> dH 2 O </td><td> ~ </td></tr><tr><td> Totale: </td><td> 100 ul </td></tr><tr><td> gDNA PS312 </td><td> 10 mcg </td></tr><tr><td> Tampone 10x </td><td> 10 microlitri </td></tr><tr><td> Pst I e Sal I (10 U / mL) </td><td> 8 microlitri </td></tr><tr><td> dH 2 O </td><td> ~ </td></tr><tr><td> Totale: </td><td> 100 ul </td></tr></tbody></table> Tabella 1. Miscela di enzimi di restrizione Digest. <ol start="5"><li> DNA precipitazioni e preparazione <ol><li> Aggiungi acetato di sodio 1:10 (3 M NaCOOCH 3 pH 5,2) e 2,5 volte il volume del 100% di etanolo al al prodotto digerito; aggiungere 20 ng / l di glicogeno per vedere pellet più facile. </li><li> Centrifugare a14.000 rpm per 15 minuti a 4 ° C. </li><li> Decantare il surnatante tramite ribaltamento lentamente la provetta a testa in giù e toccando su una carta velina, facendo in modo che il pellet è sicuro nell&#39;angolo in basso del tubo e privo di etanolo. </li><li> Aggiungere 1 ml di etanolo 75%. </li><li> Immediatamente girare a 14.000 giri al minuto per 5 minuti a 4 ° C. </li><li> Decantare il surnatante tramite lentamente ribaltamento della provetta a testa in giù e toccando su una carta velina facendo in modo che il pellet è custodito l&#39;angolo inferiore del tubo e privo di etanolo. </li><li> Secco pellet in centrifuga a vuoto a 14.000 rpm per 5 minuti a 25-30 ° C, fino a secco e completamente privo di etanolo. </li><li> Aggiungere 30 ml di TE o sterili dH 2 O e lasciare per una notte a 4 ° C prima brevemente mescolando con un vortice. </li><li> La tabella 2 descrive come creare la miscela di iniezione per ogni DNA purificato per l&#39;iniezione. </li><li> Conservare a -20 ° C. Dopo lo scongelamento la miscela nelproiezione, spin down del tubo a 14.000 rpm per 5 minuti per eliminare particelle di detriti che potrebbero ostruire l&#39;ago di iniezione. </li></ol></li></ol><table border="1"><tbody><tr><td> Materiale genetico </td><td> Concentrazione </td></tr><tr><td> Ppa-EGL-4p:: GFP (Sali) </td><td> &gt; 5 ng / mL (0.1-10ng/μl) </td></tr><tr><td> pRL3 (PstI) </td><td> &gt; 1 ng / mL </td></tr><tr><td> gDNA PS312 (PstI + Sali) </td><td> &gt; 60 ng / mL </td></tr><tr><td> dH 2 O </td><td> ~ </td></tr><tr><td> Totale: </td><td> 30 microlitri </td></tr></tbody></table> Tabella 2. Miscela ppa-PRL-1 iniezione 2. RNA Interference: In sintesi in vitro di RNA a doppio filamento <ol><li> Usa PCR per amplificare il gene di interesse. RHL091 5&#39;-agtggatccGAAGGTCCATACGGGAGC-3 &#39;(Sito BamHI) e RHL092 5&#39;-tatctgcagGTGAGGAGTACCAGGAGAG-3 &#39;(sito PstI) sono stati utilizzati per amplificare un frammento di 464 bp genomico (posizione 3-466) dal vettore pRL3 contenente il PPA-PRL-1 (tu92) allele. </li><li> Digerire il prodotto di PCR con BamHI e PstI. </li><li> Legare il frammento BamHI / PstI digerito pL4440 RNAi alimentazione vettore 2. </li><li> Trasformare il vettore inserito in cellule competenti e farle crescere in piastre di LB ampicillina mezzi di comunicazione (50 mcg / ml). </li><li> Selezionare il vettore inserito con successo mediante PCR utilizzando i primer T7. </li><li> Utilizzare il T7 affiancato prodotto di PCR contenente il PRL-1 gene per fare RNA a doppio filamento dsRNA) utilizzando il BLOCCO-IT RNAi sintesi kit (Invitrogen). </li><li> La concentrazione dsRNA è meglio se&gt; 200 ng / mL, fino a 1 mg / mL per l&#39;iniezione. </li><li> Conservare a -20 ° C. Dopo lo scongelamento la miscela per iniezione, rotazione verso il basso il tubo a 14.000 rpm per 5 minuti per liberarsi di particelle di detriti che potrebbero ostruire il injection ago. </li></ol> 3. Microiniezione: protocollo per l&#39;iniezione dei transgeni e / o dsRNA <ol><li> Aghi di iniezione <ol><li> Impostare il Narishige ago estrattore (modello #: PC-10) temperatura: <ol><li> Ruotare la manopola in basso a &quot;Heater No.2&quot;. </li><li> Girare il &quot;Adj riscaldatore No.2&quot;. manopola fino a pannello legge 55,0-60,0 ° C per la punta dell&#39;ago conici di varie lunghezze. </li></ol></li><li> Ruotare la manopola in basso torna a &quot;Fase 1&quot; (Tira ago in un passo). </li><li> Carico ago nella camera e assicurarsi che sia sicuro. </li><li> Premere il pulsante &quot;Start&quot; (tasto rosso) e consentire l&#39;ago di essere tirato con tutti i pesi (questo dovrebbe fare due aghi identiche in direzioni opposte). </li><li> Rimuovere l&#39;ago dalla camera e conservare in una piastra di coltura di plastica fissata in posizione di Play-Doh per evitare l&#39;accumulo di polvere sugli aghi. </li></ol></li><li> Pad di montaggio <ol><li> In una provetta da 1,5 ml centrifuga, fare una solutio 2% Agar Noblen con l&#39;aggiunta di 0,02 g di Agar Noble a 1 ml di H 2 O. L&#39;agar possono essere conservati a 4 ° C per un massimo di un anno. </li><li> Mescolare accuratamente e si fondono agar in un blocco di calore impostato&gt; 88 ° C. </li><li> Usando una micropipetta da 1 ml, una goccia di liquido Agar 2% Nobile al centro del vetrino (Fisher, 12-544-F). </li><li> Appiattire mettendo un&#39;altra lastra di vetro sulla parte superiore della goccia. </li><li> Ripetere il &quot;Passo Drop&quot; fino a quando non vi è uno strato di cinque vetrini. </li><li> Rimuovere ogni vetrino facendo scorrere il vetro scivola a vicenda. </li><li> Secco appiattire pad agar su vetrini in un forno sotto vuoto a 70 ° C per 4 ore per notte (oa temperatura ambiente per una notte). </li><li> Fai pastiglie di iniezione multipla e conservare per un uso futuro. </li></ol></li><li> Microiniezione in gonadi ermafrodite </li></ol><img alt="Figura 1" src="/files/ftp_upload/3270/3270fig1.jpg" /> Figura 1. Un disegno schematico della P. pacificus Anatnomia. Le cellule chiare nucleate sono cellule germinali femminili (ovociti) e la parte in grigio è l&#39;intestino. Solo una gonade distale anteriore è mostrato a meno di notare le due gonadi distale si incrociano dorsalmente vicino a metà del corpo. <img alt="Figura 2" src="/files/ftp_upload/3270/3270fig2.jpg" /> Figura 2. Immagini di microiniezione. (A) La punta dell&#39;ago iniezione è a fuoco con la linea più a destra del pezzo tirato capillare. Con una leggera toccando la punta dell&#39;ago al bordo, la punta dell&#39;ago si dovesse rompere, pur mantenendo una punta acuminata. (B) Gli inserti ago nella parte superiore gonade appena sopra la curvatura budella per la prima iniezione. (C) Gli inserti ago nel fondo gonade appena sotto la curvatura budella per la seconda iniezione. <ol start="3"><li><ol><li> Assicurarsi che il contrasto e la tosatura DIC impostazione è ideale per visualizzare le gonadi ermafrodita. </li><li> Carico 1,0-2,0 ml di miscela di iniezione nella tiratocapillare ago. </li><li> Posizionare l&#39;ago nel manipolatore microiniezione, misuratori di pressione del serbatoio di azoto deve essere impostato a condizioni ideali (10-20 psi per 0,5-1 sec). </li><li> Rompere la punta dell&#39;ago toccandolo leggermente a bordo di un tubo sottile tirato capillare posto su un vetrino (figura 2A). <ol><li> Ago scorrere interruttore: la parte più sottile di un capillare tirato posto su un vetrino in una goccia d&#39;olio </li></ol></li><li> Una volta che l&#39;ago è rotto può essere posto ad una posizione del minimo, mentre si scegli il tuo verme al pad (P. pacificus hanno una finestra 5 minuti per l&#39;iniezione prima che si secchi-out e morire). </li><li> Utilizzando un piatto pieno di J4-giovani vermi adulti, versare l&#39;olio di paraffina abbastanza (pesante) per coprire appena tutti i vermi in OP50 prato batterica. </li><li> Scegli un verme e posizionarlo sulla rampa di iniezione. </li><li> Posizionare il vetrino con il worm sul microscopio; posizione delle gonadi del verme in direzione dell&#39;ago e la vulva dalla necessitàle (Figura 2B, 2C). </li><li> Abbassare l&#39;ago in a posizione della vista microscopio. </li><li> Posizione della gonade in alto e l&#39;ago sullo stesso piano focale (Figura 2B). </li><li> Poke il worm delicatamente e la pompa nella miscela di iniezione; ooctyes separa verso la punta distale indicano una iniezione di successo (Per ottimizzare il transgene o consegna dsRNA, si dovrebbe vedere un seguito di iniezione ondata di espansione della gonade che continua fino a quando non è vicino al distale punta). </li><li> Riposizionare l&#39;ago per iniettare alla gonade inferiore e iniezioni ripetute, ma poiché questo gonade è sotto l&#39;intestino la diffusione degli ovociti non sarà visto ed è quindi più difficile da raggiungere (Figura 2C). </li><li> Normale verme alla ricerca indica una iniezione di successo dopo l&#39;iniezione, non ci dovrebbe essere alcun gonadi danneggiato (che sporge fuori dalla vulva o schioccato all&#39;interno della cavità del corpo). </li><li> Posizionare l&#39;ago in posizione di riposo. </li><li> Preparatevi a salvare il worm. </li><li> Aggiungere uncalo (0,5 mL) di tampone M9 sul tampone dove si trova il worm iniettato. </li><li> Utilizzando alcuni OP50 batteri sulla tua scelta, toccare il worm con l&#39;OP50, e dovrebbe bastone. Non usiamo pipetta a bocca. </li><li> Posizionare il worm sulla piastra seminata con 50 punti l di OP50, 2-4 worm possono essere salvati su piatti del genere. </li></ol></li><li> Post-iniezione di stoccaggio e di screening per transgenesi <ol><li> Conservare iniettato vermi (P 0) a 20 ° C per ~ 4 giorni di tempo per deporre le uova e crescono F 1. </li><li> Vermi schermo il quarto giorno di fenotipo selezionato (Per schermo per transgenesi, la ricerca di animali rotolamento esprimere la ppa-PRL-1 marcatore dominante). </li><li> Scegli singolo indipendente F 1 &#39;animale s con il fenotipo osservato alla piastra nuove teste di serie e conservarlo a 25 ° C, questa temperatura favorisce la formazione di linee stabili da F 1&#39; s. </li><li> F singolo 2 da indipendente F 1 linee. Individuale F 2 </subLinee di&gt; avere simili tassi di trasmissione transgene. Noi spesso ottenere&gt; 15 F 2 linee per trasformare F 1 animale. </li><li> Conservare le generazioni successive a 25 ° C. </li><li> La velocità di trasmissione dei rollers di solito rimane costante dopo la generazione F 4, per cui è necessario in questo momento di determinare il livello di espressione del transgene bersaglio (PPA-EGL-4p:: GFP) per ogni singola linea. Espressione di GFP non è necessariamente correlato l&#39;entità della penetranza del fenotipo dominante rullo. </li></ol></li><li> Post-iniezione di stoccaggio e di screening per fenotipi RNAi <ol><li> Conservare iniettato vermi (P 0) a 20 ° C per ~ 4 giorni per definire e far crescere F 1. </li><li> Schermo F 1 vermi il quarto giorno di fenotipo selezionato (Per schermo per fenotipo knockdown RNAi, ricerca di fenotipi penetranti che possono essere associati con la perdita di attività del gene bersaglio, nel nostro caso la perdita della PPA-PRL-1 fenotipo). </li><li> Nella nostra esperienza, il non-roller fenotipo knockdown si perde in&gt; 97% della F 2. </li></ol></li></ol> 4. Rappresentante dei risultati: 1. Risultato della transgenesi <table border="1"><tbody><tr><td> Sessione </td><td> Iniettato P 0 </td><td> F 1 Rulli </td><td> % Transgenico F 2 </td><td> % Medio di trasmissione dopo la F 2 </td></tr><tr><td> 1 </td><td> 60 </td><td> 2 </td><td> (Linea 1) 0% (Linea 2) 13% </td><td> NA 13% ± 10 </td></tr><tr><td> 2 </td><td> 40 </td><td> 1 * </td><td> (Linea 3) 0% </td><td> NA </td></tr><tr><td> 3 </td><td> 40 </td><td> 0 </td><td> 0% </td><td> NA </td></tr><tr><td> 4 </td><td> 40 </td><td> 1 </td><td> (Linea 4) 0% </td><td> NA </td&gt; </tr><tr><td> 5 </td><td> 20 </td><td> 0 </td><td> 0% </td><td> NA </td></tr><tr><td> 6 </td><td> 40 </td><td> 1 </td><td> (Linea 5) 26% </td><td> 22% ± 10 </td></tr></tbody></table> * Un rullo maschio che non ha attraversato; NA: Non applicabile Risultati della tabella 3 PS312 iniettato con PPA-EGL-4p.:: GFP (Sal ho digerito), pRL3 (rullo) (Pst ho digerito), e gDNA PS312 (PstI e Sali digerito). <img alt="Figura 3" src="/files/ftp_upload/3270/3270fig3.jpg" /> Figura 3 (A) tipo selvatico (B, C) ​​Una stabile F 4 pRL3; ppa-EGL-4p.:: GFP linea transgenica mostrando espressione testa neurone GFP. 2. Risultato di interferenza dell&#39;RNA <img alt="Figura 4" src="/files/ftp_upload/3270/3270fig4.jpg" /> Figura 4. RNA iniettato prl-1 mutantimostrano knockdown completa di locomozione &quot;rolling&quot;. (A) PRL-1 a rulli guadagno di funzione mutante tu92. (B) &quot;abbattuto&quot; prl-1 mutante postura tipo selvatico normale esporre e locomozione. (C) iniettata PRL-1 (in basso) è anche più lungo corpo di prl-1 mutante (in alto). (D) Il corpo più del iniettato PRL-1 (in basso) è anche più lungo del PS312 tipo selvatico (in alto). Il fenotipo corpo più a lungo è stata osservata anche nel rol-5 (SQT-1) knockdown RNAi di C. elegans N2 (dati non riportati). <table border="1"><tbody><tr><td></td><td> rotolo </td><td> non-roll </td></tr><tr><td> A prl-1 dsRNA (200 ng / mL) </td><td> 13 </td><td> 21 </td></tr><tr><td> B prl-1 dsRNA (1000 ng / mL) </td><td> 9 </td><td> 16 </td></tr><tr><td> controllo </td><td> 20 </td><td> 2 </td></tr></tbody></table> Tabella 4. </sTrong&gt; Riepilogo della PPA-PRL-1 dsRNA iniezioni. (A) [dsRNA] = 200 ng / mL; (B) [dsRNA] = 1000 ng / mL. P = 0,0019 e 0,041 per il test esatto di Fisher, a due code, per 200 e 1000 iniezioni ng / mL, rispettivamente.

<ol>
	<li>Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., Mello, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+Genetics.">Reverse Genetics.</a> Wormbook. , (2006).</li><li>Mello, C. C., Kramer, J. M., Stinchcomb, D., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+in+C.+elegans:+extrachromosomal+maintenance+and+integration+of+transforming+sequences.">Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO J. 10, 3959-3970 (1991).</li><li>Junio, A. J., Li, X., Massery, H. C., Nolan, T. J., Lamitina, S. T., Sundaram, M. V., Lok, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strongyloides+stercoralis:+cell-+and+tissue-specific+transgene+expression+and+co-transformation+with+vector+constructs+incorporating+a+common+multifunctional+3'+UTR.">Strongyloides stercoralis: cell- and tissue-specific transgene expression and co-transformation with vector constructs incorporating a common multifunctional 3' UTR.</a> Exp. Parasitology. 118, 253-265 (2008).</li><li>Schlager, B., Wang, X., Braach, G., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+dominant+roller+mutant+and+establishment+of+DNA-mediated+transformation+in+the+nematode+Pristionchus+pacificus.">Molecular cloning of a dominant roller mutant and establishment of DNA-mediated transformation in the nematode Pristionchus pacificus.</a> Genesis. 47, 300-304 (2009).</li><li>Hong, R. L., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pristionchus+pacificus:+a+well-rounded+nematode.">Pristionchus pacificus: a well-rounded nematode.</a> Bioessays. 28, 651-659 (2006).</li><li>Hong, R. L., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoattraction+in+Pristionchus+nematodes+and+implications+for+insect+recognition.">Chemoattraction in Pristionchus nematodes and implications for insect recognition.</a> Curr. Biol. 16, 2359-2365 (2006).</li><li>Rudel, D., Riebesell, M., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadogenesis+in+Pristionchus+pacificus+and+organ+evolution:+development,+adult+morphology+and+cell-cell+interactions+in+the+hermaphrodite+gonad.">Gonadogenesis in Pristionchus pacificus and organ evolution: development, adult morphology and cell-cell interactions in the hermaphrodite gonad.</a> Dev. Biol. 277, 200-221 (2005).</li><li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+marker+for+gene+expression.">Green fluorescent protein as a marker for gene expression.</a> Science. 11, 802-805 (1994).</li><li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Introduction+to+Worm+Lab:+from+Culturing+Worms+to+Mutagenesis.">An Introduction to Worm Lab: from Culturing Worms to Mutagenesis.</a> J. Vis. Exp. (47), e2293-e2293 (2011).</li><li>Evans, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+and+Microinjection.">Transformation and Microinjection.</a> Wormbook. , (2006).</li></ol>

Nessun conflitto di interessi dichiarati.

P. popolazioni pacificus si trovano in stretta associazione con varie specie scarabeo tutto il mondo ed è un nematode modello intermedio tra il vivere libero e nematodi parassiti. La forza del P. pacificus come organismo modello emergente laici nella integrazione delle sue mappe genetiche e fisiche che promuovono la mappatura della posizione di mutanti isolati da imparziali schermi in avanti genetico (cioè non solo per geni candidati precedentemente caratterizzato in C. elegans)...

<table border="1"><tbody><tr><th> Prodotto: </th><th> Catalog #: </th><th> Fornitore: </th></tr><tr><td> DMI3000 iniezione microscopio </td><td> DMI3000 </td><td> Leica </td></tr><tr><td> Microinjector manipolatore (in presa diretta) </td><td> Narashige congiunto AC-3 palle </td><td> Tritech di ricerca </td></tr><tr><td> Ago Puller </td><td> Narashige PC-10 </td><td> Tritech di ricerca </td></tr><tr><td> MicroInjector ™ All-Digital Multi-pressione di sistema </td><td> Narashige Minj-D </td><td> Tritech di ricerca </td></tr><tr><td> GeneElute ™ mammiferi Genomic DNA Kit Miniprep </td><td> G1N70 </td><td> Sigma </td></tr><tr><td> pJet Clonazione Jet kit </td><td> K1231 </td><td> Fermentas </td></tr><tr><td> GeneJET plasmide Miniprep kit </td><td> K0502 </td><td> Fermentas </td></tr><tr><td> DNA Clean &amp; Concentrator ™ </td><td> D4005 </td><td> ZYMO ricerca </td></tr><tr><td> BLOCK-IT ™ RNAi TOPO ® kit di trascrizione </td><td> K3500 &amp; K3650-01-01 </td><td> Invitrogen </td></tr><tr><td> Difco Agar ™ Noble </td><td> DF0142-15-2 </td><td> Fisher scientifi </td></tr><tr><td> Microscopio vetro di copertura (1,5 - 0,16 a 0,19 millimetri di spessore; Dimensioni: 50 x 45mm) </td><td> 12-554-F </td><td> Fisher Scientific </td></tr><tr><td> Vetro capillari (filamento) </td><td> 615000 </td><td> AM sistemi </td></tr><tr><td> Olio di paraffina (pesante) </td><td> O122-1 </td><td> Fisher Scientific </td></tr><tr><td> KH 2 PO 4 </td><td> P386-500 </td><td> Fisher Scientific </td></tr><tr><td> Na 2 HPO 4 </td><td> AC20651-5000 </td><td> Fisher Scientific </td></tr><tr><td> NaCl </td><td> BP3581 </td><td> Fisher Scientific </td></tr><tr><td> MgSO 4 </td><td> M80-500 </td><td> Fisher Scientific </td></tr></tbody></table>

rnai mediated gene knockdown and transgenesis by microinjection in the necromenic nematode pristionchus pacificus

Anche se è sempre più accessibili per gli organismi modello emergente di ottenere genomi completamente sequenziati, approfondita la funzione del gene e analisi di espressione di RNA interference e transgenesi stabile rimanere limitata in molte specie a causa della particolare anatomia e biologia cellulare molecolare dell&#39;organismo. Per esempio, al di fuori del Caenorhabditis gruppo corona che comprende Caenorhabditis elegans 3, stabilmente trasmesse in linee transgeniche non Caenorhabditis specie non sono stati riportati in questo phylum specioso (Nematodi), con l&#39;eccezione di Strongyloides stercoralis Pristionchus pacificus 4 e 5. Per facilitare il ruolo crescente del P. pacificus nello studio di sviluppo, l&#39;evoluzione e il comportamento 6-7, si descrivono qui i metodi attuali di utilizzare microiniezione per la produzione degli animali transgenici e gene abbattere da RNAi. Come le gonadi di <em&gt; C. elegans e la maggior parte di altri nematodi, le gonadi di P. pacificus è syncitial e capace di incorporare DNA e RNA in ovociti al momento della consegna da microiniezione diretta. A differenza di C. elegans tuttavia, l&#39;ereditarietà transgene stabile e di espressione somatica in P. pacificus richiede l&#39;aggiunta di DNA genomico digerito con endonucleasi di sé complementari alle estremità dei transgeni bersaglio e marcatori co-iniezione 5. L&#39;aggiunta di DNA genomico vettore è analoga a quella prescritta per l&#39;espressione del transgene in Strongyloides stercoralis 4 e nelle cellule germinali di C. elegans. Tuttavia, non è chiaro se il requisito specifico per il DNA genomico proprio gli animali &#39;è perché P. pacificus soma è molto efficiente a tacere non complessi multi-copia dei geni o che gli array extracromosomico in P. pacificus richiedono sequenze genomiche per l&#39;assemblaggio cinetocoro corretto durante la mitosi. La migrazione ventrale dei due bracci (didelphic) gonadi in ermafroditi complica ulteriormente la possibilità di iniettare in entrambe le gonadi vermi individuale 8. Abbiamo anche dimostrare l&#39;uso di microiniezione per knockdown un mutante dominante (rullo, tu92) iniettando RNA a doppio filamento (dsRNA) nelle gonadi di ottenere non rotolamento F 1 progenie. A differenza di C. elegans, ma come la maggior parte di altri nematodi, P. pacificus PS312 non è ricettivo a sistemico RNAi attraverso l&#39;alimentazione e ammollo e quindi dsRNA deve essere somministrato con microiniezione nelle gonadi syncitial. In questo studio, speriamo di descrivere il processo di microiniezione necessari per trasformare un PPA-EGL-4 promotore:: reporter di fusione GFP e knockdown un rullo dominante PRL-1 (tu92) mutante in un protocollo visivamente informativo.

Watch this Scientific Journal Video about RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus at JoVE.com

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In organismi modello, transgenesi in grado di manipolare le funzioni del gene, mentre RNAi può knockdown trascritti di mRNA specifico 1-2. Questo protocollo si propone di illustrare le tecniche necessarie per introdurre in modo stabile il DNA trasmessi e transitori di RNA a doppio filamento nel nematode necromenic<em&gt; Pristionchus pacificus</em&gt; Per gli studi in biologia evolutiva dello sviluppo, e comportamentali.

RNAi mediata Knockdown Gene e Transgenesi dalla microiniezione nel nematode Necromenic Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

rnai-mediated-gene-knockdown-transgenesis-microinjection-necromenic

Research

JoVE Journal

Biology

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

16.4K Views.  California State University. In organismi modello, transgenesi in grado di manipolare le funzioni del gene, mentre RNAi può knockdown trascritti di mRNA specifico 1-2. Questo protocollo si propone di illustrare le tecniche necessarie per introdurre in modo stabile il DNA trasmessi e transitori di RNA a doppio filamento nel nematode necromenic Pristionchus pacificus Per gli studi in biologia evolutiva dello sviluppo, e comportamentali.

Video: RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

Localizzato RNAi e di espressione genica ectopica nel Leech medicinali

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

Ricerca

Biologia

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Microiniezione di embrioni di zebrafish per analizzare la funzione del gene

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

Consegna batterica di RNAi per DJ: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Attivazione di apoptosi da Microiniezione citoplasmatici del Citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentivirali-mediata Knockdown Durante Ex Vivo</emEritropoiesi> di cellule staminali emopoietiche

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Mediata da Agrobacterium Virus-Induced tacere Assay Gene In Cotone

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi-mediato Knockdown Gene e<em&gt; In Vivo</em&gt; Saggio La diuresi in femmina adulta<em&gt; Aedes aegypti</em&gt; Le zanzare

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Genome-wide gene delezioni in<em&gt; Streptococcus sanguinis</em&gt; Da Throughput PCR ad alta

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Utilizzo di RNA-mediata strategia di alimentazione di interferenza secondo schermo per geni coinvolti nella regolazione Dimensione del corpo nel nematode<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...