Questo lavoro è stato sostenuto da un G. Harold e Leila Y. Mathers Premio per HL e una sovvenzione NIH (R24 GM080527) per RESCEJ stato un NRSA Postdoctoral Fellow (NIH F32GM9037222) ed è attualmente supportato da un Mentored Research Scientist Development Award dal National Institute on Aging (K01AG04435). Vorremmo ringraziare i revisori per utili commenti.

1 Preparazione di DNA plasmidico, aghi, e piatti iniezione <ol><li> Preparare il DNA plasmidico utilizzando un kit Maxi alta velocità o Midi e concentrare il DNA a 2,5 mg / ml mediante precipitazione in etanolo. Il pellet di DNA deve essere sciolto in acqua deionizzata priva di nucleasi. Conservare il DNA in 10-20 microlitri aliquote a -20 ° C. (Vedi Tabella supplementare 1 per un elenco di vettori disponibili) </li><li> Fare un piatto di iniezione utilizzando agarosio per costruire un trogolo per lo svolgimento di embrioni in una capsula di Petri di 100 x 15 mm. <ol><li> Posizionare un vetrino da microscopio 75 x 50 vetro in un piatto 100 x 15 mm Petri in un angolo. Versare circa 50 ml di 2% agarosio sciolto in Hydra medio 24 nella piastra di Petri. In alternativa, versare l&#39;agarosio nel piatto prima e galleggiare un &quot;Microiniezione pesce Mold&quot; in alto. </li><li> Dopo l&#39;agarosio si è solidificato, rimuovere il vetrino o lo stampo. Se è stato utilizzato un vetrino, tagliare via l&#39;eccesso di agarosio con un bla rasoiode per formare un muro. Versare Hydra media nel piatto e utilizzare immediatamente o conservarlo a 4 ° C fino al momento dell&#39;uso. Nota: i piatti iniezione possono essere riutilizzati se conservato a 4 ° C tra usi. </li></ol></li><li> Tirare aghi da capillari in vetro borosilicato con un filamento sul estrattore micropipetta con le seguenti condizioni: scaldare 525, tirare 75, velocità 100, tempo 50. Conservare gli aghi di loro premendo in una stretta striscia di argilla in una capsula di Petri tale che siano tenuto parallelo al fondo del piatto. </li><li> Per preparare la soluzione iniettabile, combinare 3 ml di / soluzione di DNA plasmidico ml 2,5 mg con 2 ml di 10% di rosso fenolo. Vortex la soluzione brevemente e poi girare per 10 min alla massima velocità in una microcentrifuga per rimuovere eventuali particelle che potrebbero intasare l&#39;ago. </li><li> Sotto un microscopio da dissezione, agganciare l&#39;ago a pochi millimetri dalla punta con una pinza per creare una piccola apertura. Nota: Vi è una certa flessibilità nella dimensione appropriata del apertozione perché la pressione può essere regolata nel passaggio 3.3 per accogliere questa variazione. </li><li> Porre la capsula di Petri in modo che sta tenendo l&#39;ago in posizione verticale con la punta verso il basso. Caricare l&#39;ago con circa 0,5 ml di soluzione iniettabile pipettando liquido nell&#39;estremità posteriore dell&#39;ago. Lasciare azione capillare per tirare la soluzione nella punta dell&#39;ago. </li></ol> 2 Preparazione di embrioni per iniezione <ol><li> Su base giornaliera eseguire la scansione della cultura ceppo Hydra AEP per polipi producono uova. Raccogliere questi polipi e metterli da parte in un piatto di cultura separata. Osservare questi polipi ogni poche ore durante il giorno per monitorare l&#39;andamento della formazione dell&#39;uovo. Quando le uova si rompono attraverso l&#39;ectoderma e si siedono su un anello di cellule ectodermiche retratti sono pronti per l&#39;iniezione 25. Posizionare questi polipi in un piatto con diversi Hydra che hanno testicoli per almeno 1 ora prima dell&#39;iniezione per consentire la fecondazione. Hydra &lt;/ Em&gt; maschi rilascerà spermatozoi senza alcuna manipolazione speciale. </li><li> Se le femmine si trovano nella colonia AEP con uova completamente formate, che sono circa 400 micron di diametro 25, o da 1 a 8 celle embrioni stadio, raccogliere anche questi per l&#39;iniezione. Iniettare immediatamente gli embrioni che hanno iniziato scissione. Nota: Non è possibile capire la differenza tra le uova completamente formate e zigoti unicellulari, quindi questi devono essere incubate con i maschi prima dell&#39;iniezione. </li><li> Prima dell&#39;iniezione, rimuovere la maggior parte del tessuto parentale sopra e sotto l&#39;embrione utilizzando un bisturi con lama # 15. Lasciare solo l&#39;embrione con un piccolo pezzo di colonna corpo attaccato, da utilizzare per manipolare l&#39;embrione con una pinza se necessario. Nota: Il tessuto che viene sezionato dalla embrione si rigenera e dovrebbe essere salvato per garantire che le femmine rimangono nella colonia di Hydra. </li><li> Usando una pipetta Pasteur, spostare embrioni al piatto di iniezione, disponendole parallele alla parete dellaattraverso agarosio. </li></ol> 3. Microiniezione di DNA plasmidi <ol><li> Montare il microinjector su un supporto magnetico che si siede su una lastra di ferro. Montare il supporto di iniezione, che è la parte del microinjector che contiene l&#39;ago, su un micromanipolatore joystick. Montare il manipolatore joystick per la piastra di ferro, con un supporto magnetico. Posizionare questo intero set-up sul lato destro di un microscopio da dissezione e posizionare il supporto di iniezione in modo che sia visibile nel campo di osservazione. </li><li> Porre la capsula iniezione con embrioni al microscopio da dissezione, orientato in modo tale che la parete verticale della vasca agarosio è a sinistra. Inserire l&#39;ago nel supporto di iniezione e abbassare la punta dell&#39;ago nel mezzo Hydra nel piatto. </li><li> Ruotare lentamente la manopola della siringa in senso orario fino olio minerale riempie la parte superiore dell&#39;ago e un flusso continuo di soluzione iniettabile si osserva uscire l&#39;ago nel Hydrun mezzo. Se il flusso è troppo forte, abbassare la pressione ruotando la manopola in senso antiorario. Pratica questo passo per ottenere ordinariamente una pressione adeguata, che dipende dalle dimensioni dell&#39;apertura dell&#39;ago (cioè, come l&#39;ago è stato fermato nella fase 1.5). Nota: Se il flusso è troppo forte, l&#39;embrione non sopravviverà l&#39;iniezione. </li><li> Durante la visualizzazione attraverso il microscopio da dissezione, spostare il piatto iniezione, in modo che il primo embrione è al centro del campo. Spostare l&#39;ago in modo che sia a contatto con quella dell&#39;embrione. Utilizzare il micromanipolatore per perforare l&#39;embrione con l&#39;ago. Nota: La parete verticale della vasca agarosio manterrà l&#39;embrione di essere messo da parte dal ago. </li><li> Lasciare che la soluzione iniettabile di fluire in embrione 1 o 2 secondi e poi rimuovere l&#39;ago rapidamente. Se l&#39;embrione ha più di una cella, iniettare ogni cella singolarmente. Usare pinze per riorientare l&#39;embrione per allineare la cella successiva con la punta dell&#39;ago. </li><li> Dopo il primo Embryo viene iniettato, spostare il piatto in modo che la prossima embrione è nella posizione di iniezione e ripetere la procedura; continuare fino a quando sono state iniettate tutti gli embrioni. </li></ol> 4. coltura e cova di embrioni <ol><li> Spostare tutti gli embrioni iniettati in un piatto con un paio di Hydra che hanno testicoli (questo è quello di garantire che tutti gli embrioni sono fecondati). Incubare gli embrioni in un 18 ° C incubatore mentre continuano embriogenesi. </li><li> Una volta raggiunta la fase della cuticola, spostare ogni embrione iniettato ad un pozzo individuale di una piastra da 24 pozzetti riempiti con Hydra medie. </li><li> Incubare gli embrioni iniettati per 2 settimane al buio a 18 ° C. </li><li> Dopo il periodo di incubazione 2 settimane, controllare ogni embrione iniettato sotto il microscopio da dissezione. Nota casi in cui l&#39;embrione cuticola stadio ha staccati dal piccolo pezzo di tessuto parentale e il tessuto dei genitori ha rigenerati. Rimuovere questo polipo rigenerato immediatamente in modo che non è mistaken per un nuovo cucciolo. </li><li> Spostare il piatto di embrioni iniettati sotto una luce acquario a temperatura ambiente. Controllare le piastre ogni giorno e raccogliere larve. Le nuove larve saranno bianco perché il tipico colore rosa di polipi Hydra nasce dalla Artemia di cui si nutrono. </li><li> Osservare i piccoli sotto un microscopio a fluorescenza per rilevare l&#39;espressione del transgene. </li></ol>

<ol>
	<li>Wittlieb, J., Khalturin, K., Lohmann, J. U., Anton-Erxleben, F., Bosch, 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=Transgenic+Hydra+allow+in+vivo+tracking+of+individual+stem+cells+during+morphogenesis.">Transgenic Hydra allow in vivo tracking of individual stem cells during morphogenesis.</a> Proc Natl Acad Sci U S A. 103, 6208-6211 (2006).</li><li>Glauber, K. M., Dana, C. E., Steele, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydra.">Hydra.</a> Curr Biol. 20, 964-965 (2010).</li><li>Holstein, T. W., Hobmayer, E., David, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+of+epithelial+cell+cycling+in+hydra.">Pattern of epithelial cell cycling in hydra.</a> Dev Biol. 148, 602-611 (1991).</li><li>Dubel, S., Hoffmeister, S. A., Schaller, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+pathways+of+ectodermal+epithelial+cells+in+hydra.">Differentiation pathways of ectodermal epithelial cells in hydra.</a> Differentiation. 35, 181-189 (1987).</li><li>Khalturin, 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=Transgenic+stem+cells+in+Hydra+reveal+an+early+evolutionary+origin+for+key+elements+controlling+self-renewal+and+differentiation.">Transgenic stem cells in Hydra reveal an early evolutionary origin for key elements controlling self-renewal and differentiation.</a> Dev Biol. 309, 32-44 (2007).</li><li>Bosch, T., David, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cells+of+Hydra+magnipapillata+can+differentiate+into+somatic+cells+and+germ+line+cells.">Stem Cells of Hydra magnipapillata can differentiate into somatic cells and germ line cells.</a> Dev Biol. 121, 182-191 (1987).</li><li>David, C. N., Murphy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+interstitial+stem+cells+in+hydra+by+cloning.">Characterization of interstitial stem cells in hydra by cloning.</a> Dev Biol. 58, 372-383 (1977).</li><li>Chapman, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+genome+of+Hydra.">The dynamic genome of Hydra.</a> Nature. 464, 592-596 (2010).</li><li>Boehm, A. M., Bosch, 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=Migration+of+multipotent+interstitial+stem+cells+in+Hydra.">Migration of multipotent interstitial stem cells in Hydra.</a> Zoology (Jena). 115, 275-282 (2012).</li><li>Glauber, K. 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=A+small+molecule+screen+identifies+a+novel+compound+that+induces+a+homeotic+transformation+in+Hydra.">A small molecule screen identifies a novel compound that induces a homeotic transformation in Hydra.</a> Development. 140, 4788-4796 (2013).</li><li>Siebert, S., Anton-Erxleben, F., Bosch, 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=Cell+type+complexity+in+the+basal+metazoan+Hydra+is+maintained+by+both+stem+cell+based+mechanisms+and+transdifferentiation.">Cell type complexity in the basal metazoan Hydra is maintained by both stem cell based mechanisms and transdifferentiation.</a> Dev Biol. 313, 13-24 (2008).</li><li>Anton-Erxleben, F., Thomas, A., Wittlieb, J., Fraune, S., Bosch, 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=Plasticity+of+epithelial+cell+shape+in+response+to+upstream+signals:+a+whole-organism+study+using+transgenic+Hydra.">Plasticity of epithelial cell shape in response to upstream signals: a whole-organism study using transgenic Hydra.</a> Zoology (Jena). 112, 185-194 (2009).</li><li>Hemmrich, G., 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=Molecular+signatures+of+the+three+stem+cell+lineages+in+Hydra+and+the+emergence+of+stem+cell+function+at+the+base+of+multicellularity.">Molecular signatures of the three stem cell lineages in Hydra and the emergence of stem cell function at the base of multicellularity.</a> Mol Biol Evol. , (2012).</li><li>Juliano, C. E., 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=PIWI+proteins+and+PIWI-interacting+RNAs+function+in+Hydra+somatic+stem+cells.">PIWI proteins and PIWI-interacting RNAs function in Hydra somatic stem cells.</a> Proc Natl Acad Sci U S A. 111, 337-342 (2014).</li><li>Nishimiya-Fujisawa, C., Kobayashi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+stem+cells+and+sex+determination+in+Hydra.">Germline stem cells and sex determination in Hydra.</a> Int J Dev Biol. 56, 499-508 (2012).</li><li>Milde, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+taxonomically+restricted+genes+in+a+phylum-restricted+cell+type.">Characterization of taxonomically restricted genes in a phylum-restricted cell type.</a> Genome Biol. 10, 8 (2009).</li><li>Nakamura, Y., Tsiairis, C. D., Ozbek, S., Holstein, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoregulatory+and+repressive+inputs+localize+Hydra+Wnt3+to+the+head+organizer.">Autoregulatory and repressive inputs localize Hydra Wnt3 to the head organizer.</a> Proc Natl Acad Sci U S A. 108, 9137-9142 (2011).</li><li>Gee, 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=beta-catenin+plays+a+central+role+in+setting+up+the+head+organizer+in+hydra.">beta-catenin plays a central role in setting up the head organizer in hydra.</a> Dev Biol. 340, 116-124 (2010).</li><li>Fraune, S., 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=In+an+early+branching+metazoan,+bacterial+colonization+of+the+embryo+is+controlled+by+maternal+antimicrobial+peptides.">In an early branching metazoan, bacterial colonization of the embryo is controlled by maternal antimicrobial peptides.</a> Proc Natl Acad Sci U S A. 107, 18067-18072 (2010).</li><li>Bridge, D., 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=FoxO+and+stress+responses+in+the+cnidarian+Hydra+vulgaris.">FoxO and stress responses in the cnidarian Hydra vulgaris.</a> PLoS One. 5, e11686 (2010).</li><li>Boehm, A. 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=FoxO+is+a+critical+regulator+of+stem+cell+maintenance+in+immortal+Hydra.">FoxO is a critical regulator of stem cell maintenance in immortal Hydra.</a> Proc Natl Acad Sci U S A. 109, 19697-19702 (2012).</li><li>Franzenburg, S., 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=MyD88-deficient+Hydra+reveal+an+ancient+function+of+TLR+signaling+in+sensing+bacterial+colonizers.">MyD88-deficient Hydra reveal an ancient function of TLR signaling in sensing bacterial colonizers.</a> Proc Natl Acad Sci U S A. 109, 19374-19379 (2012).</li><li>Dana, C. E., Glauber, K. M., Chan, T. A., Bridge, D. M., Steele, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+a+horizontally+transferred+gene+into+an+operon+during+cnidarian+evolution.">Incorporation of a horizontally transferred gene into an operon during cnidarian evolution.</a> PLoS One. 7, e31643 (2012).</li><li>Lenhoff, H. M., Lenhoff, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hydra: Research Methods. , 29-34 (1983).</li><li>Miller, M. A., Technau, U., Smith, K. M., Steele, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+development+in+Hydra+involves+selection+from+competent+precursor+cells.">Oocyte development in Hydra involves selection from competent precursor cells.</a> Dev Biol. 224, 326-338 (2000).</li><li>Lenhoff, H. M., Lenhoff, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hydra: Research Methods. , 53-62 (1983).</li></ol>

The authors have nothing to disclose

Hydra riproduce asessualmente di routine, ma richiede stimoli ambientali per iniziare la produzione di gameti. Questi stimoli non sono ben definiti, per la maggior parte delle specie Hydra e possono differire da ceppo a ceppo. Un ostacolo significativo alla produzione di transgenico Hydra è l&#39;ottenimento di embrioni su base regolare, perché può essere difficile in un ambiente di laboratorio per indurre Hydra per diventare sessuale. Il ceppo AEP 25, tuttavia, produce ...

Hydra è stato utilizzato per studiare la rigenerazione, formazione di pattern, e cellule staminali per circa 250 anni 2 Hydra ha un piano corpo semplice che consiste di tre linee cellulari:. Epiteliale ectodermica, endodermico epiteliale, e interstiziale. Il corpo tubolare è formato dalla ectodermica e linee dell&#39;epitelio endodermico, ciascuno dei quali è un singolo strato di cellule. Tutte le cellule epiteliali della colonna corpo sono mitotico. Quando le cellule epiteliali vengono spostati nelle estremità 3, la testa (bocca e tentacoli) alla fine della bocca o il piede (disco basale) alla fine aborale, arrestano nella fase G2 del ciclo cellulare e cambiate destino cellulare 4. Le cellule della linea interstiziale risiedono negli interstizi tra le cellule epiteliali. Questa linea è sostenuta da cellule staminali multipotenti che si trovano nello strato epiteliale ectodermica della colonna corpo 5. Le cellule staminali interstiziali danno origine a tre cel somaticaTipi L (nervi, cellule della ghiandola, e nematocytes) e le cellule germinali 6,7. Come membro del phylum Cnidari, il gruppo sorella a tutti bilaterians, Hydra può far luce sui processi biologici fondamentali condivisi tra gli animali multicellulari. Fino a poco tempo, questi sforzi sono stati ostacolati dalla mancanza di metodi affidabili per la perturbazione della funzione del gene. Tuttavia, con lo sviluppo della metodologia transgenica 1, siamo ora in grado di sfruttare appieno Hydra per ottenere una migliore comprensione dei meccanismi di base comuni ad animali pluricellulari, come ad esempio la funzione delle cellule staminali, la rigenerazione, e patterning. Linee transgeniche Hydra sono stabiliti mediante iniezione di DNA plasmidico in embrioni, che si traduce in integrazione casuale ed espressione chimerico in frequenza sostanziale di larve. Una linea con l&#39;espressione uniforme in un particolare lignaggio può essere stabilita per propagazione asessuata. La capacità di propagare clonale transgENIC Hydra righe è un vantaggio rispetto alla maggior parte dei modelli animali, che possono essere propagate solo da riproduzione sessuale. Inoltre, cellule transgeniche possono essere seguiti facilmente in vivo a causa della trasparenza dell&#39;animale e l&#39;assenza di proteine ​​fluorescenti endogene 8. Nei sette anni dalla prima linea Hydra transgeniche sono state effettuate 1, tali linee sono stati utilizzati per una varietà di applicazioni. Espressione di proteine ​​fluorescenti in diversi tipi di cellule ha permesso di monitorare il movimento delle cellule, osservare cambiamenti di forma delle cellule, e tenere traccia destini cellulari sia in condizioni di tipo selvatico e dopo perturbazione chimica 1,5,9-12. Inoltre, l&#39;espressione di proteine ​​fluorescenti differenti nelle diverse linee consente FACS isolamento di specifiche popolazioni cellulari. Questa tecnica è stata utilizzata per la sequenza di mRNA specifici di cellule staminali e le specifiche del lineage piccoli RNA 13,14. Mentre il promotore diuno dei due geni di actina Hydra è stato più utilizzato, a poche cellule di tipo promotori specifici sono stati identificati e utilizzati per guidare l&#39;espressione di GFP in transgenici Hydra 9,11,15,16. In futuro, i promotori specifici delle cellule di tipo permetteranno l&#39;osservazione e la raccolta di qualsiasi tipo di cellula specifico. Inoltre, un approccio transgenico è stato usato con successo per definire cis-agenti elementi regolatori del promotore Wnt3 17. Lo sviluppo di metodi transgenici in Hydra fornisce un valido approccio per testare la funzione dei geni dall&#39;espressione ectopica, sovraespressione, e atterramento. Gli animali transgenici sono stati fatti che esprimono proteine ​​fluorescenti-taggato, al fine di esaminare sia la funzione e la localizzazione cellulare 18-20. Inoltre, l&#39;espressione di forcine RNA nel 3&#39;UTR di un transgene GFP conduce atterramento di geni bersaglio 21,22. In questi approcci è necessaria GFP per identificaree monitorare il tessuto transgenico durante la creazione della linea transgenica. Tuttavia, è probabile che in alcuni casi la molecola GFP potrebbe interferire con la funzione della proteina tag. Uno studio recente dimostra che i geni Hydra possono essere organizzati in una configurazione operone, cioè, trascrizioni policistronici sono fatti, che vengono poi separati da trans-impiombato Inoltre leader e tradotto separatamente 23. Inserendo un gene codificante una proteina o una forcina RNA nella posizione a monte di un operone e un gene della proteina fluorescente nella posizione a valle, si può tracciare tessuto transgenico senza dover codificare il gene codificante la proteina o RNA hairpin. Questo metodo è stato utilizzato per esprimere un tornante RNA in una configurazione operone con DsRed2 al fine di raggiungere gene knockdown 14.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AEP Hydra Strain</td>
			<td style="width:112px;">NA</td>
			<td style="width:119px;">NA</td>
			<td style="width:576px;">Email: Celina Juliano at celina.juliano@yale.edu or Hiroshi Shimizu bubuchin2006@yahoo.co.jp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High Speed Maxi Kit</td>
			<td style="width:112px;">Qiagen</td>
			<td align="right" style="width:119px;">12662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 x 15 mm Petri Dishes</td>
			<td style="width:112px;">BD Falcon</td>
			<td align="right" style="width:119px;">351029</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">75 x 50 mm Glass Microscope Slide</td>
			<td style="width:112px;">Sigma</td>
			<td>CLS294775X50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microinjection Fish Mold</td>
			<td style="width:112px;">IBI Scientific</td>
			<td style="width:119px;">FM-600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Borosilicate Glass with Filament</td>
			<td style="width:112px;">Sutter Instrument</td>
			<td style="width:119px;">BF100-50-10</td>
			<td>O.D.: 1.0 mm, I.D.: 0.50 mm, 10 cm length</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flaming/Brown Micropipette Puller</td>
			<td style="width:112px;">Sutter Instrument</td>
			<td style="width:119px;">P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phenol Red</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">P3532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jewelers Forceps, Durmont #5</td>
			<td style="width:112px;">Sigma</td>
			<td>F6521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel Blade #15</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:119px;">50-822-421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mineral oil</td>
			<td style="width:112px;">Sigma</td>
			<td style="width:119px;">M8410-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microinjector</td>
			<td style="width:112px;">Narishige</td>
			<td style="width:119px;">IM-9B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic Stand</td>
			<td style="width:112px;">Narishige</td>
			<td style="width:119px;">GJ-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iron Plate</td>
			<td style="width:112px;">Narishige</td>
			<td style="width:119px;">IP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Joystick Micromanipulator</td>
			<td style="width:112px;">Narishige</td>
			<td style="width:119px;">MN-151</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of transgenic hydra by embryo microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Stabilire linee transgeniche Hydra Alimentare larve transgeniche ogni 2-3 giorni con Artemia nauplii. I piccoli a volte non mangiano per un giorno o due dopo la schiusa. Alcuni nuovi nascituri saranno mai mangiare, e quindi non sopravviverà. Se il cucciolo è transgenico sia nel ectodermica (Figura 1A) o tessuto epiteliale endodermico, permettere all&#39;animale di germoglio e raccogliere le gemme che hanno il tessuto più transgenico (Figura 1B, C).<...

Watch this Scientific Journal Video about Generation of Transgenic Hydra by Embryo Microinjection at JoVE.com

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

Generazione di transgenici<em&gt; Hydra</em&gt; Da Embryo microiniezione

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

generation-of-transgenic-hydra-by-embryo-microinjection

Research

JoVE Journal

Biology

Generation of Transgenic Hydra by Embryo Microinjection

13.9K Views.  Yale University School of Medicine. Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi. 

Video: Generation of Transgenic Hydra by Embryo Microinjection

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

Microiniezione di A. aegypti embrioni per ottenere zanzare transgeniche

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

Ricerca

Biologia

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Generazione di Stabile transgenici C. elegans Utilizzando Microiniezione

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Tecniche di microiniezione per Studiare Mitosi in Drosophila melanogaster Embryo sinciziale

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Produzione di transgenici Xenopus laevis Integrazione mediata da enzima di restrizione e trapianto nucleare

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Generazione di transgenici C. elegans Di Trasformazione Biolistic

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

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

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

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

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

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

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

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

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

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

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 ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generazione di Myospheres Da hESCs di epigenetica Riprogrammazione

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

Methods Article

Generazione di transgenici