Name: biolistic transformation of a fluorescent tagged gene into the opportunistic fungal pathogen cryptococcus neoformans
Uploaded: 2015-03-19T16:00:00.000+00:00
Duration: 7 min 32 s
Description: Watch this Scientific Journal Video about Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans at JoVE.com

Questo lavoro è stato sostenuto da premi dalla National Science Foundation (Award # 0.920.274) e l&#39;esperimento South Carolina Progetto Stazione SC-1.700.340. Questo documento isTechnical Contributo No. 6283 del Experiment Station Clemson University. Gli autori ringraziano il Dr. Lukasz Kozubowski per i suoi utili consigli per lo sviluppo di questo protocollo finale e il dottor Cheryl Ingram-Smith, Katie Glenn, e Grace Kisirkoi per la lettura critica del manoscritto.

NOTA: Lo schema generale di questo protocollo è descritto nella Figura 1. 1. C. neoformans Preparazione <ol><li> Per ogni reazione di trasformazione, crescere un 2-3 ml O / N cultura di C. neoformans in YPD medio a 30 ° C in agitazione a 250 rpm. </li><li> Centrifugare la / cultura O N per 5 minuti a 900 xga 10 ° C e scartare il surnatante. </li><li> Risospendere ogni pellet cellulare in 300 ml di lievito Peptone destrosio (YPD) medio. </li><li> Utilizzando perle di vetro, si sviluppa lentamente 300 ml di sospensione cellulare lavato su agar YPD contenente 1 M sorbitolo. </li><li> Lasciare le piastre ad asciugare a temperatura ambiente per 3-4 ore. </li></ol> 2. Oro microcarrier Preparazione <ol><li> Risospendere 0,25 g di 0,6 micron perle d&#39;oro in 1 ml di DDH 2 O, centrifugare per 1 min a 900 xg a pellet le perline, e rimuovere il surnatante. </li><li> Risospendere le perle d&#39;oro in 1 ml di etanolo al 100%. &lt;/ Li&gt; <li> Distribuire le perline in 4 tubi, 250 microlitri ciascuna, e aggiungere 750 ml di etanolo al 100%. </li><li> Conservare aliquote perline oro a 4 ° C. </li></ol> Preparazione DNA 3. <ol><li> Preparare i dischi macrocarrier Biolistic arancione da loro immergendo in 100% etanolo usando pinze. Dischi Mettere in un grande piatto di Petri contenente Drierite asciugare (assicurarsi che il Drierite non tocchi i dischi). </li><li> Una volta asciutto, premere i dischi macrocarrier nei supporti disco d&#39;argento (precedentemente puliti con 100% di etanolo). </li><li> Perline Vortex oro (preparato come al punto 2) e aliquote di 12 ml in 1,5 ml provetta, un tubo per la trasformazione. </li><li> Aggiungere ad ogni provetta per: 2 mg di DNA (preferibilmente 2 ml di 1 mg / mL di DNA), 10 microlitri 2,5 M CaCl 2, e 2 ml 1 M spermidine base libera. </li><li> Impostare un controllo negativo come nel passaggio 3.4 ma senza DNA. </li><li> Vortex ciascuna provetta e incubare a temperatura ambiente per5 min. Flick delicatamente ogni tubo di tanto in tanto per risospendere le sfere regolate durante questa incubazione. </li><li> Tubi Spin a 225 g per 30 sec a pellet perline oro rivestite di DNA. Rimuovere con attenzione il surnatante (pipettando o aspirazione) e scartare. </li><li> Perline risospendere completamente in 600 ml di etanolo al 100% da lentamente pipettando su e giù. </li><li> Spin provette a 225 xg per 30 secondi per far sedimentare le perline senza imballaggio. Rimuovere con cautela e scartare il surnatante. </li><li> Risospendere le sfere d&#39;oro rivestite di DNA a 8 ml di etanolo al 100% lentamente pipettando su e giù. </li><li> Pipettare le perline oro rivestite di DNA sul centro del disco biolistic in un diametro di 1 cm e lasciare asciugare. NOTA: Un cerchio oro essiccati visibile sul centro del disco biolistic indica che una concentrazione sufficiente di perline oro è presente. NOTA: I dischi macrocarrier caricati con perle oro rivestite di DNA sono ora pronti per l&#39;uso con la pistola gene. </li></ol> 4. OPerating il Gene Gun <ol><li> Accendere la pompa a vuoto. </li><li> Accendere il gas elio ruotando la manopola in senso antiorario fino ad una pressione di circa 2200 psi viene raggiunto sul manometro. </li><li> Accendere la pistola gene spostando l&#39;interruttore rosso sulla sinistra. </li><li> Assicurarsi che le portate per il vuoto e la ventola sono regolati in modo che il vuoto raggiungerà 28 pollici Hg entro 15 sec. </li><li> Assicurarsi che la distanza tra il disco di rottura e macrocarrier è di circa 3/8 di pollice. </li><li> Pulire l&#39;intera camera pulendo giù con etanolo. </li><li> Immergere i dischi di rottura di 100% di etanolo. Lasciare asciugare su una superficie sterile (ad esempio, Petri piatto). </li><li> Utilizzare una chiave dinamometrica per allentare il supporto disco di rottura. Inserire un disco di rottura pulito nel supporto. Avvitare il supporto disco di rottura al suo posto e stringere con la chiave coppia girandolo una volta a destra. NOTA: I dischi di rottura saranno sostituiti dopo ogni tiro. </li><li> Immergere the schermi di maglia in 100% di etanolo. Lasciare asciugare su una superficie sterile (ad esempio, Petri piatto). </li><li> Una volta asciutto, mettere un setaccio a maglie lavato sulla piastra di montaggio in plastica bianca. Posizionare il lato titolare DNA disco macrocarrier giù nella camera del disco. Avvitare il tappo in argento, e posizionare la piastra di montaggio nel più alto slot. NOTA: Lo schermo di maglia sarà sostituito dopo ogni tiro. </li><li> Posizionare un piatto YPD agar contenente 1 M sorbitolo sul fondo piatto. </li><li> Chiudi porta della camera e bloccare in posizione. </li><li> Premere e tenere premuto il tasto centrale rosso fino a coinvolgere il vuoto e permettere il vuoto di raggiungere 28 pollici Hg. Una volta che il livello di vuoto corretta viene raggiunto, spostare l&#39;interruttore in posizione abbassata. Quando è pronto, tenere premuto il tasto rosso sulla destra per sparare. Quando il disco di rottura si apre, rilasciare immediatamente il pulsante di fuoco e premere l&#39;interruttore centrale rossa in posizione centrale per sfogare la camera a 0 psi. </li><li> Pulire i detriti disco di rottura e spegnere la pistola gene. Quindi spegnere il helium gas ruotando la manopola in senso orario, e, infine, spegnere la pompa a vuoto. </li></ol> 5. Cellule placcatura trasformate <ol><li> Lasciare le piastre di trasformazione a sedersi a temperatura ambiente per 4 ore per consentire alle cellule di recuperare. </li><li> Pipettare 700 ml di YPD sulla piastra. Utilizzare un raschietto cellulare per raschiare delicatamente le cellule fuori del liquido agar e trasferirli in una sterile 1,5 ml provetta da microcentrifuga. Ripetere questo passaggio per assicurare tutte le cellule sono state recuperate dalla piastra. </li><li> Agglomerare cellule a 225 xg per 30 sec. Rimuovere e scartare il surnatante. </li><li> Risospendere il pellet in 500 ml di YPD. </li><li> Dispensare 100 l di sospensione cellulare sul centro dei piatti + antibiotici YPD e diffondere con perle di vetro. </li><li> Lasciare le piastre capovolte a temperatura ambiente per 3-4 giorni. Come appaiono colonie, cerotto sulla nuova YPD + piastre antibiotici. </li></ol> 6. Isolamento DNA genomico per PCR NOTA: Questa è una versione modificata utilizzando reagentis da un kit di purificazione del DNA (Vedi Tabella dei Materiali). <ol><li> Crescere una cultura 5 ml di ciascuno dei C. neoformans trasformanti in YPD liquido a 30 ° C in agitazione a 250 rpm O / N. </li><li> Pellet 3 ml di cellule a 900 xg, e risospendere in 600 ml di soluzione di lisi dei nuclei. </li><li> Aggiungere la sospensione ad una nuova provetta da 1,5 ml microcentrifuga con 200 ml di acido 0,5 millimetri lavati perline di vetro. </li><li> Omogeneizzare per 45 secondi in un mini beadbeater a temperatura ambiente, tubo freddo sul ghiaccio, e ripetere. </li><li> Lasciare che il campione di stabilirsi in ghiaccio per 2 minuti e trasferire il surnatante in una nuova provetta da 1,5 ml. Aggiungere 200 ml di soluzione proteica precipitazioni a ciascun tubo, (100 ml per ogni 600 ml di surnatante recuperato) e vortex energicamente per 20 sec. </li><li> Lasciare che i campioni di stabilirsi in ghiaccio per 5 minuti, e centrifugare a 11.000 xg per 3 min. </li><li> Trasferire il surnatante in una provetta da 1,5 ml pulito contenente 300 ml di RT isopropanolo. Mescolare delicatamente per inversione. </li><li>Centrifugare i campioni a 11.000 xg per 2 minuti, rimuovere con attenzione il surnatante, e drenano i tubi su tovaglioli di carta. </li><li> Aggiungere 300 ml di RT 70% di etanolo a ciascuna provetta, e capovolgere delicatamente per lavare il pellet. </li><li> Centrifugare i campioni a 11.000 xg per 2 minuti, e con attenzione rimuovere tutti i etanolo. </li><li> Svuotare il tubo sulla carta assorbente pulita, e permettere il pellet asciugare per 10-15 minuti. </li><li> Aggiungere 50 ml di soluzione di reidratazione DNA e 1,5 ml di soluzione di RNase ad ogni pellet e vortice. </li><li> Campioni di centrifugazione per 5 secondi per rimuovere tutto il liquido dal tappo. </li><li> Incubare i campioni a 37 ° C per 15 min. </li><li> Reidratare il DNA incubando i campioni a 65 ° C per 1 ora. </li><li> Quantificare DNA spettrofotometricamente misurando l&#39;assorbanza a 260 nm (un A 260 lettura di 1.0 è equivalente a ~ 50 ug / ml di DNA a doppio filamento), e utilizzare fino a 200 ng in ciascuna reazione PCR. </li></ol> 7. RNA Isolation perReverse Transcriptase-PCR. <ol><li> Utilizzando un kit di purificazione RNA (Vedi Tabella dei Materiali), seguire le istruzioni del produttore per isolare RNA da cellule di lievito con un minibeadbeater. </li><li> Quantificare la concentrazione dell&#39;RNA misurando l&#39;assorbanza a 260 nm (un A 260 lettura di 1.0 è equivalente a ~ 40 ug / ml di RNA a singolo filamento). </li><li> L&#39;utilizzo di un kit di RT-PCR (vedi Tabella dei Materiali), seguire le istruzioni del produttore di creare reazioni RT-PCR, con circa 1 mg di RNA. Per i risultati ottenuti in questo studio, utilizzare i primer elencati nella Tabella 1. </li></ol>

<ol>
	<li>Price, M. 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=Cryptococcusneoformans+requires+a+functional+glycolytic+pathway+for+disease+but+not+persistence+in+the+host.">Cryptococcusneoformans requires a functional glycolytic pathway for disease but not persistence in the host.</a> MBio. 2, e00103-e00111 (2011).</li><li>Bubb, W. 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=Heteronuclear+NMR+studies+of+metabolites+produced+by+Cryptococcusneoformans+in+culture+media:+identification+of+possible+virulence+factors.">Heteronuclear NMR studies of metabolites produced by Cryptococcusneoformans in culture media: identification of possible virulence factors.</a> Magn Reson Med. 42, 442-453 (1999).</li><li>Himmelreich, U., 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=Identification+of+metabolites+of+importance+in+the+pathogenesis+of+pulmonary+cryptococcoma+using+nuclear+magnetic+resonance+spectroscopy.">Identification of metabolites of importance in the pathogenesis of pulmonary cryptococcoma using nuclear magnetic resonance spectroscopy.</a> Microbes Infect. 5, 285-290 (2003).</li><li>Wright, 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=Metabolites+released+by+Cryptococcusneoformans+var.+neoformans+and+var.+gattii+differentially+affect+human+neutrophil+function.">Metabolites released by Cryptococcusneoformans var. neoformans and var. gattii differentially affect human neutrophil function.</a> Microbes Infect. 4, 1427-1438 (2002).</li><li>Hu, G., Cheng, P. Y., Sham, A., Perfect, J. R., Kronstad, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+adaptation+in+Cryptococcusneoformans+during+early+murine+pulmonary+infection.">Metabolic adaptation in Cryptococcusneoformans during early murine pulmonary infection.</a> Mol Microbiol. 69, 1456-1475 (2008).</li><li>Ingram-Smith, C., Martin, S. R., Smith, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetate+kinase:+not+just+a+bacterial+enzyme.">Acetate kinase: not just a bacterial enzyme.</a> Trends Microbiol. 14, 249-253 (2006).</li><li>Davidson, R. C., 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=Gene+disruption+by+biolistic+transformation+in+serotype+D+strains+of+Cryptococcus+neoformans.">Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans.</a> Fungal Genet Biol: FG &amp; B. 29, 38-48 (2000).</li><li>Toffaletti, D. L., Rude, T. H., Johnston, S. A., Durack, D. T., Perfect, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+in+Cryptococcusneoformans+by+use+of+biolistic+delivery+of+DNA.">Gene transfer in Cryptococcusneoformans by use of biolistic delivery of DNA.</a> J. Bacteriol. 175, 1405-1411 (1993).</li><li>Edman, J. C., Kwon-Chung, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+the+URA5+gene+from+Cryptococcusneoformans+var.+neoformans+and+its+use+as+a+selective+marker+for+transformation.">Isolation of the URA5 gene from Cryptococcusneoformans var. neoformans and its use as a selective marker for transformation.</a> Mol Cell Biol. 10, 4538-4544 (1990).</li><li>Chang, Y. C., Kwon-Chung, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementation+of+a+capsule-deficient+mutation+of+Cryptococcus+neoformans+restores+its+virulence.">Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence.</a> Mol Cell Biol. 14, 4912-4919 (1994).</li><li>Del Poeta, 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=Topoisomerase+I+is+essential+in+Cryptococcusneoformans:+role+In+pathobiology+and+as+an+antifungal+target.">Topoisomerase I is essential in Cryptococcusneoformans: role In pathobiology and as an antifungal target.</a> Genetics. 152, 167-178 (1999).</li><li>McClelland, C. M., Chang, Y. C., Kwon-Chung, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+transformation+of+Cryptococcusneoformans+and+Cryptococcusgattii+by+Agrobacteriumtumefaciens.">High frequency transformation of Cryptococcusneoformans and Cryptococcusgattii by Agrobacteriumtumefaciens.</a> Fungal Genet Biol:FG &amp; B. 42, 904-913 (2005).</li><li>Gan, W. B., Grutzendler, J., Wong, W. T., Wong, R. O., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+'DiOlistic'+labeling+of+the+nervous+system+using+lipophilic+dye+combinations.">Multicolor 'DiOlistic' labeling of the nervous system using lipophilic dye combinations.</a> Neuron. 27, 219-225 (2000).</li><li>Nicola, A. 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We have nothing to disclose.

Utilizing this protocol, biolistic transformation can be accomplished in which linear DNA is integrated into a desired locus in the Cryptococcus neoformans genome by homologous recombination. Certain steps in the protocol can have a dramatic effect on the effectiveness/efficiency of the transformation. For a successful transformation, it is imperative that the DNA utilized in the shoot has a concentration of at least 1 &#181;g. However, the volume of DNA added to the gold beads can be increased in the chance the...

Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis resulting in more than 625,000 deaths per year worldwide 1. Acetate has been shown to be a major fermentation product during cryptococcal infection 2,3,4, and genes encoding enzymes from three putative acetate-producing pathways have been shown to be upregulated during infection 5. This suggests that acetate production and transport may be a necessary and required part of the pathogenic process; however, the significance of this is not yet understood. One possible pathway for acetate production is the xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) - acetate kinase (Ack), a pathway previously thought to be present only in bacteria but recently identified in both euascomycete as well as basidiomycete fungi, including&#160;C. neoformans&#160;6.
To determine the localization of these enzymes of this pathway in the cell, a construct carrying a neomycin resistance gene downstream of an ACK gene fusion to the fluorescent tag mCherry (ACK:mCherry:Neo) will be introduced into C. neoformans using the well-established method of biolistic transformation 7,8. Although electroporation is an efficient method for transformation of plasmids that will be maintained as episomes into Cryptococcus 9, it is not useful in creating stable homologous transformants 8. Only&#160;biolistic&#160;delivery using a gene gun&#160;provides&#160;an effective means to transform&#160;linear&#160;DNAs&#160;that will be integrated into the genome by homologous recombination. For example, Edman et al. showed that of the transformants resulting from electroporation of a plasmid-borne URA5 selectable marker into C. neoformansura5 mutants, just 0.001 to 0.1% of transformants were stable 9. Chang et al. achieved just a 0.25% stable transformation efficiency using electroporation to reconstitute capsule production in an acapsular mutant 10. Unlike electroporation, biolistic transformation has been shown to result in stable transformation efficiency of 2-50% depending on the gene that is being altered 7,8,11. 
This visual experiment will provide a step-by-step demonstration of biolistic transformation of the linear ACK:mCherry:Neo DNA construct into C. neoformans, and will describe how to confirm its proper integration via homologous recombination into the&#160;ack&#160;locus. The protocol demonstrated here is a modification of the method developed in the Perfect laboratory 8.

<table><tbody><tr><td>0.6 &amp;mu;m gold beads</td><td>Bio-Rad</td><td>165-2262</td><td>http://www.bio-rad.com</td></tr><tr><td>Spermadine-free base</td><td>Sigma- Aldrich</td><td>S0266</td><td>https://www.sigmaaldrich.com</td></tr><tr><td>G418 - Sulfate (Neomycin)</td><td>Gold Biotechnology</td><td>G-418-10</td><td>www.goldbio.com</td></tr><tr><td>Hygromycin</td><td>Gold Biotechnology</td><td>H-270-1</td><td>www.goldbio.com</td></tr><tr><td>1350 psi Rupture Discs</td><td>Bio-Rad</td><td>165-2330</td><td>http://www.bio-rad.com</td></tr><tr><td>Stopping Screens</td><td>Bio-Rad</td><td>165-2336</td><td>http://www.bio-rad.com</td></tr><tr><td>Macrocarriers discs</td><td>Bio-Rad</td><td>165-2335</td><td>http://www.bio-rad.com</td></tr><tr><td>YPD Broth</td><td>Becton Dickinson &amp;amp; Co.</td><td>242820</td><td>www.bd.com</td></tr><tr><td>Agar</td><td>Becton Dickinson &amp;amp; Co.</td><td>214530</td><td>www.bd.com</td></tr><tr><td>Sorbitol</td><td>Fisher Scientific</td><td>BP439</td><td>http://www.fishersci.com</td></tr><tr><td>PDS-1000/He System</td><td>Bio-Rad</td><td>165-2257</td><td>http://www.bio-rad.com</td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Axio</td><td>http://www.zeiss.com/microscopy</td></tr><tr><td>KOD One Step PCR Kit</td><td>EMD Millipore</td><td>71086-4</td><td>http://www.emdmillipore.com</td></tr><tr><td>One Step RT-PCR Kit</td><td>Qiagen</td><td>210212</td><td>www.qiagen.com</td></tr><tr><td>Wizard Genomic DNA Purification Kit</td><td>Promega</td><td>A1120</td><td>www.promega.com</td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td>www.qiagen.com</td></tr><tr><td>Mini Beadbeater - 1</td><td>BioSpecs</td><td>3110BX</td><td>http://www.biospec.com</td></tr><tr><td>Microfuge 18 Centrifuge</td><td>Beckman Coulter</td><td>F241.5P</td><td>www.beckmancoulter.com</td></tr><tr><td>Microplate Spectrophotometer</td><td>BioTek</td><td>EPOCH</td><td>www.biotek.com</td></tr></tbody></table>

biolistic transformation of a fluorescent tagged gene into the opportunistic fungal pathogen cryptococcus neoformans

Il basidiomicete Cryptococcus neoformans, un patogeno opportunista invasivo del sistema nervoso centrale, è la causa più frequente di meningite fungina con conseguente tutto il mondo in più di 625.000 morti all&#39;anno in tutto il mondo. Sebbene elettroporazione è stato sviluppato per la trasformazione di plasmidi in Cryptococcus, soltanto la consegna biolistic fornisce un mezzo efficace per trasformare DNA lineare che può essere integrato nel genoma mediante ricombinazione omologa. Acetato ha dimostrato di essere un importante prodotto di fermentazione durante l&#39;infezione criptococcica, ma il significato di questa non è ancora noto. Un percorso batterica composta da enzimi xilulosio-5-fosfato / di fruttosio-6-fosfato phosphoketolase (Xfp) e chinasi acetato (ACK) è uno dei tre possibili percorsi per la produzione di acetato in C. neoformans. Qui, dimostriamo la trasformazione biolistic di un costrutto,che ha il gene che codifica per Ack fuso al tag fluorescenti mCherry, in C. neoformans. Abbiamo poi Confermiamo l&#39;integrazione della fusione ACK -mCherry nel locus ACK.

Un successo trasformazione biolistic di C. neoformans possono essere ottenute seguendo questo schema di protocollo (Figura 1). Con trasformazione biolistica, un germoglio successo delle perle oro rivestite è indicato da un anello oro visibile sulla piastra dopo il DNA è girato (Figura 2A). Colonie dovrebbero apparire entro 4 o 5 giorni se lasciato a temperatura ambiente dopo la placcatura le cellule recuperate dalle piastre sorbitolo YPD + 1M su terreni selettivi. Trasformand...

Watch this Scientific Journal Video about Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans at JoVE.com

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Trasformazione Biolistic è un metodo utilizzato per generare stabile integrazione del DNA nel genoma delle opportunistiche neoformans patogeno Cryptococcus tramite ricombinazione omologa. Dimostreremo trasformazione biolistica di un costrutto, che ha l&#39;acetato chinasi gene codificante fuso al tag mCherry fluorescente in C. neoformans.

Trasformazione Biolistic di una fluorescente Tagged Gene nel Opportunistic Fungal patogeni<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

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Immunology and Infection

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

9.9K Views.  Clemson University.  Trasformazione Biolistic è un metodo utilizzato per generare stabile integrazione del DNA nel genoma delle opportunistiche neoformans patogeno Cryptococcus tramite ricombinazione omologa. Dimostreremo trasformazione biolistica di un costrutto, che ha l'acetato chinasi gene codificante fuso al tag mCherry fluorescente in C. neoformans. 

Video: Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Generation of Fluorescent Protein Fusions in Candida Species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

Generation of Fluorescent Protein Fusions in Candida Species

PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis.

Generazione di fusioni proteina fluorescente in<em&gt; Candida</em&gt; Specie

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

Ricerca

Genetica

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Un metodo basato su fluorescenza per studiare la regolazione genica batterica nei tessuti infetti

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

Il canale di Excretory c. elegans come un modello per intracellulare Lumen morfogenesi e biogenesi di membrana polarizzata In Vivo in una singola cella: etichettatura di GFP-fusioni, schermata di interazione di RNAi e Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Biologia dello sviluppo

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Trasformazione genomica del Picoeukaryote<em&gt; Ostreococcus Tauri</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Biologia

Integrazione genica stabile in Chlorella vulgaris utilizzando Agrobacterium tumefaciens

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

Autore in primo piano: Protocollo di trasformazione ottimizzato per la Chlorella vulgaris utilizzando l'Agrobacterium tumefaciens 

This protocol outlines the utilization of Agrobacterium tumefaciens-mediated transformation (AMT) for integrating gene(s) of interest into the nuclear genome of the green microalgae Chlorella vulgaris, leading to the production of stable transformants.

Ingegneria genetica mediata da Agrobacterium tumefaciens di microalghe verdi, Chlorella vulgaris

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit ...

Bioingegneria

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Effetto di proteine fluorescenti su Fusion partner utilizzando saggi di tossicità di Polyglutamine in lievito

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae AD&#916;&#916; is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae AD&#916;&#916;, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

This article presents a small-scale plasma membrane isolation protocol for the characterization of Candida albicans ABC (ATP-binding cassette) protein Cdr1, overexpressed in Saccharomyces cerevisiae. A protease-cleavable C-terminal mGFPHis double tag with a 16-residue linker between Cdr1 and the tag was designed to facilitate the purification and detergent-screening of Cdr1.

Preparazione della membrana plasmatica su piccola scala per l'analisi di Candida albicans Cdr1-mGFPHis

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

Trasformazione naturale, espressione proteica e crioconservazione del cianobatterio filamentoso Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...

Trasformazione Biolistic di una fluorescente Tagged Gene nel Opportunistic Fungal patogeni

Riepilogo

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Capitoli in questo video

Generazione di fusioni proteina fluorescente in

Un metodo basato su fluorescenza per studiare la regolazione genica batterica nei tessuti infetti

Il canale di Excretory c. elegans come un modello per intracellulare Lumen morfogenesi e biogenesi di membrana polarizzata In Vivo in una singola cella: etichettatura di GFP-fusioni, schermata di interazione di RNAi e Imaging

Trasformazione genomica del Picoeukaryote

Ingegneria genetica mediata da Agrobacterium tumefaciens di microalghe verdi, Chlorella vulgaris

Ingegneria genetica mediata da Agrobacterium tumefaciens di microalghe verdi, Chlorella vulgaris

Ingegneria genetica mediata da Agrobacterium tumefaciens di microalghe verdi, Chlorella vulgaris

Effetto di proteine fluorescenti su Fusion partner utilizzando saggi di tossicità di Polyglutamine in lievito

Preparazione della membrana plasmatica su piccola scala per l'analisi di Candida albicans Cdr1-mGFPHis

Trasformazione naturale, espressione proteica e crioconservazione del cianobatterio filamentoso Phormidium lacuna