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

Diese Arbeit wurde durch Auszeichnungen von der National Science Foundation (Preis # 0.920.274) und dem South Carolina Experiment Station Projekt SC-1.700.340 unterstützt. Dieses Papier isTechnical Beitrag Nr 6283 der Clemson University Experiment Station. Die Autoren danken Dr. Lukasz Kozubowski für seine hilfreiche Ratschläge bei der Entwicklung dieser letzten Protokoll und Dr. Cheryl Ingram-Smith, Katie Glenn, und Grace Kisirkoi für die kritische Durchsicht des Manuskripts.

HINWEIS: Das Gesamtkonzept dieses Protokolls ist in Abbildung 1 dargestellt. 1. C. neoformans Vorbereitung <ol><li> Für jedes Transformationsreaktion, wachsen einem 2-3 ml O / N-Kultur von C. neoformans in YPD-Medium bei 30 ° C unter Schütteln bei 250 rpm. </li><li> Zentrifugieren Sie die O / N-Kultur für 5 Minuten bei 900 · g bei 10 ° C und den Überstand verwerfen. </li><li> Resuspendieren Zellpellets in 300 &amp; mgr; l Hefe-Pepton-Dextrose (YPD) Medium. </li><li> Mit Glasperlen, sanft verteilen 300 ul der gewaschenen Zellsuspension auf YPD-Agar, 1 M Sorbitol. </li><li> Zulassen Platten bei Raumtemperatur für 3-4 Stunden trocknen. </li></ol> 2. Gold Mikro Vorbereitung <ol><li> Resuspendieren 0,25 g von 0,6 um Gold Perlen in 1 ml ddH 2 O, Zentrifuge für 1 min bei 900 xg pelletiert die Perlen, und entfernen Sie den Überstand. </li><li> Resuspendieren der Gold Perlen in 1 ml 100% Ethanol. &lt;/ Li&gt; <li> Verteilen Sie die Perlen in 4 Röhrchen wurden 250 ul jeder, und fügen Sie 750 ul 100% Ethanol. </li><li> Shop Goldperle Aliquots bei 4 ° C. </li></ol> 3. DNA-Präparation <ol><li> Bereiten Orange Makroträger biolistischen Scheiben durch Eintauchen sie in 100% Ethanol mit einer Pinzette. Platz Scheiben in eine große Petrischale Drierite zu trocknen, die (sicherstellen, dass der Drierite nicht die Scheiben berühren). </li><li> Nach dem Trocknen, drücken Sie die Makroträger Scheiben in die Silberplattenhalter (bisher mit 100% Ethanol abgewischt). </li><li> Vortex Gold Perlen (wie in Schritt 2 hergestellt) und aliquote 12 ul in ein 1,5 ml Mikrozentrifugenröhrchen, eine Röhre pro Transformation. </li><li> Hinzufügen zu jedem Röhrchen, um: 2 ug DNA (vorzugsweise 2 &amp; mgr; l von 1 &amp; mgr; g / &amp; mgr; l DNA), 10 ul 2,5 M CaCl 2 und 2 ul 1 M Spermidin freie Base. </li><li> Eine negative Kontrolle, wie in Schritt 3.4, aber ohne DNA festgelegt. </li><li> Vortex jedes Röhrchen und Inkubation bei Raumtemperatur5 min. Flick vorsichtig jedes Röhrchen gelegentlich die siedelt Perlen Während dieser Inkubation resuspendieren. </li><li> Spin-Röhrchen bei 225 g für 30 Sekunden bis zu pelletieren Sie die DNA-beschichteten Goldperlen. Den Überstand (durch Pipettieren oder Aspiration) Entfernen und entsorgen. </li><li> Resuspendieren Perlen vollständig in 600 ul 100% Ethanol durch langsames Auf- und Abpipettieren. </li><li> Spin-Röhrchen bei 225 xg für 30 Sekunden, um die Kügelchen zu pelletieren ohne Verpackung. Entfernen und entsorgen Sie den Überstand. </li><li> Resuspendieren der DNA-beschichteten Goldperlen in 8 ul 100% Ethanol durch langsames Auf- und Abpipettieren. </li><li> Pipettieren Sie die DNA-beschichteten Goldperlen auf die Mitte des biolistischen Scheibe in einer 1 cm Durchmesser und trocknen lassen. HINWEIS: Eine getrocknete gold Kreis in der Mitte des biolistischen Scheibe sichtbar zeigt an, dass eine ausreichende Konzentration von Gold Perlen vorliegt. HINWEIS: Die Makroträger Scheiben mit DNA-beschichteten Goldkügelchen beladen sind jetzt bereit für den Einsatz mit der Genkanone. </li></ol> 4. O-perating die Gene Gun <ol><li> Einschalten der Vakuumpumpe. </li><li> Schalten Sie das Heliumgas durch Drehen des Knopfes gegen den Uhrzeigersinn, bis ein Druck von etwa 2200 psi wird am Manometer erreicht. </li><li> Schalten Sie den Gen-Kanone, indem Sie den roten Schalter auf der linken Seite. </li><li> Achten Sie darauf, dass die Durchflussraten für das Vakuum und die Entlüftung werden so eingestellt, das Vakuum 28 Zoll Hg innerhalb von 15 Sekunden zu erreichen. </li><li> Seien Sie sicher, dass der Abstand zwischen der Berstscheibe und Makroträger ist ungefähr 3/8 Zoll. </li><li> Die gesamte Kammer Reinigen durch Abwischen mit Ethanol. </li><li> Tauchen Sie die Berstscheiben in 100% Ethanol. Zulassen, um eine sterile Oberfläche trocknen auf (zB Petrischale). </li><li> Verwenden Sie einen Drehmomentschlüssel, um die Berstscheibe Halter lockern. Legen Sie eine saubere Berstscheibe in die Halterung. Schrauben Sie die Berstscheibe Halter zurück an ihren Platz und ziehen Sie mit einem Drehmomentschlüssel durch einmaliges Drehen Sie ihn nach rechts. HINWEIS: Berstscheiben wird nach jedem Shooting ersetzt werden. </li><li> Tauchen Sie the Maschensieben in 100% Ethanol. Zulassen, um eine sterile Oberfläche trocknen auf (zB Petrischale). </li><li> Einmal trocken, legen Sie ein gewaschen Sieb auf dem weißen Kunststoff-Montageplatte. Setzen Sie den Macrocarrier Scheibenhalter DNA Seite nach unten in das Disc-Kammer. Schrauben Sie die silberne Kappe und legen Sie die Montageplatte in der höchsten Steckplatz. HINWEIS: Das Sieb wird nach jedem Shooting ersetzt werden. </li><li> Legen Sie eine YPD-Agar-Platte mit 1 M Sorbit auf der Bodenplatte. </li><li> Halt die Kammertür und einrasten. </li><li> Drücken und halten Sie den mittleren roten Schalter bis zu Vakuum engagieren und das Vakuum bis 28 Zoll Hg erreichen. Sobald geeignete Vakuumniveau erreicht ist, bewegen Sie den Schalter in die untere Position. Wenn Sie bereit sind, halten Sie den roten Schalter auf der rechten Seite, um Feuer. Wenn die Berstscheibe öffnet, lassen Sie sofort den Feuerknopf und drücken Sie den mittleren roten Schalter in die mittlere Position, um die Kammer auf 0 psi entlüften. </li><li> Reinigen Sie Berstscheibe Schutt und schalten Sie den Gen-Kanone. Schalten Sie dann den helium Gas durch Drehen des Knopfes im Uhrzeigersinn, und schließlich, schalten Sie die Vakuumpumpe. </li></ol> 5. Überzug transformierten Zellen <ol><li> Lassen Sie die Transformationsplatten bei RT 4 Stunden ruhen lassen, damit die Zellen sich zu erholen. </li><li> Pipette 700 ul YPD auf die Platte. Verwenden Sie einen Zellschaber, um sanft kratzen die Zellen aus der Agar und Pipette Flüssigkeit in ein steriles 1,5 ml Mikrozentrifugenröhrchen. Wiederholen Sie diesen Schritt, um sicherzustellen, dass alle Zellen von der Platte wiederhergestellt. </li><li> Pelle Zellen bei 225 · g für 30 sec. Entfernen Sie den Überstand. </li><li> Das Pellet in 500 ul YPD. </li><li> Je 100 ul der Zellsuspension in die Mitte der YPD + Antibiotikum Platten und verbreiten mit Glasperlen. </li><li> Lassen umgekehrten Platten bei Raumtemperatur für 3-4 Tage. Wie Kolonien erscheinen, Patch auf neue YPD + Antibiotikum Platten. </li></ol> 6. Die genomische DNA Isolierung für die PCR Hinweis: Dies ist eine modifizierte Version mit Reagenzs aus einem DNA-Reinigungskits (Siehe Tabelle der Materialien). <ol><li> Wachsen Sie eine 5-ml-Kultur von jedem der C neoformans -Transformanten in YPD Flüssigkeit bei 30 ° C unter Schütteln bei 250 rpm O / N. </li><li> Pelle 3 ml Zellen bei 900 · g und Resuspension in 600 &amp; mgr; l der Zellkerne Lyselösung. </li><li> Fügen Sie die Suspension in ein neues 1,5 ml Mikrozentrifugenröhrchen mit 200 ul 0,5 mm säuregewaschene Glasperlen. </li><li> Homogenisierung für 45 Sekunden in einem Mini BeadBeater bei Umgebungstemperatur, kühle Röhrchen auf Eis, und wiederholen. </li><li> Lassen Sie Probe auf Eis für 2 min absetzen und übertragen Stand in ein neues 1,5-ml-Tube. Gib 200 ul Proteinfällungslösung zu jedem Röhrchen (100 &amp; mgr; l pro 600 &amp; mgr; l Überstand gewonnen) und Vortex kräftig 20 sec. </li><li> Werden die Proben auf Eis für 5 min absitzen und Zentrifugieren bei 11.000 g für 3 min. </li><li> Den Überstand in ein sauberes 1,5 ml Röhrchen mit 300 ul RT Isopropanol. Vorsichtig mischen durch Umdrehen. </li><li>Zentrifugieren Sie die Proben bei 11.000 g für 2 min, entfernen Sie vorsichtig den Überstand, und entleeren Sie die Rohre auf Papiertücher. </li><li> Hinzufügen von 300 &amp; mgr; l RT 70% Ethanol zu jedem Röhrchen und vorsichtig umgedreht, um das Pellet zu waschen. </li><li> Zentrifugieren Sie die Proben bei 11.000 g für 2 min, und entfernen Sie vorsichtig die gesamte Ethanol. </li><li> Lassen Sie das Rohr auf die saubere Papierhandtücher, und lassen Sie das Pellet an der Luft trocknen für 10-15 min. </li><li> In 50 ul DNA Rehydrationslösung und 1,5 ul RNase-Lösung zu jedem Pellet und Wirbel. </li><li> Zentrifuge Proben für 5 sec bis die gesamte Flüssigkeit aus der Kappe zu entfernen. </li><li> Proben bei 37 ° C für 15 min. </li><li> Rehydrieren die DNA durch Inkubation der Proben bei 65 ° C für 1 Stunde. </li><li> Quantifizierung DNA spektrophotometrisch durch Messung der Absorption bei 260 nm (A 260 ein Lesen von 1,0 ist äquivalent zu ~ 50 &amp; mgr; g / ml doppelsträngige DNA), und Verwendung von bis zu 200 ng in jeder PCR-Reaktion. </li></ol> 7. RNA-Isolierung fürReverse Transkriptase-PCR. <ol><li> Mit Hilfe eines RNA-Reinigungskits (Siehe Tabelle der Materialien), folgen Sie den Anweisungen des Herstellers, um RNA aus Hefezellen mit einem minibeadbeater isolieren. </li><li> Quantifizierung der Konzentration der RNA durch Messung der Absorption bei 260 nm (A 260 ein Lesen von 1,0 ist äquivalent zu ~ 40 &amp; mgr; g / ml Einzelstrang-RNA). </li><li> Die Verwendung eines RT-PCR-Kit (siehe Tabelle der Materialien), folgen Sie den Anweisungen des Herstellers, um die RT-PCR-Reaktionen mit ungefähr 1 ug RNA einzurichten. Für die in dieser Studie erhaltenen Ergebnisse, die in Tabelle 1 aufgelisteten Primer. </li></ol>

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	<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. 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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. 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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

Die Basidiomyceten Cryptococcus neoformans, eine invasive opportunistische Erreger des zentralen Nervensystems, ist die häufigste Ursache für Pilzmeningitis weltweiten was zu mehr als 625.000 Todesfälle pro Jahr weltweit. Obwohl Elektroporation zur Transformation von Plasmiden in Cryptococcus entwickelt, liefert nur biolistischen Anlieferung ein wirksames Mittel, um eine lineare DNA, die in das Genom durch homologe Rekombination integriert werden kann, zu transformieren. Acetat wurde gezeigt, ein Hauptfermentationsprodukt während cryptococcal Infektion sein, aber die Bedeutung dieser Tatsache wird noch nicht bekannt. Eine bakterielle Wegs der Enzyme Xylulose-5-Phosphat / Fructose-6-phosphat Phosphoketolase (XFP) und Acetatkinase (Ack) zusammengesetzt ist, eines von drei möglichen Pfade für Acetatproduktion in C. neoformans. Hier zeigen wir die biolistische Transformation eines Konstrukts,was hat die Ack kodierende Gen in die fluoreszierende Markierung verschmolzen mCherry, in C. neoformans. Wir bestätigen dann die Integration des ACK -mCherry Fusion in die ACK-Locus.

Eine erfolgreiche biolistische Transformation von C. neoformans kann durch Anschluss an diese Protokollschema (Abbildung 1) erreicht werden. Mit biolistische Transformation, ist ein erfolgreicher Shooting der beschichteten Goldkügelchen von einem goldenen Ring auf der Platte sichtbar angezeigt, nachdem die DNA Aufnahme (Abbildung 2A). Kolonien sollte innerhalb von 4 bis 5 Tage angezeigt werden, wenn bei Raumtemperatur nach der Plattierung der gewonnenen Zellen von den YPD + 1 ...

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

Biolistische Transformation ein Verfahren verwendet werden, um eine stabile Integration der DNA in das Genom der opportunistisches Pathogen Cryptococcus neoformans durch homologe Rekombination zu erzeugen. Wir biolistische Transformation eines Konstrukts, das den kodierenden Gens Acetatkinase zur Fluoreszenzmarkierung mCherry in C. neoformans fusioniert hat demonstrieren.

Biolistischen Transformation einer Leuchtstoff Tagged-Gens in den Opportunistic Pilzpathogenentwicklung<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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JoVE Journal

Immunology and Infection

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

9.9K Aufrufe.  Clemson University.  Biolistische Transformation ein Verfahren verwendet werden, um eine stabile Integration der DNA in das Genom der opportunistisches Pathogen Cryptococcus neoformans durch homologe Rekombination zu erzeugen. Wir biolistische Transformation eines Konstrukts, das den kodierenden Gens Acetatkinase zur Fluoreszenzmarkierung mCherry in C. neoformans fusioniert hat demonstrieren. 

Serie: 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.

Die Erzeugung von Fluorescent Protein Fusions in<em&gt; Candida</em&gt; Arten

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

Forschung

Genetik

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.

Eine Fluoreszenz basierende Methode, bakterielle Genregulation in infizierten Gewebe zu studieren

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.

C. Elegans Excretory Kanal als Modell für die intrazelluläre Lumen Morphogenese und In Vivo polarisiert Membrane Biogenesis in einer einzelnen Zelle: Kennzeichnung von GFP-Fusionen, RNAi Interaktion Bildschirm und 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 ...

Entwicklungsbiologie

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.

Genomic Transformation des 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 ...

Biologie

Stabile Genintegration in Chlorella vulgaris mit 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.

Autor im Rampenlicht: Optimiertes Transformationsprotokoll für Chlorella vulgaris mit 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.

Agrobacterium tumefaciens-vermittelte Gentechnik von Grünen Mikroalgen, Chlorella vulgaris

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

Biotechnik

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.

Wirkung von fluoreszierenden Proteinen auf Fusion Partner mit Trinukleotiderkrankungen Toxizität Assays in Hefe

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.

Plasmamembranpräparation im kleinen Maßstab zur Analyse von 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.

Natürliche Transformation, Proteinexpression und Kryokonservierung des filamentösen Cyanobakteriums Phormidium lacuna

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

Biolistischen Transformation einer Leuchtstoff Tagged-Gens in den Opportunistic Pilzpathogenentwicklung

Zusammenfassung

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Die Erzeugung von Fluorescent Protein Fusions in

Eine Fluoreszenz basierende Methode, bakterielle Genregulation in infizierten Gewebe zu studieren

C. Elegans Excretory Kanal als Modell für die intrazelluläre Lumen Morphogenese und In Vivo polarisiert Membrane Biogenesis in einer einzelnen Zelle: Kennzeichnung von GFP-Fusionen, RNAi Interaktion Bildschirm und Imaging

Genomic Transformation des Picoeukaryote

Agrobacterium tumefaciens-vermittelte Gentechnik von Grünen Mikroalgen, Chlorella vulgaris

Agrobacterium tumefaciens-vermittelte Gentechnik von Grünen Mikroalgen, Chlorella vulgaris

Agrobacterium tumefaciens-vermittelte Gentechnik von Grünen Mikroalgen, Chlorella vulgaris

Wirkung von fluoreszierenden Proteinen auf Fusion Partner mit Trinukleotiderkrankungen Toxizität Assays in Hefe

Plasmamembranpräparation im kleinen Maßstab zur Analyse von Candida albicans Cdr1-mGFPHis

Natürliche Transformation, Proteinexpression und Kryokonservierung des filamentösen Cyanobakteriums Phormidium lacuna