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

Bu çalışma, Ulusal Bilim Vakfı (Ödül # 0.920.274) ve Güney Carolina Deney İstasyonu Projesi SC-1.700.340 dan ödülle tarafından desteklenmiştir. Clemson Üniversitesi Deney Station Bu kağıt isTechnical Katkı sayılı 6283. Yazarlar bu son protokol ve yazının eleştirel okuma Dr Cheryl Ingram-Smith, Katie Glenn, Grace Kisirkoi gelişiminde onun yararlı tavsiyeler Dr. Lukasz Kozubowski teşekkür ederim.

NOT: Bu protokolün genel şeması Şekil 1&#39;de özetlenmiştir. 1. C. neoformans Hazırlık <ol><li> Her bir dönüşüm reaksiyonu için, C 2-3 mi O / N kültürünün yetiştirilmesi 30 ° C&#39;de YPD besiyeri içinde neoformans 250 rpm &#39;de çalkalanarak. </li><li> 10 ° C&#39;de 900 x g&#39;de 5 dakika boyunca O / N kültürü santrifüj ve süpernatant atılır. </li><li> Maya Peptonlu Dekstroz (YPD) orta 300 ul her hücre pelletini....

<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. M., Frases, S., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipophilic+dye+staining+of+Cryptococcusneoformans+extracellular+vesicles+and+capsule.">Lipophilic dye staining of Cryptococcusneoformans extracellular vesicles and capsule.</a> Eukaryot Cell. 8, 1373-1380 (2009).</li></ol>

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.

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			<td height="21" style="height:21px;width:273px;">Product</td>
			<td style="width:171px;">Company</td>
			<td style="width:99px;">Catalog #</td>
			<td style="width:255px;">Website</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.6 &#956;m gold beads</td>
			<td>Bio-Rad</td>
			<td>165-2262</td>
			<td>http://www.bio-rad.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spermadine-free base</td>
			<td>Sigma- Aldrich</td>
			<td>S0266</td>
			<td>https://www.sigmaaldrich.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">G418 - Sulfate (Neomycin)</td>
			<td>Gold Biotechnology</td>
			<td>G-418-10</td>
			<td>www.goldbio.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hygromycin</td>
			<td>Gold Biotechnology</td>
			<td>H-270-1</td>
			<td>www.goldbio.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1350 psi Rupture Discs</td>
			<td>Bio-Rad</td>
			<td>165-2330</td>
			<td>http://www.bio-rad.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stopping Screens</td>
			<td>Bio-Rad</td>
			<td>165-2336</td>
			<td>http://www.bio-rad.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Macrocarriers discs</td>
			<td>Bio-Rad</td>
			<td>165-2335</td>
			<td>http://www.bio-rad.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">YPD Broth</td>
			<td>Becton Dickinson &#38; Co.</td>
			<td>242820</td>
			<td>www.bd.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Becton Dickinson &#38; Co.</td>
			<td>214530</td>
			<td>www.bd.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorbitol</td>
			<td>Fisher Scientific</td>
			<td>BP439</td>
			<td>http://www.fishersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDS-1000/He System</td>
			<td>Bio-Rad</td>
			<td>165-2257</td>
			<td>http://www.bio-rad.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td>Zeiss</td>
			<td>Axio</td>
			<td>http://www.zeiss.com/microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KOD One Step PCR Kit</td>
			<td>EMD Millipore</td>
			<td>71086-4</td>
			<td>http://www.emdmillipore.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">One Step RT-PCR Kit</td>
			<td>Qiagen</td>
			<td>210212</td>
			<td>www.qiagen.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wizard Genomic DNA Purification Kit</td>
			<td>Promega</td>
			<td>A1120</td>
			<td>www.promega.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>www.qiagen.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini Beadbeater - 1</td>
			<td>BioSpecs</td>
			<td>3110BX</td>
			<td>http://www.biospec.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microfuge 18 Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>F241.5P</td>
			<td>www.beckmancoulter.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

basidiyomiset Cryptococcus neoformans, merkezi sinir sisteminin bir invaziv fırsatçı patojen, dünya çapında, dünya çapında yılda fazla 625.000 ölümle sonuçlanan mantar menenjitin en sık nedenidir. Elektroporasyon Cryptococcus plasmidlerin dönüştürülmesi için geliştirilmiş olmasına rağmen, sadece biyolistik homolog rekombinasyon ile genomun içine entegre edilebilir lineer DNA dönüşümü için etkili bir araç sağlar. Asetat HIV enfeksiyonu sırasında büyük bir fermentasyon ürünü olduğu gösterilmiştir, ancak bu önemi henüz bilinmemektedir. Enzimler ksiluloz-5-fosfat / fruktoz-6-fosfat fosfoketolaz (Xfp) ve asetat kinaz (ACK) oluşan bir bakteriyel yol C&#39;de asetat üretimi için üç potansiyel geçiş yollarından biri olup neoformans. Burada, bir yapının Biyolistik dönüşüm gösterir,Ack kodlayan gen, MCherry floresan etiketi kaynaşmış C geri olan neoformans. Daha sonra ACK mahaline içine ACK -mCherry füzyon entegrasyonu onaylayın.

C başarılı bir biolistik transformasyon neoformans bu protokol şeması (Şekil 1) takip edilerek elde edilebilir. DNA, püskürtmenin (Şekil 2A) sonra biyolistik dönüşüm ile kaplanmış altın parçacıkları başarılı bir filiz plaka üzerinde görünür bir altın ile gösterilir. Seçici ortama YPD + 1M sorbitol plakaları kurtarıldı hücreleri kaplama sonra oda sıcaklığında sol Koloniler 4 ila 5 gün içinde görünmelidir. DNA 2 ug Dönüşümü 2...

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

Biyolistik dönüşüm homolog yeniden bağlama aracılığıyla fırsatçı patojen Cryptococcus neoformans genomuna DNA&#39;nın kararlı bütünleşmesinin oluşturmak için kullanılan bir yöntemdir. Biz C.neoformans içine floresan etiket mCherry kaynaşmış kodlayan gen asetat kinaz olan bir yapı, biolistik dönüşümü gösterecektir.

Fırsatçı mantar patojenlere bir Floresan etiketli geninin biyolistik transformasyonu<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 ...

Research

JoVE Journal

Immunology and Infection

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

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Mikobakteriler canlılık Tedbir Fonksiyonel Tam Kan Testi, Reporter Gen BCG veya M. Tb (BCG Takip edilen konu kullanarak Lüks / M. Tb Lüks)

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Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

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Sigara litik Ekzositoz görselleştirme<em&gt; Cryptococcus neoformans</em&gt; Dijital Işık mikroskopi ile Makrofagların gelen

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

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Makrofaj Kolesterol tükenmesi ve Fagositoz Üzerine Etkisi<em&gt; Cryptococcus neoformans</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

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