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

وأيد هذا العمل من الجوائز من مؤسسة العلوم الوطنية (جائزة # 0920274) وساوث كارولينا تجربة مشروع محطة SC-1700340. هذه الورقة isTechnical مساهمة رقم 6283 من محطة تجربة جامعة كليمسون. الكتاب أشكر الدكتور لوكاس Kozubowski للحصول على المشورة المفيدة له في تطوير هذا البروتوكول النهائي والدكتور شيريل انجرام سميث، كاتي جلين، وغريس Kisirkoi لقراءة نقدية لها من المخطوطة.

ويرد المخطط العام لهذا البروتوكول في الشكل رقم 1: ملاحظة. 1. C. إعداد الأدعومية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لكل رد فعل التحول، وتنمو 2-3 مل O / N ثقافة C. الأدعومية في YPD المتوسطة في 30 ° C تهتز ?...

<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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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

وbasidiomycete المستخفية المورمة، وهو ممرض الانتهازية الغازية من الجهاز العصبي المركزي، هي السبب الأكثر شيوعا لالتهاب السحايا الفطرية الناتجة في جميع أنحاء العالم في أكثر من 625،000 حالة وفاة سنويا في جميع أنحاء العالم. وعلى الرغم من وضعت Electroporation للللتحول من البلازميدات في المستخفية، ويوفر تسليم biolistic فقط وسيلة فعالة لتحويل DNA الخطية التي يمكن دمجها في الجينوم عن طريق إعادة التركيب مثلي. وقد تبين خلات أن يكون المنتج التخمير كبير خلال العدوى بالمستخفيات، ولكن أهمية هذا ومن غير المعروف حتى الان. وهناك مسار البكتيريا تتكون من الانزيمات زايلولوز-5-فوسفات / الفركتوز-6-فوسفات فسفوكيتولاز (XFP) وكيناز خلات (ACK) هو واحد من ثلاثة مسارات محتملة لإنتاج خلات في C. الأدعومية. هنا، علينا أن نظهر التحول biolistic لبناء،الذي الجين ترميز ACK تنصهر إلى العلامة الفلورسنت mCherry، إلى C. الأدعومية. نحن بعد تأكيد تكامل الانصهار ACK -mCherry في موضع ACK.

وتحول biolistic الناجح لC. ويمكن الحصول على الأدعومية باتباع هذا المخطط بروتوكول (الشكل 1). مع التحول biolistic، يشار إلى تبادل لاطلاق النار الناجح لحبات الذهب المغلفة من قبل خاتم من الذهب واضحة على لوحة بعد DNA هو النار (الشكل 2A). يجب أن تظهر المستعمرا...

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

التحول Biolistic هو طريقة تستخدم لتوليد التكامل مستقر من الحمض النووي في الجينوم من الانتهازية الأدعومية الممرض المستخفية من خلال إعادة التركيب مثلي. وسوف نظهر التحول biolistic من بناء، والتي لديها خلات كيناز ترميز الجينات تنصهر إلى العلامة فلوري mCherry إلى C. الأدعومية.

التحول Biolistic من نيون معلم جين إلى القناص الفطرية مسببات الأمراض<em&gt; المستخفية المورمة</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

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وظيفية فحص الدم لقياس الجامعة صلاحية مايكوباكتيريا ، وذلك باستخدام الجينات المرتبطة دلاليا مراسل بي سي جي إم أو السل (BCG لوكس / M. التربيوم لوكس)

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

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تصور الإيماس غير التحللي من<em&gt; الأدعومية المستخفية</em&gt; من الضامة الرقمية الخفيفة عن طريق الميكروسكوب

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

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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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مسببات الأمراض المنقولة عن طريق الأغذية الفحص باستخدام المغناطيسية-فلوري نانوسينسور: الكشف السريع عن O157:H7 كولاي

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