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

This work was supported by awards from the National Science Foundation (Award# 0920274) and the South Carolina Experiment Station Project SC-1700340. This paper isTechnical Contribution No. 6283 of the Clemson University Experiment Station. The authors thank Dr. Lukasz Kozubowski for his helpful advice in development of this final protocol and Dr. Cheryl Ingram-Smith, Katie Glenn, and Grace Kisirkoi for their critical reading of the manuscript.

NOTE: The overall scheme of this protocol is outlined in Figure 1.
1. C. neoformans Preparation
<ol>
	<li>For each transformation reaction, grow a 2-3 ml&#160;O/N culture of C. neoformans in YPD medium at 30 &#176;C shaking at 250 rpm.</li>
	<li>Centrifuge the O/N culture for 5 min at 900 x g at 10 &#176;C and discard the supernatant.</li>
	<li>Resuspend each cell pellet in 300 &#956;l of Yeast Peptone Dextrose (YPD) medium.</li>
	<li>Using glass beads, gen...

<ol>
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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. 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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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		<tr height="21">
			<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

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

A successful biolistic transformation of C. neoformans can be obtained by following this protocol scheme (Figure 1). With biolistic transformation, a successful shoot of the coated gold beads is indicated by a gold ring visible on the plate after the DNA is shot (Figure 2A). Colonies should appear within 4 to 5 days when left at room temperature after plating the recovered cells from the YPD + 1M sorbitol plates onto selective media. Transforming 2 &#181;g of DNA should result i...

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

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

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

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

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