Biolistische Transformatie van een TL Tagged Gene in de Opportunistische schimmelpathogeen<em> Cryptococcus neoformans</em
Summary
Biolistische transformatie een methode om stabiele integratie van DNA genereren in het genoom van de opportunistische pathogeen Cryptococcus neoformans via homologe recombinatie. We zullen biolistische transformatie van een construct, waarin het gen codeert acetaat kinase gefuseerd met de fluorescerende tag mCherry in C. neoformans is aangetoond.
Abstract
De basidiomycete Cryptococcus neoformans, een invasieve opportunistische pathogeen van het centrale zenuwstelsel, is de meest voorkomende oorzaak van fungale meningitis wereldwijd resulteert in meer dan 625.000 doden per jaar wereldwijd. Hoewel elektroporatie is ontwikkeld voor de transformatie van plasmiden Cryptococcus, alleen biolistische levering een effectief middel om lineair DNA die kunnen worden geïntegreerd in het genoom door homologe recombinatie transformeren.
Acetaat is aangetoond dat een belangrijk fermentatieproduct tijdens cryptococcal infectie, maar de betekenis hiervan is nog niet bekend. Een bacteriële pad bestaande uit enzymen xylulose-5-fosfaat / fructose-6-fosfaat fosfoketolase (Xfp) en acetaat kinase (Ack) is een van drie manieren waarop acetaat productie C. neoformans. Hier tonen we de biolistische transformatie van een construct,waarin het gen codeert Ack is gefuseerd met de fluorescente tag mCherry, in C. neoformans. Wij bevestigen dan is integratie van de ACK -mCherry fusie in de ACK locus.
Introduction
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 C. neoformans 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 biolistic delivery using a gene gun provides an effective means to transform linear DNAs 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 ack locus. The protocol demonstrated here is a modification of the method developed in the Perfect laboratory 8.
Protocol
Representative Results
Discussion
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 µg. However, the volume of DNA added to the gold beads can be increased in the chance the…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dit werk werd ondersteund door de awards van de National Science Foundation (Award # 0.920.274) en de Zuid-Carolina Experiment Station Project SC-1.700.340. Deze paper isTechnical Bijdrage No. 6283 van de Clemson University Experiment Station. De auteurs danken dr Lukasz Kozubowski voor zijn nuttig advies bij de ontwikkeling van deze laatste protocol en Dr. Cheryl Ingram-Smith, Katie Glenn, en Grace Kisirkoi voor hun kritische lezing van het manuscript.
Materials
| Product | Company | Catalog # | Website |
| 0.6 μm gold beads | Bio-Rad | 165-2262 | http://www.bio-rad.com |
| Spermadine-free base | Sigma- Aldrich | S0266 | https://www.sigmaaldrich.com |
| G418 – Sulfate (Neomycin) | Gold Biotechnology | G-418-10 | www.goldbio.com |
| Hygromycin | Gold Biotechnology | H-270-1 | www.goldbio.com |
| 1350 psi Rupture Discs | Bio-Rad | 165-2330 | http://www.bio-rad.com |
| Stopping Screens | Bio-Rad | 165-2336 | http://www.bio-rad.com |
| Macrocarriers discs | Bio-Rad | 165-2335 | http://www.bio-rad.com |
| YPD Broth | Becton Dickinson & Co. | 242820 | www.bd.com |
| Agar | Becton Dickinson & Co. | 214530 | www.bd.com |
| Sorbitol | Fisher Scientific | BP439 | http://www.fishersci.com |
| PDS-1000/He System | Bio-Rad | 165-2257 | http://www.bio-rad.com |
| Microscope | Zeiss | Axio | http://www.zeiss.com/microscopy |
| KOD One Step PCR Kit | EMD Millipore | 71086-4 | http://www.emdmillipore.com |
| One Step RT-PCR Kit | Qiagen | 210212 | www.qiagen.com |
| Wizard Genomic DNA Purification Kit | Promega | A1120 | www.promega.com |
| RNeasy Mini Kit | Qiagen | 74104 | www.qiagen.com |
| Mini Beadbeater – 1 | BioSpecs | 3110BX | http://www.biospec.com |
| Microfuge 18 Centrifuge | Beckman Coulter | F241.5P | www.beckmancoulter.com |
| Microplate Spectrophotometer | BioTek | EPOCH | www.biotek.com |
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