Transformation biolistique d'un fluorescent Tagged Gene dans la opportuniste pathogène fongique<em> Cryptococcus neoformans</em
Summary
Transformation biolistique est une méthode utilisée pour générer une intégration stable de l'ADN dans le génome de l'agent pathogène Cryptococcus neoformans opportunistes par recombinaison homologue. Nous démontrerons transformation biolistique d'une construction, qui a la kinase acétate de codage de gène fusionné au marqueur fluorescent mCherry en C. neoformans.
Abstract
Le basidiomycète de Cryptococcus neoformans, un pathogène opportuniste invasive du système nerveux central, est la cause la plus fréquente de méningite fongique dans le monde entier résultant dans plus de 625 000 décès par an dans le monde. Bien que l'électroporation a été développé pour la transformation de plasmides dans Cryptococcus, ne délivrance biolistique fournit un moyen efficace pour transformer ADN linéaire qui peut être intégré dans le génome par recombinaison homologue.
Acétate a été montré pour être un produit de fermentation majeure lors de l'infection à Cryptococcus, mais l'importance de ce ne est pas encore connu. Une voie bactérienne composé des enzymes xylulose-5-phosphate / fructose-6-phosphate phosphocétolase (Xfp) et l'acétate kinase (ACK) est l'une des trois voies possibles pour la production d'acétate de C. neoformans. Ici, nous démontrons la transformation biolistique d'une construction,qui a le gène codant Ack fusionné au marqueur fluorescent mCherry, en C. neoformans. Nous confirmons donc l'intégration de la fusion ACK -mCherry dans le locus ACK.
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
Ce travail a été soutenu par des prix de la National Science Foundation (Award # 0920274) et l'expérience de Caroline du Sud Projet de station de SC-1700340. Ce document isTechnical Contribution n ° 6283 de la station expérimentale de l'Université Clemson. Les auteurs remercient le Dr Lukasz Kozubowski pour ses conseils utiles dans le développement de ce protocole final et le Dr Cheryl Ingram-Smith, Katie Glenn, et Grace Kisirkoi pour leur lecture critique du manuscrit.
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 |
Referências
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