Trasformazione Biolistic di una fluorescente Tagged Gene nel Opportunistic Fungal patogeni<em> Cryptococcus neoformans</em
Summary
Trasformazione Biolistic è un metodo utilizzato per generare stabile integrazione del DNA nel genoma delle opportunistiche neoformans patogeno Cryptococcus tramite ricombinazione omologa. Dimostreremo trasformazione biolistica di un costrutto, che ha l'acetato chinasi gene codificante fuso al tag mCherry fluorescente in C. neoformans.
Abstract
Il basidiomicete Cryptococcus neoformans, un patogeno opportunista invasivo del sistema nervoso centrale, è la causa più frequente di meningite fungina con conseguente tutto il mondo in più di 625.000 morti all'anno in tutto il mondo. Sebbene elettroporazione è stato sviluppato per la trasformazione di plasmidi in Cryptococcus, soltanto la consegna biolistic fornisce un mezzo efficace per trasformare DNA lineare che può essere integrato nel genoma mediante ricombinazione omologa.
Acetato ha dimostrato di essere un importante prodotto di fermentazione durante l'infezione criptococcica, ma il significato di questa non è ancora noto. Un percorso batterica composta da enzimi xilulosio-5-fosfato / di fruttosio-6-fosfato phosphoketolase (Xfp) e chinasi acetato (ACK) è uno dei tre possibili percorsi per la produzione di acetato in C. neoformans. Qui, dimostriamo la trasformazione biolistic di un costrutto,che ha il gene che codifica per Ack fuso al tag fluorescenti mCherry, in C. neoformans. Abbiamo poi Confermiamo l'integrazione della fusione ACK -mCherry nel 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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Questo lavoro è stato sostenuto da premi dalla National Science Foundation (Award # 0.920.274) e l'esperimento South Carolina Progetto Stazione SC-1.700.340. Questo documento isTechnical Contributo No. 6283 del Experiment Station Clemson University. Gli autori ringraziano il Dr. Lukasz Kozubowski per i suoi utili consigli per lo sviluppo di questo protocollo finale e il dottor Cheryl Ingram-Smith, Katie Glenn, e Grace Kisirkoi per la lettura critica del manoscritto.
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 |
References
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