Biolistisk Transformation av en fluorescerande taggade gen i Opportunistic svamppatogen<em> Cryptococcus neoformans</em
Summary
Biolistisk omvandling är en metod som används för att generera en stabil integrering av DNA i genomet hos de opportunistiska patogenen Cryptococcus neoformans genom homolog rekombination. Vi kommer att visa biolistisk transformation av en konstruktion, som har den gen som kodar acetatkinas smält till den fluorescerande taggen mCherry in C. neoformans.
Abstract
Den basidiomycet Cryptococcus neoformans, en invasiv opportunistisk patogen av det centrala nervsystemet, är den vanligaste orsaken till svampmeningit globalt ledde till mer än 625.000 dödsfall per år i hela världen. Även elektroporering har utvecklats för omvandlingen av plasmider i Cryptococcus, ger bara biolistisk leverans ett effektivt medel för att omvandla linjära DNA som kan integreras i genomet genom homolog rekombination.
Acetat har visat sig vara en stor jäsningsprodukt under krypto infektion, men betydelsen av detta är ännu inte känt. En bakteriell väg bestående av enzymerna xylulos-5-fosfat / fruktos-6-fosfat phosphoketolase (Xfp) och acetatkinas (ACK) är en av tre möjliga vägar för acetat produktion i C. neoformans. Här visar vi den biolistiska transformationen av en konstruktion,som har den gen som kodar Ack fuserad till den fluorescerande taggen mCherry, in C. neoformans. Vi bekräfta sedan integrationen av ACK -mCherry fusion i 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
Detta arbete stöddes av utmärkelser från National Science Foundation (Award # 0.920.274) och South Carolina Experiment Station Project SC-1.700.340. Denna uppsats isTechnical bidrag nr 6283 i Clemson University Experiment Station. Författarna tackar Dr Lukasz Kozubowski för hans goda råd i utvecklingen av denna slutliga protokollet och Dr. Cheryl Ingram-Smith, Katie Glenn, och Grace Kisirkoi för deras kritisk läsning av manuskriptet.
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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