Biolistic Transformasjon av en Fluorescent Tagged Gene inn i Opportunistic Fungal Patogen<em> Cryptococcus neoformans</em
Summary
Biolistic transformasjon er en metode som brukes til å generere stabil integrering av DNA inn i genomet til de opportunistiske patogener Cryptococcus neoformans gjennom homolog rekombinasjon. Vi vil demonstrere biolistic transformasjon av en konstruksjon, som har genet som koder acetat kinase kondensert til den fluorescerende tag mCherry inn C. neoformans.
Abstract
Den basidiomycet Cryptococcus neoformans, en invasiv opportunistisk patogen av sentralnervesystemet, er den hyppigste årsaken til sopp hjernehinnebetennelse på verdensbasis som resulterer i mer enn 625 000 dødsfall per år på verdensbasis. Selv elektroporering har blitt utviklet for omdanning av plasmider i Cryptococcus, gir bare biolistic levering av en effektiv måte å omdanne lineært DNA som kan bli integrert inn i genomet ved homolog rekombinasjon.
Acetat har vist seg å være en stor fermenteringsprodukt under cryptococcal infeksjon, men betydningen av dette er ennå ikke kjent. En bakteriell vei består av enzymene xylulose-5-fosfat / fruktose-6-fosfat phosphoketolase (Xfp) og acetat kinase (ACK) er en av tre mulige veier for acetat produksjon i C. neoformans. Her demonstrerer vi biolistic transformasjon av en konstruksjon,som har genet som koder Ack fusjonert til det fluorescerende tag mCherry, inn C. neoformans. Vi deretter bekrefte integrering av ACK -mCherry fusjon inn 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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbeidet ble støttet av utmerkelser fra National Science Foundation (Award # 0920274) og South Carolina Experiment Station Prosjekt SC-1700340. Dette papiret isTechnical bidrag nr 6283 av Clemson University Experiment Station. Forfatterne takker Dr. Lukasz Kozubowski for hans nyttige råd i utviklingen av denne endelige protokollen og Dr. Cheryl Ingram-Smith, Katie Glenn, og Grace Kisirkoi for deres kritisk lesing 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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