Transformación biolística de un fluorescente Tagged gen en el oportunista por hongos patógenos<em> Cryptococcus neoformans</em
Summary
Transformación biolística es un método utilizado para generar la integración estable de ADN en el genoma de los oportunistas neoformans patógeno Cryptococcus través de la recombinación homóloga. Vamos a demostrar la transformación biolística de una construcción, que tiene el gen que codifica acetato quinasa fusionado a la mCherry etiqueta fluorescente en C. neoformans.
Abstract
El basidiomycete Cryptococcus neoformans, un patógeno oportunista invasivo del sistema nervioso central, es la causa más frecuente de meningitis fúngica en todo el mundo como resultado más de 625.000 muertes al año en todo el mundo. Aunque la electroporación se ha desarrollado para la transformación de los plásmidos en Cryptococcus, sólo entrega biolística proporciona un medio eficaz para transformar ADN lineal que se puede integrar en el genoma por recombinación homóloga.
Acetato ha demostrado ser un importante producto de fermentación durante la infección criptocócica, pero la importancia de esto todavía no se conoce. Una vía bacteriana integrada por los enzimas xilulosa-5-fosfato / fructosa-6-fosfato fosfocetolasa (Xfp) y acetato quinasa (ACK) es una de las tres vías posibles para la producción de acetato en C. neoformans. Aquí, nos demuestran la transformación biolística de una construcción,que tiene el gen que codifica Ack fusionado a la etiqueta fluorescente mCherry, en C. neoformans. Entonces Confirmamos integración de la fusión ACK -mCherry en el 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
Este trabajo fue apoyado por los premios de la Fundación Nacional de Ciencia (Premio # 0920274) y el Experimento de Carolina del Sur Estación Proyecto SC-1700340. Este papel isTechnical Contribución No. 6283 de la Estación Experimental de la Universidad de Clemson. Los autores agradecen al Dr. Lukasz Kozubowski por su útil asesoramiento en el desarrollo de este protocolo final y el Dr. Cheryl Ingram-Smith, Katie Glenn, y Grace Kisirkoi por su lectura crítica del manuscrito.
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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