Biolistic transformação é um método usado para gerar a integração estável de ADN no genoma das oportunistas Cryptococcus neoformans patogénio através de recombinação homóloga. Iremos demonstrar transformação biolística de uma construção, o qual tem o gene que codifica a quinase de etilo fundido com o marcador fluorescente mCherry em C. neoformans.
O basidiomycete Cryptococcus neoformans, um patógeno oportunista invasiva do sistema nervoso central, é a causa mais freqüente de meningite fúngica em todo o mundo, resultando em mais de 625 mil mortes por ano em todo o mundo. Embora electroporação foi desenvolvida para a transformação de plasmídeos em Cryptococcus, apenas entrega biolística fornece um meio eficaz para transformar o ADN linear que pode ser integrado no genoma por recombinação homóloga.
Acetato tem sido demonstrado ser um importante produto de fermentação durante a infecção criptocócica, mas o significado de tal ainda não é conhecida. Uma via bacteriana composto das enzimas xilulose-5-fosfato / frutose-6-fosfato phosphoketolase (Xfp) e cinase de etilo (ACK) é uma das três vias possíveis para a produção de etilo em C. neoformans. Aqui, demonstramos a transformação biolística de uma construção,que o gene que codifica Ack fundido com o marcador fluorescente mCherry, em C. neoformans. Nós, então, confirmar a integração da fusão ACK -mCherry no locus ACK.
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.
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…
The authors have nothing to disclose.
Este trabalho foi apoiado por prêmios da Fundação Nacional de Ciência (Award # 0920274) e da Carolina do Sul Experiment Station Projeto SC-1700340. Este papel isTechnical Contribuição No. 6283 da Estação Experimental da Universidade Clemson. Os autores agradecem ao Dr. Lukasz Kozubowski por seu conselho útil no desenvolvimento deste protocolo final e Dr. Cheryl Ingram-Smith, Katie Glenn, e Grace Kisirkoi por sua leitura crítica do manuscrito.
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 |