日和見菌病原体への蛍光タグ付き遺伝子のバイオリスティック変容<em>クリプトコックス·ネオフォルマンス</em
Summary
バイオリスティック形質転換は、相同組換えによる日和見病原体クリプトコッカス·ネオフォルマンスのゲノムへのDNAの安定な組み込みを生成するために使用される方法である。我々 は 、C。 ネオフォルマンスへの蛍光タグmCherryをに融合をコードする遺伝子の酢酸キナーゼを持つ構築物のバイオリスティック形質転換のデモンストレーションを行います。
Abstract
担子菌クリプトコッカスネオフォルマンス 、中枢神経系の侵襲的な日和見病原体は、世界的に全世界で年間以上の625000人が死亡、その結果、真菌性髄膜炎の最も頻繁な原因である。エレクトロポレーションは、 クリプトコッカスにおけるプラスミドの形質転換のために開発されてきたが、唯一のバイオリスティック送達は、相同組換えによりゲノム中に組み込むことができる直鎖状DNAを形質転換する有効な手段を提供する。
酢酸クリプトコッカス感染中の主要な発酵産物であることが示されているが、この重要性はまだ知られていない。酵素がキシルロース-5-リン酸/フルクトース-6-リン酸ホスホケトラーゼ（XFP）及び酢酸キナーゼ（肯定応答）からなる細菌の経路はC.で酢酸を製造するための3つの潜在的な経路の一つであるネオフォルマンス 。ここでは、構築物の微粒子銃形質転換を実証する、のAckをコードする遺伝子は、mCherryを蛍光タグに融合C.にしたネオフォルマンス 。次に、ACK座にACK -mCherry融合の統合を確認する。
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
この作品は、国立科学財団（賞＃0920274）とサウスカロライナ州の試験場プロジェクトSC-1700340からの賞によってサポートされていました。クレムソン大学試験場の本論文isTechnical貢献号6283。著者は、原稿の彼らの重要な読書のためのこの最後のプロトコルおよび博士シェリルイングラム·スミス、ケイティグレン、とグレースKisirkoiの開発に彼の有益な助言のために博士はLukasz Kozubowskiに感謝。
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 |
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