편의적 곰팡이 병원균에 형광 태그 유전자의과 유전자 변형<em> 크립토 콕 쿠스 네오 포르</em
Summary
유전자 총 변형은 상동 재조합을 통하여 기회 병원체 크립토 콕 쿠스 네오 포르 만 스의 게놈 DNA 내로 안정적으로 통합을 생성하는 데 사용되는 방법이다. 우리는 C. 콕 쿠스 네오 포르 만 스에 mCherry 형광 태그에 융합 된 유전자 부호화 아세테이트 키나아제를 갖는 구조, 유전자 총의 변화를 설명 할 것이다.
Abstract
담자균 크립토 콕 쿠스 네오 포르 만, 중추 신경계의 침입 기회 병원균은 전 세계적으로 전 세계적으로 연간 이상 625,000 명이 사망 진균 성 수막염의 가장 흔한 원인이다. 전기 천공은 크립토에서 플라스미드의 형질 전환을 위해 개발되었지만, 오직과 유전자 전달은 상동 재조합에 의해 게놈 내로 통합 될 수있는 선형 DNA를 변환하는 효과적인 수단을 제공한다.
아세트산 크립토 감염시 중요한 발효 산물 것으로 도시 하였지만, 이것의 의미는 아직 알려져 있지 않다. 효소 크실 로스 -5- 포스페이트 / 프 룩토 오스 -6- 포스페이트 phosphoketolase (XFP 광고) 및 아세테이트 키나아제 (ACK)로 구성된 세균 통로 C. 아세테이트의 제조를위한 세 개의 가능한 경로 중 하나이다 콕 쿠스 네오 포르 만. 여기, 우리는 구조의과 유전자 변형을 보여,긍정 응답을 코딩하는 유전자는 mCherry 형광 태그에 융합시킨 C. 콕 쿠스 네오 포르 만. 우리는 그 다음 ACK 궤적에 ACK -mCherry 융합의 통합을 확인합니다.
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
이 작품은 국립 과학 재단 (National Science Foundation 상 # 0920274)와 사우스 캐롤라이나 실험 역 프로젝트 SC-1700340에서 수상에 의해 지원되었다. 클렘 슨 대학 시험장이 종이 isTechnical 공헌 호 6283. 저자는 마지막 프로토콜과 원고의 비판적 읽기 박사 셰릴 잉그램 스미스, 케이티 글렌, 그레이스 Kisirkoi의 개발에 그의 도움이 조언 박사 루카스 Kozubowski 감사합니다.
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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