שינוי Biolistic של פלורסנט Tagged Gene לOpportunistic פטרייתיים פתוגן<em> Neoformans קריפטוקוקוס</em
Summary
שינוי Biolistic הוא שיטה המשמשת ליצירת אינטגרציה יציבה של DNA לתוך הגנום של neoformans הפתוגן קריפטוקוקוס האופורטוניסטי באמצעות רקומבינציה ההומולוגית. נדגים שינוי biolistic של מבנה, שבו יש קינאז אצטט גן המקודד התמזג mCherry תג הניאון לneoformans ג.
Abstract
Basidiomycete neoformans קריפטוקוקוס, הפתוגן האופורטוניסטי פולשנית של מערכת העצבים המרכזית, הוא הסיבה השכיחה ביותר לדלקת קרום המוח פטרייתי בעולם וכתוצאה מכך יותר מ 625,000 מקרי מוות בשנה ברחבי העולם. למרות electroporation פותח להפיכתו של פלסמידים בקריפטוקוקוס, רק משלוח biolistic מספק אמצעי יעיל כדי להפוך את ה- DNA ליניארי שניתן לשלב לתוך הגנום ידי רקומבינציה ההומולוגית.
אצטט הוכח להיות מוצר תסיסה גדול בזיהום cryptococcal, אבל המשמעות של זה עדיין לא ידוע. מסלול חיידקים המורכב מהאנזימים xylulose-5-פוספט / פרוקטוז-6-פוספט phosphoketolase (XFP) וקינאז אצטט (Ack) הוא אחד משלושה מסלולים אפשריים לייצור אצטט בג neoformans. הנה, אנחנו מדגימים את שינוי biolistic של מבנה,שגן קידוד Ack שהתמזג לתג פלורסנט mCherry, לג neoformans. לאחר מכן, אנו מאשרים שילוב של היתוך ACK -mCherry למוקד 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
עבודה זו נתמכה על ידי מענקים מהקרן הלאומית למדע (פרס # 0,920,274) וניסוי הדרום קרוליינה תחנת פרויקט SC-1,700,340. נייר תרומת מס 'זה isTechnical 6283 של תחנת ניסיונות אוניברסיטת קלמסון. המחברים מודים לד"ר לוקאש Kozubowski לעצות מועילות שלו בפיתוח של פרוטוקול זה סופי ושריל ד"ר אינגרם-סמית, קייטי גלן, וגרייס Kisirkoi לקריאה הביקורתית שלהם של כתב היד.
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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