Optogenetic Tilfældig mutagenese ved anvendelse histon-miniSOG i<em> C. elegans</em
Summary
Genetisk kodet histon-miniSOG inducerer genom-dækkende arvelige mutationer i et blåt lys-afhængig måde. Denne mutagenese metode er enkel, hurtig, fri for giftige kemikalier, og velegnet til frem genetisk screening og transgen integration.
Abstract
Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.
Introduction
Fremad genetiske skærme er ofte blevet brugt til at generere genetiske mutanter forstyrre forskellige biologiske processer i modelorganismer såsom Caenorhabditis elegans (C. elegans) 1. Analyser af sådanne mutanter føre til opdagelsen af funktionelt vigtige gener og deres signalveje 1,2. Traditionelt mutagenese i C. elegans opnås ved anvendelse af mutagene kemikalier, stråling eller transposoner 2. Kemikalier såsom -ethylmethansulfonat (EMS) og N-ethyl-N -nitrosourea (ENU) er giftige for mennesker; gamma-ray eller ultraviolet (UV) stråling mutagenese kræver særligt udstyr; og transposon-aktiv stammer, såsom mutatorstammer 3, kan forårsage unødvendige mutationer under vedligeholdelse. Vi har udviklet en enkel fremgangsmåde til at fremkalde arvelige mutationer under anvendelse af en genetisk kodet fotosensibilisator 4.
Reaktive Oxygen Species (ROS) kan skade DNA 5.Mini Singlet Oxygen Generator (miniSOG) er et grønt fluorescerende protein af 106 aminosyrer, som blev manipuleret fra LOV (Light, oxygen og spænding) domæne af Arabidopsis phototropin 2 6. Ved udsættelse for blåt lys (~ 450 nm), miniSOG genererer ROS herunder singlet oxygen med fl avin mononukleotid som en cofaktor 6-8, som er til stede i alle celler. Vi konstruerede et His-mSOG fusionsproteinet ved tagging miniSOG til C-terminalen af histon-72, en C. elegans variant af histon 3. Vi genererede et enkelt eksemplar transgen 9 til at udtrykke His-mSOG i kimcellelinje C. elegans. Under normale dyrkningsbetingelser i mørke, de His-mSOG transgene orme har normal yngel størrelse og levetid 4. Ved udsættelse for blå LED lys, His-mSOG orme producerer arvelige mutationer blandt deres afkom 4. Spektret af inducerede mutationer omfatter nukleotidændringer, såsom G: C til T: A og G: C til C: G, og små chromosomig deletioner 4. Denne mutagenese procedure er enkel at udføre, og kræver minimal opsætning lab sikkerhed. Her beskriver vi LED belysning setup og procedurer for optogenetic mutagenese.
Protocol
Representative Results
Discussion
Her beskriver vi en detaljeret procedure for optogenetic mutagenese hjælp His-mSOG udtryk i kimcellelinje og giver et eksempel på en lille skala fremad genetisk skærm. Sammenlignet med standard kemisk mutagenese, denne optogenetic mutagenese metode har flere fordele. For det første er ikke-toksisk og dermed holde lab personale væk fra toksiske kemiske mutagener, såsom EMS, ENU og trimethylpsoralen med ultraviolet lys (TMP / UV). For det andet kan den eksperimentelle fremgangsmåde for mutagenese anvendes til et li…
Declarações
The authors have nothing to disclose.
Acknowledgements
The work is supported by HHMI. We thank our lab members for their help with testing the protocol and revising the manuscript.
Materials
| Ultra High Power LED light source | Prizmatix | UHP-mic-LED-460 | 460 ± 5 nm |
| LED controller | Prizmatix | UHPLCC-01 | |
| Digital function generator/amplifier | PASCO | PI-9587C | PI-9587C is no longer available. The replacement is PI-8127. |
| BNC cable male/male | THORLABS | CA3136 | |
| USB-TTL interface | Prizmatix | Optional | |
| Photometer | THORLABS | PM50 and Model D10MM | |
| Filter paper | Whatman | 1001-110 | |
| Copper chloride dihydrate (CuCl2∙2H2O) | Sigma | C6641 | |
| Stereomicroscope | Leica | MZ95 | |
| NGM plate | Dissolve 5g NaCl, 2.5g Peptone, 20g Agar, 10 µg/ml cholesterol in 1 L H2O. After autoclaving, add 1 mM CaCl2, 1 mM MgSO4, 25 mM KH2PO4(pH6.0). | ||
| Lysis solution | 10 mM Tris(pH8.8), 50 mM KCl, 0.1% Triton X100, 2.5 mM MgCl2, 100 µg/ml Proteinase K |
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