Name: optogenetic random mutagenesis using histone-minisog in c. elegans
Uploaded: 2016-11-14T15:00:00
Description: Watch this Scientific Journal Video about Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans at JoVE.com

The work is supported by HHMI. We thank our lab members for their help with testing the protocol and revising the manuscript.

1. Konstruktion av LED-lampan OBS: Den erforderliga LED utrustning sammanfattas i förteckningen över material. Hela LED installationen är liten och kan placeras var som helst i labbet, men vi rekommenderar det placeras i ett mörkt rum för att styra ljusexponering av maskar 4. <ol><li> Anslut LED till en styrenhet med kablar (Figur 1A, D). </li><li> Anslut kontrollenheten till en digital funktionsgenerator / förstärkare med en BNC-kabel (Figur 1A, C, D). </li>...

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Här beskriver vi ett detaljerat förfarande för optogenetic mutagenes med hjälp av His-marknadsövervakningsgruppen uttryckt i könsceller och ge ett exempel på en småskalig framåt genetisk skärm. Jämfört med standard kemisk mutagenes, har denna optogenetic mutagenes metod flera fördelar. För det första är det giftfritt, varigenom laboratoriepersonal från giftiga kemiska mutagener, såsom EMS, ENU och trimetylpsoralen med ultraviolett ljus (TMP / UV). För det andra kan den experimentella proceduren för m...

Framåt genetiska skärmar har använts i stor utsträckning för att generera genetiska mutanter störa olika biologiska processer i modellorganismer såsom Caenorhabditis elegans (C. elegans) 1. Analyser av sådana mutanter att leda till upptäckten av funktionellt viktiga gener och deras signalvägar 1,2. Traditionellt, mutagenes i C. elegans uppnås med hjälp av mutagena kemikalier, strålning eller transposoner 2. Kemikalier såsom etylmetansulfonat (EMS) och N-etyl-N -nitrosourea (ENU) är giftiga för människor; gamma-ray eller ultraviolett (UV) strålning mutagenes kräver speciell utrustning; och transposon-aktiva stammar, såsom mutatorstammar 3, kan orsaka onödiga mutationer vid underhåll. Vi har utvecklat en enkel metod för att orsaka ärftliga mutationer med hjälp av en genetiskt kodad fotosensibiliserare 4. Reaktiva syreradikaler (ROS) kan skada DNA 5.Mini Singlet Oxygen Generator (miniSOG) är ett grönt fluorescerande protein av 106 aminosyror som var konstruerad från LOV (ljus, syre och spänning) domän av Arabidopsis phototropin 2 6. Vid exponering för blått ljus (~ 450 nm), miniSOG genererar ROS inklusive singlettsyre med fl avin mononukleotiden som en kofaktor 6-8, som är närvarande i alla celler. Vi konstruerade en His-marknadsövervakningsgruppen fusionsprotein genom att märka miniSOG till C-terminalen av histon-72, ett C. elegans variant av Histon 3. Vi genererade en enda kopia transgen 9 att uttrycka His-marknadsövervakningsgruppen i nedärvda C. elegans. Under normala odlingsförhållanden i mörker, de His-marknadsövervakningsgruppen transgena maskarna har normal kull storlek och livslängd 4. Vid exponering för blått LED-ljus, de His-marknadsövervakningsgruppen maskar producera ärftliga mutationer bland deras avkomma 4. Spektrumet av inducerade mutationer inkluderar nukleotid förändringar, såsom G: C till T: A och G: C till C: G, och små chromosomig deletioner 4. Denna mutagenes Proceduren är enkel att utföra och kräver minimal lab säkerhetsinställning. Här beskriver vi den LED-belysning setup och förfaranden för optogenetic mutagenes.

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		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Ultra High Power LED light source</td>
			<td style="width:71px;">Prizmatix</td>
			<td style="width:115px;">UHP-mic-LED-460</td>
			<td style="width:184px;">460 &#177; 5 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">LED controller</td>
			<td style="width:71px;">Prizmatix</td>
			<td style="width:115px;">UHPLCC-01</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:256px;">Digital function generator/amplifier</td>
			<td style="width:71px;">PASCO</td>
			<td style="width:115px;">PI-9587C</td>
			<td style="width:184px;">PI-9587C is no longer available. The replacement is PI-8127.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">BNC cable male/male</td>
			<td style="width:71px;">THORLABS</td>
			<td style="width:115px;">CA3136</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">USB-TTL interface</td>
			<td style="width:71px;">Prizmatix</td>
			<td style="width:115px;"></td>
			<td style="width:184px;">Optional</td>
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			<td height="42" style="height:42px;width:256px;">Photometer</td>
			<td style="width:71px;">THORLABS</td>
			<td style="width:115px;">PM50 and Model D10MM</td>
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			<td height="21" style="height:21px;width:256px;">Filter paper</td>
			<td style="width:71px;">Whatman</td>
			<td style="width:115px;">1001-110</td>
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			<td height="46" style="height:46px;width:256px;">Copper chloride dihydrate (CuCl2&#8729;2H2O)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:115px;">C6641</td>
			<td style="width:184px;"></td>
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			<td height="21" style="height:21px;width:256px;">Stereomicroscope</td>
			<td style="width:71px;">Leica</td>
			<td style="width:115px;">MZ95</td>
			<td style="width:184px;"></td>
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			<td height="163" style="height:163px;width:256px;">NGM plate</td>
			<td style="width:71px;"></td>
			<td style="width:115px;"></td>
			<td style="width:184px;">Dissolve 5g NaCl, 2.5g Peptone, 20g Agar, 10 &#181;g/ml cholesterol in 1 L H2O. After autoclaving, add 1 mM CaCl2, 1 mM MgSO4, 25 mM KH2PO4(pH6.0).&#160;</td>
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			<td height="88" style="height:88px;width:256px;">Lysis solution</td>
			<td style="width:71px;"></td>
			<td style="width:115px;"></td>
			<td style="width:184px;">10 mM Tris(pH8.8), 50 mM KCl, 0.1% Triton X100, 2.5 mM MgCl2, 100 &#181;g/ml Proteinase K</td>
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optogenetic random mutagenesis using histone-minisog in c. elegans

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.

Vi gjorde en framåt genetisk skärm med CZ20310 juSi164 UNC-119 (ED3) med användning av 30 min ljusexponering efter standardprotokoll som beskrivs i avsnitten 2 och 3. Bland 60 F 1 plattor motsvarande 120 muterade haploida genomet har vi hittat 8 F 1 plattor med F 2 maskar visar synliga fenotyper såsom kroppsstorlek defekt (figur 3B), okoordinerade rörelser (figur 3C), och vuxna dödlighet (Figur 3D)....

Watch this Scientific Journal Video about Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans at JoVE.com

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetiskt kodade histon-miniSOG inducerar genomet hela ärftliga mutationer i en blå ljusberoende sätt. Denna mutagenes Metoden är enkel, snabb, fri från giftiga kemikalier, och lämpar sig väl för fram genetisk screening och transgen integration.

Optogenetic Slumpmässig mutagenes med användning Histon-miniSOG i<em&gt; C. elegans</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

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Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

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