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. Bygging av LED-lys MERK: Den nødvendige LED utstyr er oppsummert i materialliste. Hele LED oppsettet er liten og kan plasseres hvor som helst i laboratoriet, men vi anbefaler den plasseres i et mørkt rom til å styre lys eksponering av ormer 4. <ol><li> Koble LED til en kontroller med kabler (figur 1A, D). </li><li> Koble kontrolleren til en digital funksjon generator / forsterker med en BNC-kabel (figur 1A, C, D). </li><li> Fest LED lys 10 cm over scenen ved h...

<ol>
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Her beskriver vi en detaljert fremgangsmåte for optogenetic mutagenese bruker Hans-mSOG uttrykt i germline og gi et eksempel på en liten skala frem genetisk skjermen. Sammenlignet med standard kjemisk mutagenese, har dette optogenetic mutagenese fremgangsmåte flere fordeler. For det første er det ikke giftig, og dermed holde lab personell unna giftige kjemiske mutagener som EMS, ENU og trimethylpsoralen med ultrafiolett lys (TMP / UV). For det andre kan den eksperimentelle prosedyren ifølge mutagenese benyttes for ...

Termin genetiske skjermer har blitt mye brukt til å generere genetiske mutanter forstyrre ulike biologiske prosesser i modellorganismer som Caenorhabditis elegans (C. elegans) 1. Analyser av slike mutanter føre til oppdagelse av funksjonelt viktige gener og deres signalveier 1,2. Tradisjonelt mutagenese i C. elegans oppnås ved bruk av mutagene kjemikalier, stråling, eller transposoner 2. Kjemikalier som etylmetansulfonat (EMS) og N-etyl-N -nitrosourea (ENU) er giftige for mennesker; gamma-ray eller ultrafiolett (UV) stråling mutagenese krever spesialutstyr; og transposon-aktive stammer som Mutator stammer 3, kan føre til unødvendige mutasjoner under vedlikehold. Vi har utviklet en enkel tilnærming til å indusere arve mutasjoner ved hjelp av en genetisk kodet fotosensibiliserende 4. Reaktive oksygenforbindelser (ROS) kan skade DNA fem.Mini Singlet Oxygen generator (miniSOG) er et grønt fluorescerende protein på 106 aminosyrer som ble konstruert fra LOV (lys, oksygen og spenning) domene av Arabidopsis phototropin 2 6. Ved eksponering for blått lys (~ 450 nm), miniSOG genererer ROS inkludert singlet oksygen med fl avin mononukleotid som en kofaktor 6-8, som er til stede i alle celler. Vi har konstruert en His-mSOG fusjonsproteinet ved å merke miniSOG til den C-terminale ende av histon-72, en C. elegans variant av Histone 3. Vi ga en løssalgs transgen 9 til å uttrykke Hans-mSOG i germline av C. elegans. Under normale dyrkningsforhold i mørke, Hans-mSOG transgene ormer har normal kullstørrelse og levetid fire. Ved eksponering for blå LED lys, Hans-mSOG ormer produserer arve mutasjoner blant deres avkom 4. Spekteret av induserte mutasjoner omfatter nucleotide endringer, for eksempel G: C til T: A og G: C til C: G og små chromosomeg slettinger fire. Dette mutagenese prosedyren er enkel å utføre, og krever minimalt med lab sikkerhet oppsett. Her beskriver vi LED-belysning oppsett og prosedyrer for optogenetic mutagenese.

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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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		<tr height="42">
			<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>
			<td style="width:184px;"></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>
			<td style="width:184px;"></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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		<tr height="21">
			<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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		<tr height="163">
			<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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		<tr height="88">
			<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>
		</tr>
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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 utførte en frem genetisk skjerm med CZ20310 juSi164 unc-119 (DE3) med 30 min lyseksponering etter standard protokoll beskrevet i punkt 2 og 3. Blant 60 F 1 plater som tilsvarer 120 mutageniserte haploide genomer, fant vi 8 F 1 plater med F 2 ormer viser synlige fenotyper som kroppsstørrelse defekt (figur 3B), ukoordinert bevegelse (figur 3C), og voksen dødelighet (Figur 3D). Vi gjorde ikke ...

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

Genetisk kodet histon-miniSOG induserer genom-wide arve mutasjoner i et blått lys avhengig måte. Dette mutagenese metoden er enkel, rask, fri for giftige kjemikalier, og godt egnet for videre genetisk screening og transgenet integrasjon.

Optogenetic Random Mutagenese Bruke Histone-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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