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. Construction de l&#39;illuminateur LED REMARQUE: L&#39;équipement LED requis est résumée dans la liste des matériaux. La configuration LED ensemble est petit et peut être placé n&#39;importe où dans le laboratoire, bien que nous recommandons qu&#39;il soit placé dans une pièce sombre pour contrôler l&#39; exposition la lumière des vers 4. <ol><li> Branchez le LED à un contrôleur avec des câbles (Figure 1A, D). </li><li> Connectez le contrôleur à une fonction gén?...

<ol>
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Ici, nous décrivons une procédure détaillée de la mutagenèse optogenetic utilisant His-MSOG exprimé dans la lignée germinale et fournir un exemple d&#39;un écran avant génétique à petite échelle. Par rapport à une mutagenèse chimique standard, ce procédé de mutagenèse optogenetic présente plusieurs avantages. Tout d&#39;abord, il est non toxique, gardant ainsi le personnel de laboratoire loin de mutagènes chimiques toxiques tels que EMS, ENU et triméthylpsoralène avec une lumière ultraviolette (TMP...

Écrans génétiques à terme ont été largement utilisés pour générer des mutants génétiques perturbant divers processus biologiques dans des organismes modèles tels que Caenorhabditis elegans (C. elegans) 1. Les analyses de ces mutants conduisent à la découverte de gènes fonctionnellement importants et de leur voies de signalisation 1,2. Traditionnellement, la mutagénèse en C. elegans est obtenue en utilisant des produits chimiques mutagènes, le rayonnement, ou transposons 2. Les produits chimiques tels que le méthanesulfonate d&#39;éthyle (EMS) et N - éthyl - N -nitrosourea (ENU) sont toxiques pour l&#39; homme; rayons gamma ou ultraviolet (UV) mutagenèse de rayonnement nécessite un équipement spécial; et les souches de transposons-actifs tels que des souches mutantes 3, peuvent causer des mutations inutiles lors de l&#39; entretien. Nous avons développé une approche simple pour induire des mutations héréditaires utilisant un photosensibilisant génétiquement codés 4. Reactive Oxygen Species (ROS) peuvent endommager l&#39; ADN 5.Le générateur de mini - Singlet Oxygen (miniSOG) est une protéine fluorescente verte de 106 acides aminés qui a été conçu à partir de la LOV (Light, oxygène et tension) domaine Arabidopsis phototropin 2 6. Lors de l&#39; exposition à la lumière bleue (~ 450 nm), miniSOG génère ERO comprenant de l&#39; oxygène singulet avec fl avine mononucléotide comme cofacteur 6-8, qui est présent dans toutes les cellules. Nous avons construit une protéine de fusion His-MSOG miniSOG par le marquage à l&#39; extrémité C-terminale de l&#39; histone-72, un C. elegans variante de Histone 3. Nous généré une seule copie transgène 9 pour exprimer His-MSOG dans la lignée germinale de C. elegans. Dans des conditions normales de culture dans l&#39;obscurité, les vers transgéniques His-MSOG ont la taille des couvées normale et la durée de vie 4. Lors de l&#39; exposition à la lumière LED bleue, les vers His-MSOG produisent des mutations héréditaires chez leur progéniture 4. Le spectre des mutations induites inclut des changements nucléotidiques, tels que G: C à T: A et G: C à C: G et petit chromosome suppressions 4. Cette procédure de mutagenèse est simple à réaliser et nécessite une configuration minimale de sécurité en laboratoire. Ici, nous décrivons la configuration et les procédures d&#39;éclairage LED pour la mutagenèse optogenetic.

<table border="0" cellpadding="0" cellspacing="0" width="626">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="46">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
	</tbody>
</table>

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.

Nous avons effectué un criblage génétique avant avec CZ20310 juSi164 unc-119 (ed3) en utilisant une exposition lumineuse de 30 min suivant le protocole standard décrit dans les sections 2 et 3. Parmi les 60 F 1 plaques correspondant à 120 génomes haploïdes mutagénisées, nous avons trouvé 8 F 1 plaques avec F 2 vers l&#39; affichage des phénotypes visibles comme défaut de la taille du corps (figure 3B), le mouvement no...

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

Génétiquement codés histone-miniSOG induit des mutations héréditaires du génome entier d&#39;une manière dépendant de la lumière bleue. Cette méthode de mutagenèse est simple, rapide, exempt de produits chimiques toxiques, et bien adapté pour le dépistage génétique avant et l&#39;intégration du transgène.

Optogénétiques mutagenèse aléatoire Utilisation Histone-miniSOG dans<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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