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. Costruzione del illuminatore LED NOTA: L&#39;attrezzatura necessaria a LED è riassunto nella lista dei materiali. L&#39;intera impostazione del LED è piccolo e può essere posizionato in qualsiasi punto in laboratorio, anche se si consiglia di riporla in una stanza buia per controllare la luce l&#39;esposizione di vermi 4. <ol><li> Collegare il LED ad un controllore con cavi (Figura 1A, D). </li><li> Collegare il controller per una funzione generatore / amplificatore digitale c...

<ol>
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Qui, descriviamo una procedura dettagliata di mutagenesi optogenetic usando la sua-mSOG espresso nella linea germinale e fornire un esempio di uno screening genetico in avanti su piccola scala. Rispetto alla mutagenesi chimica standard, questo metodo di mutagenesi optogenetic ha diversi vantaggi. In primo luogo, non è tossico, mantenendo in tal modo il personale di laboratorio da mutageni chimici tossici come SME, ITA e trimethylpsoralen con la luce ultravioletta (TMP / UV). In secondo luogo, la procedura sperimentale ...

Schermi genetici a termine sono stati ampiamente utilizzati per generare mutanti genetici che perturbano vari processi biologici in organismi modello come il Caenorhabditis elegans (C. elegans) 1. Le analisi di questi mutanti portano alla scoperta dei geni funzionalmente importanti e loro vie di segnalazione 1,2. Tradizionalmente, mutagenesi in C. elegans si ottiene utilizzando prodotti chimici mutageni, radiazioni, o trasposoni 2. Sostanze chimiche come metanosolfonato etile (EMS) e N -ethyl- N -nitrosourea (ITA) sono tossici per l&#39;uomo; raggi gamma o raggi ultravioletti (UV) mutagenesi richiede attrezzature speciali; e ceppi trasposoni-attivi, come ad esempio ceppi mutatori 3, possono causare mutazioni inutili durante la manutenzione. Abbiamo sviluppato un approccio semplice per indurre mutazioni ereditarie con un fotosensibilizzante geneticamente codificato 4. Specie reattive dell&#39;ossigeno (ROS) possono danneggiare il DNA 5.Il generatore mini ossigeno singoletto (miniSOG) è una proteina fluorescente verde di 106 aminoacidi che è stato progettato dalla LOV (luce, ossigeno, e tensione) del dominio di Arabidopsis phototropin 2 6. Dopo l&#39;esposizione alla luce blu (~ 450 nm), miniSOG genera ROS compreso l&#39;ossigeno singoletto con fl avin mononucleotide come cofattore 6-8, che è presente in tutte le cellule. Abbiamo costruito una proteina di fusione His-mSOG etichettando miniSOG al C-terminale dell&#39;istone-72, un C. elegans variante dell&#39;istone 3. Abbiamo generato una singola copia transgene 9 per esprimere la sua-mSOG nella linea germinale di C. elegans. In condizioni di coltura normali nel buio, i vermi transgenici His-mSOG hanno dimensioni normali covata e durata 4. Al momento l&#39;esposizione alla luce blu del LED, i vermi His-mSOG producono mutazioni ereditarie tra loro progenie 4. Lo spettro di mutazioni indotte include cambiamenti nucleotide, come ad esempio G: C a T: A e G: C a C: G, e piccoli chromosoMi eliminazioni 4. Questa procedura mutagenesi è semplice da eseguire, e richiede l&#39;installazione minima di sicurezza del laboratorio. Qui, descriviamo l&#39;installazione di illuminazione a LED e le procedure per la mutagenesi optogenetic.

<table border="0" cellpadding="0" cellspacing="0" width="626">
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	<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.

Abbiamo condotto uno screening genetico in avanti con CZ20310 juSi164 unc-119 (ED3) con l&#39;esposizione alla luce 30 minuti seguendo il protocollo standard descritto nelle sezioni 2 e 3. Tra 60 F 1 piastre corrispondenti a 120 genomi aploidi mutagenizzate, abbiamo trovato 8 F 1 piatti con F 2 vermi che mostrano fenotipi visibili, come difetti dimensioni del corpo (figura 3B), il movimento scoordinato (Figura 3C),</stron...

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

Geneticamente codificato istone-miniSOG induce genome-wide mutazioni ereditarie in un modo dipendente dalla luce blu. Questo metodo mutagenesi è semplice, veloce, privo di sostanze chimiche tossiche, e ben si adatta per lo screening genetico in avanti e l&#39;integrazione del transgene.

Optogenetic caso Mutagenesi Utilizzando istone-miniSOG in<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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