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.

LEDイルミの1建設注：必要なLED装置は、材料のリストにまとめられています。全体LEDセットアップは小さく、我々はそれがワーム4の露光量を制御するために、暗い部屋に置くことをお勧めしますが、ラボのどこにでも配置することができます。 <ol><li>ケーブル（ 図1A、D）でコントローラにLEDを接続します。 </li><li> BNCケーブルでデジタルファンクションジェネレー...

<ol>
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ここで、我々は彼の-mSOGを使用して、光遺伝学の突然変異誘発の詳細な手順を説明する生殖細胞系列で発現し、小規模前方遺伝画面の例を提供します。標準的な化学的突然変異誘発と比較して、この光遺伝学突然変異誘発方法は、いくつかの利点を有します。第一に、それによって離れて、このようなEMS、ENUなどの有害化学変異誘発物質からラボの人材を維持し、紫外線（TMP / UV）とトリメチ...

フォワード遺伝子スクリーニングは、広くそのような線虫（C.エレガンス）1などのモデル生物において様々な生物学的プロセスを破壊する遺伝子変異体を生成するために使用されてきました。このような変異体の分析は、機能的に重要な遺伝子およびそれらのシグナル伝達経路1,2の発見につながります。 C.で伝統的に、突然変異誘発エレガンスは、変異原性化学物質、放射線、またはトランスポゾン2を用いて達成されます。このようなメタンスルホン酸エチル（EMS）とN -エチル- Nの -nitrosourea（ENU）などの化学物質がヒトに対して毒性があります。ガンマ線または紫外線（UV）放射線変異誘発特別な装置を必要とします。そして、このような突然変異誘発株3、などのトランスポゾン活性株は、保守時に不必要な突然変異を引き起こす可能性があります。我々は遺伝的にコード光増感剤4を用いて、遺伝的変異を誘発するための簡単なアプローチを開発しました。 活性酸素種（ROS）DNA 5に損傷を与えることができます。ミニ一重項酸素発生器（miniSOG）がLOV（光、酸素、及び電圧）2 6フォトトロピンシロイヌナズナのドメインから設計された106個のアミノ酸の緑色蛍光タンパク質である。青色光（〜450 nm）のに曝されると、miniSOG ROSは、すべての細胞に存在する補因子6-8としてFL AVINモノ、と一重項酸素を含む生成されます。我々は、ヒストン-72、CのC末端にminiSOGをタグ付けすることによって彼の-mSOG融合タンパク質を構築しましたヒストン3のエレガンス変異体は、私たちはCの生殖細胞系列に彼-mSOGを表現するために単一コピーの導入遺伝子9を生成しましたエレガンス。暗闇の中で、通常の培養条件の下では、彼の-mSOGトランスジェニックワームは、通常のひなのサイズと寿命4を持っています。青色LEDの光に曝されると、彼の-mSOGワームは、それらの子孫4間の遺伝的変異を生成します。誘導された突然変異のスペクトルは、Gなどのヌクレオチド変化を含む：TからC：AとG：CからC：G、および小chromoso私は4を欠失。この突然変異誘発手順は実行が簡単で、かつ最小限のラボの安全設定が必要です。ここでは、LED照明のセットアップと光遺伝学突然変異誘発のための手順について説明します。

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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>
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		<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>
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		<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.

私たちは8 F 1のプレートを見つけ、120変異誘発半数体ゲノムに対応する60 F 1のプレートのうち、第2条及び3に記載されている標準的なプロトコルに従って、30分間の露光を用いて、（ED3）CZ20310 juSi164 UNC-119とフォワード遺伝学的スクリーニングを行いましたこのようなボディサイズの欠陥（ 図3B）、非協調運動（ 図3C）、...

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

遺伝的にコードされたヒストンminiSOGは、青色光依存的にゲノムワイドな遺伝変異を誘発します。この突然変異誘発法は、高速で、単純な有毒化学物質の自由、およびフォワード遺伝子スクリーニングおよび導入遺伝子組込みに適しています。

でヒストンminiSOGを使用した光遺伝学ランダム突然変異誘発<em&gt; C。エレガンス</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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