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.在LED照明的构建注：需要的LED设备中总结材料的清单。整个LED的设置是小，可以在实验室中的任何位置，但我们建议它被放置在一个黑暗的房间，以控制蠕虫4的曝光。 <ol><li>连接LED与电缆（ 图1A，D）的控制器。 </li><li>该控制器连接到用一根BNC电缆（ 图1A，C，D）的数字函数发生器/放大器。 </li><li>固定LED灯10厘米级以上使用定制支架（ 图1A）。</str...

<ol>
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在这里，我们描述了使用光遗传学诱变的详细流程的His-mSOG在种系表达并提供一个小规模向前遗传画面的一个例子。相比标准化学诱变，此光遗传学诱变方法具有几个优点。首先，它是无毒的，从而保持实验室人员从有毒的化学诱变剂如EMS，ENU路程，用紫外光（TMP / UV）trimethylpsoralen。其次，诱变的实验程序可用于一个小数目为P 0的动物，并且可以在一小时内完成。第三，光遗传学诱变产?...

向前遗传筛选已广泛用来产生遗传突变破坏在模式生物的各种生物过程，如秀丽隐杆线虫（线虫）1。这些突变体的分析导致功能重要基因的发现及其信号通路1,2。传统上，诱变C.线虫使用诱变化学物质，辐射或转座子2来实现的。化学品，如甲磺酸乙酯（EMS）和N-乙基-N- -nitrosourea（ENU）是对人体有毒;伽玛射线或紫外线（UV）辐射诱变需要特殊的设备;和转座子活性菌株，例如突变株3，可在维护期间造成不必要的突变。我们已经开发出一种简单的方法来诱导用基因编码的光敏剂4遗传突变。 活性氧（ROS）可以破坏DNA 5。微型单线态氧发生器（miniSOG）是一个从所述LOV（光，氧气，和电压）工程106氨基酸拟南芥向光2 6的结构域的绿色荧光蛋白。在暴露于蓝光（〜450纳米），miniSOG产生活性氧包括与FL AVIN单核苷酸单线态氧作为辅因子6-8，这是存在于所有细胞中。我们通过标记miniSOG到组蛋白72，C的C末端构成一的His-mSOG融合蛋白组蛋白3. 线虫变种我们产生了一个单拷贝转基因9表达他-mSOG在C的生殖细胞线虫，在黑暗中正常培养条件下，他的-mSOG转基因蠕虫具有正常的育雏大小和寿命4。在暴露于蓝光LED灯，在他的-mSOG蠕虫产生它们的后代中4遗传突变。诱发突变的频谱包括核苷酸变化，如G：C至T的：A和G：C 1至C：G和小chromoso我删除4。这种诱变方法是简单的执行，且只需最少实验室安全设置。这里，我们描述了LED照明设备和程序为光遗传学诱变。

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

我们用下面的第2和第3，其中对应于120诱变单倍体基因组的60架F 1板中描述的标准协议，30分钟曝光进行与CZ20310 juSi164 UNC-119（ED3）正向遗传筛选，我们发现8 F 1板带F 2蠕虫显示可见的表型，如身体尺寸缺陷（ 图3B），运动不协调（ 图3C），和成人杀伤力（ 图3D）。我们没有注意到在&#39;第一天“和”第2天...

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

基因编码的蛋白，miniSOG诱导蓝光依赖性全基因组的遗传突变。这种突变方法简单，快速，免费的有毒化学物质，并非常适用于正向遗传学筛查和转基因整合。

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</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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