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 of the LED Illuminator
NOTE: The required LED equipment is summarized in the List of Materials. The entire LED setup is small and can be placed anywhere in the lab, although we recommend it be placed in a dark room to control light exposure of worms4.
<ol>
	<li>Connect the LED to a controller with cables (Figure&#160;1A,&#160;D).</li>
	<li>Connect the controller to a digital function generator/amplifier with a BNC cable (Figure 1A, C, D).</li>
	<li>Fix...

<ol>
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Here, we describe a detailed procedure of optogenetic mutagenesis using His-mSOG expressed in the germline and provide an example of a small scale forward genetic screen. Compared to standard chemical mutagenesis, this optogenetic mutagenesis method has several advantages. Firstly, it is nontoxic, thereby keeping lab personnel away from toxic chemical mutagens such as EMS, ENU and trimethylpsoralen with ultraviolet light (TMP/UV). Secondly, the experimental procedure of mutagenesis can be used for a small number of the P...

Forward genetic screens have been widely used to generate genetic mutants disrupting various biological processes in model organisms such as Caenorhabditis elegans (C. elegans)1. Analyses of such mutants lead to the discovery of functionally important genes and their signaling pathways1,2. Traditionally, mutagenesis in C. elegans is achieved using mutagenic chemicals, radiation, or transposons2. Chemicals such as ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) are toxic to humans; gamma-ray or ultraviolet (UV) radiation mutagenesis requires special equipment; and transposon-active strains, such as mutator strains3, can cause unnecessary mutations during maintenance. We have developed a simple approach to induce heritable mutations using a genetically-encoded photosensitizer4.
Reactive Oxygen Species (ROS) can damage DNA5. The mini Singlet Oxygen Generator (miniSOG) is a green fluorescent protein of 106 amino acids that was engineered from the LOV (Light, Oxygen, and Voltage) domain of Arabidopsis phototropin 2 6. Upon exposure to blue light (~450 nm), miniSOG generates ROS including singlet oxygen with &#64258;avin mononucleotide as a cofactor6-8, which is present in all cells. We constructed a His-mSOG fusion protein by tagging miniSOG to the C-terminus of histone-72, a C. elegans variant of Histone 3. We generated a single-copy transgene9 to express His-mSOG in the germline of&#160;C. elegans. Under normal culture conditions in the dark, the His-mSOG transgenic worms have normal brood size and life span4. Upon exposure to blue LED light, the His-mSOG worms produce heritable mutations among their progeny4. The spectrum of induced mutations includes nucleotide changes, such as G:C to T:A and G:C to C:G, and small chromosome deletions4. This mutagenesis procedure is simple to perform, and requires minimal lab safety setup. Here, we describe the LED illumination setup and procedures for optogenetic mutagenesis.

<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.

We performed a forward genetic screen with CZ20310 juSi164 unc-119(ed3) using 30 min light exposure following the standard protocol described in Sections 2 and 3. Among 60 F1 plates corresponding to 120 mutagenized haploid genomes, we found 8 F1 plates with F2 worms displaying visible phenotypes such as body size defect (Figure 3B), uncoordinated movement (Figure 3C), and adult lethality (Figure 3D</strong...

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

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

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 ...

The overall goal of this mutagenesis protocol is to use a simple LED set-up to mutagenize the genomic DNA of C.elegans that express blue light-dependent, reactive oxygen generator proteins. The two main advantages of this technique are one, the mutagenesis protocol is very simple and do not require the toxic chemicals. And two, the mutagenesis generator proves after a long mutation.I first have the idea for this method, and I realize that ROS-generating proteins have never been used to induce heritable mutations. For this experiment, connect the controller of the LED illuminator device to a digital function generator using a BNC cable. Using a custom holder, secure the LED lights 10 centimeters above the stage.Now, partially cover the set-up to limit exposure of the blue light to the surroundings, but not so much that heat will accumulate on the stage. A custom made, hard plastic cover with an open top and bottom is employed here. During the LED's operation, always wear laser-protective glasses or sunglasses, because the light is very bright.Now, switch the lever of the LED controller to internal, and set it to 65%of its maximum power. Using a photometer, adjust the light's power if necessary, so that on the stage, it is 2.0 milliwatts per square millimeter during continuous illumination. For this protocol, maintain the light-sensitive strain in the dark under standard conditions.Unnecessary light exposure should be avoided, but routine halogen lamp use for passage, does not result in mutations. Because accidental mutation is a concern, freeze aliquots of the strain when they are first obtained. For the mutagenesis, prepare a filter paper to fit a 60 millimeter plate with an approximately 25 millimeter square hole at the center, and place it onto an unseeded plate.Then, distribute 100 microliters of 100 millimolar copper chloride evenly over the filter paper to keep the worms from leaving the square hole. Now, from a plate of worms, that are about 12 hours after their L4 stage, pick the gravid, young adult hermaphrodites and put them into the center square on the plate. Next, turn on the LED and the function generator.Then, switch the LED controller to external to control it with the function generator, and set the power appropriately. From the function generator, produce a sine wave at 4 hertz. Now, transfer the plate to the stage and illuminate the worms with blue light for 30 minutes.Immediately after the treatment, transfer all the healthy looking worms to a seeded plate. These worms will appear uncoordinated after the light treatment, but they will recover. Keep the light-treated worms in the dark.Later, check for abnormal morphologies or behaviors in the adult F2 progeny. A forward genetic screen was performed using the described technique for 60 F1 plates, corresponding to 120 mutagenized haploid genomes. Eight of the 60 F1 plates with F2 worms displayed visible phenotypes, such as dumpy body shape, uncoordinated movement, or adult lethality.The progeny of these F2 worms retained these observable phenotypes. The number of visible mutants from this screen suggests that the strain and set-up are working properly for optogenetic mutagenesis. The mutagenesis procedure can be done within an hour.Please enjoy the fast and safe mutagenesis method.

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