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. De bouw van de LED-verlichting LET OP: De benodigde LED-apparatuur wordt samengevat in de lijst van materialen. De gehele LED setup is klein en kan overal in het laboratorium worden geplaatst, hoewel we aan in een donkere kamer aan licht blootgesteld worms 4 controle worden geplaatst. <ol><li> Sluit de LED aan een controller met kabels (Figuur 1A, D). </li><li> Sluit de stuureenheid om een digitale functiegenerator / versterker met een BNC kabel (Figuur 1A, C, D).</strong...

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Hier beschrijven we een gedetailleerde procedure van optogenetic mutagenese met behulp van His-MSOG tot uitdrukking in de kiembaan en geven een voorbeeld van een kleinschalige forward genetische screen. Vergeleken met standaard chemische mutagenese, deze optogenetic mutagenese werkwijze heeft verschillende voordelen. Ten eerste is het niet-toxisch, daarmee had het laboratoriumpersoneel verwijderd van toxische chemische mutagenen zoals EMS, ENU en trimethylpsoraleen met ultraviolet licht (TMP / UV). Ten tweede kan de exp...

Genetische screens zijn op grote schaal gebruikt om genetische mutanten verstoren diverse biologische processen in modelorganismen zoals Caenorhabditis elegans (C. elegans) 1 genereren. Analyses van dergelijke mutanten leiden tot de ontdekking van functioneel belangrijke genen en hun signaalwegen 1,2. Traditioneel mutagenese in C. elegans wordt bereikt met mutagene chemicaliën, straling of transposons 2. Chemicaliën zoals ethylmethaansulfonaat (EMS) en N-ethyl-N -nitrosourea (ENU) zijn giftig voor de mens; gammastraling of ultraviolet (UV) stralingsmutagenese vereist speciale apparatuur; en transposon-actieve stammen, zoals mutator stammen 3, kan onnodige mutaties veroorzaken tijdens onderhoud. We hebben een eenvoudige benadering van erfelijke mutaties met behulp van een genetisch-gecodeerde fotosensibilisator 4 induceren ontwikkeld. Reactive oxygen species (ROS) kan DNA 5 beschadigen.De mini-singlet zuurstof Generator (miniSOG) is een groen fluorescerend eiwit van 106 aminozuren dat werd ontworpen door de LOV (licht, zuurstof, en Voltage) domein van Arabidopsis phototropin 2 6. Bij blootstelling aan blauw licht (~ 450 nm), miniSOG genereert ROS zoals singlet zuurstof met fl avin mononucleotide als cofactor 6-8, die in alle cellen. Construeerden wij een His-fusie-eiwit door MSOG tagging miniSOG aan de C-terminus van histon-72, een C. elegans variant van Histon 3. We genereerden een losse transgene 9 tot en met His-MSOG drukken in de kiemlijn van C. elegans. Onder normale kweekomstandigheden in het donker, het His-MSOG transgene wormen normale broedsel grootte en levensduur 4. Bij blootstelling aan blauw LED-licht, het His-MSOG wormen produceren erfelijke mutaties onder hun nageslacht 4. Het spectrum van geïnduceerde mutaties omvat nucleotide veranderingen, zoals G: C tot T: A en G: C C: G, en kleine chromosome deleties 4. Dit mutagenese procedure is eenvoudig uit te voeren, en vereist een minimale veiligheid lab setup. We beschrijven hier de LED verlichting setup en procedures voor optogenetic mutagenese.

<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>
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		<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>
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			<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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			<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>
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			<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>
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			<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>
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		<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 voerden een voorwaartse genetische screen met CZ20310 juSi164 unc-119 (ED3) met behulp van 30 minuten blootstelling aan licht na het standaardprotocol in de afdelingen 2 en 3 beschreven onder 60 F 1-platen die overeenkomt met 120 gemutageniseerde haploïde genomen, vonden we 8 F 1 platen met F 2 wormen weergave zichtbaar fenotypes zoals lichaamsgrootte defect (Figuur 3B), ongecoördineerde beweging (figuur 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

Genetisch gecodeerde histon-miniSOG induceert genoom-brede erfelijke mutaties in een blauw licht-afhankelijke manier. Dit mutagenese methode is eenvoudig, snel, vrij van giftige chemicaliën, en zeer geschikt voor forward genetische screening en transgene integratie.

Optogenetische willekeurige mutagenese met behulp van histon-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 ...

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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Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

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