The work is supported by HHMI. We thank our lab members for their help with testing the protocol and revising the manuscript.

LED Aydınlatıcı 1. İnşaat NOT: Gerekli LED ekipman Malzeme Listesi&#39;nde özetlenmiştir. Tüm LED kurulum küçük ve biz solucanlar 4 ışık pozlama kontrolü için karanlık bir odada yerleştirilmesi önerilir rağmen, laboratuarda herhangi bir yere yerleştirilebilir. <ol><li> Kablolar (Şekil 1A, D) ile bir denetleyiciye LED bağlayın. </li><li> Bir BNC kablosu (Şekil 1A, C, D) ile bir dijital fonksiyon jeneratörü / amplifikatör kontrolörü bağlayın. </li><li> Özel bir tutucu (Şekil 1A) kullanarak aşamada yukarıda LED ışık 10 cm düzeltildi. NOT: Biz metal parçalar ile tutucu yaptı. </li><li> Kısmen LED aydınlatma kurulum Kapak, ancak solucanlar hasta ediyor ısı birikimini (Şekil 1B), kaçının. NOT: Açık Üst ve alt (Şekil 1A) ile bir ısmarlama sert plastik kapak kullanın. Kapak mutagenez kendisi için gerekli değildir, ancak t kullanılano çevreye mavi ışık gereksiz maruz kalma sınırı. Biz solucan aşırı ısınmasını önlemek için LED kurulumu tamamen (Şekil 1B) kapsamaz tavsiye ederiz. </li><li> lazer koruyucu gözlük kullanın. NOT: Işık çok parlak olduğu için biz lazer koruyucu gözlük. Güneş de çalışabilir. </li><li> LED kontrolör kolu Anahtarı &#39;Int.&#39; ve sürekli aydınlatma (Şekil 1D) için maksimum güç (Şekil 1C)% 65 olarak ayarlayın. </li><li> solucanlar üreticinin protokolü kullanılarak yerleştirilir sahnede mavi ışık yoğunluğunu ölçmek için bir fotometre kullanın. Kurulum sürekli aydınlatma altında 2.0 mW / mm 2 verir. </li></ol> 2. Mavi Işık Tedavisi NOT:. [CB-unc-119 (+) Pmex-5-His-72 :: miniSOG-3&#39;UTR (TBB-2) +] unc-119 (ED3) III 4 streyn mevcuttur suşu CZ20310 juSi164 kullanın Caenorhabditis Genetik Merkezi (CGC de http: //www.cgc.cbs.umn.edu/). <ol><li> E. standart nematod büyüme ortamı (NGM) karanlıkta His-MSOG gerginlik korumak plakalar E. coli OP50 standart bir protokol 10,11 aşağıdaki. NOT: Ortam ışığı altında, juSi164 ihtiva-eden suşlar kendiliğinden oranı 4 üzerinde bir mutasyon oranını görüntüler yok. genellikle de mutasyona neden olmayan bir halojen lamba ile bir diseksiyon kapsamı kullanarak rutin zorlanma geçit. Bununla birlikte, bakım standart karanlık iç inkübatör suşları tutmak veya folyo ile suşları kapsayan, örneğin ışığa maruz kalmasını gereksiz önlemek için alınmalıdır. Gereksiz mutasyonlar zamanla birikir durumda aldıktan sonra zorlanma hacimde dondurmak için tavsiye edilir. </li><li> Gebe genç yetişkin almak bir solucan (yaklaşık 12 saat sonrası L4) hayvanlar 11 seçin. NOT: Büyük hayvanların düşük kuluçka boyutuna sahip olurken genç hayvanlar, daha az mutajenik etkisi 4 göstermektedir. </li><li> Bir filtre p Cutaper 25 mm x 25 mm kare delik olan 60 mm plaka uyacak ve bir dereceye giremeyen plaka (Şekil 1E) üzerine yerleştirin. NOT: Bir kare zımba kullanılabilir, ancak bu zımbanın büyüklüğüne hassas olması gerekmez. </li><li> Plaka üzerinde kare deliğin içindeki solucanlar kısıtlamak için eşit filtre kağıdı üzerinde 100 mM CuCl 2 100 mcL bırakın. </li><li> Gebe genç yetişkin çift cinsiyetli istenilen sayıda (bir örnek için Adım 3) almak ve CuCl 2 plakanın merkezine transfer. </li><li> Lazer koruyucu gözlük kullanın. </li><li> LED ve fonksiyon jeneratörü AÇIN. </li><li> LED kontrolör kolu Anahtarı &#39;Ext.&#39; fonksiyon üreteci (Şekil 1D) tarafından kontrol etmek. </li><li> Maksimum güç% 65 denetleyicisi ve sinüs dalga 4 Hz (Şekil 1C) olarak işlev jeneratör ayarlayın. </li><li> LED (Şekil 1A &lt;altında sahnede bir kapaksız Adım 2.5 den CuCl 2 tabak yerleştirin/ Strong&gt; &#39;). </li><li> 30 dakika boyunca mavi ışıkla hayvanları aydınlatır. </li><li> P 0 olarak seribaşı plaka sağlıklı görünen solucanlar aktarın. NOT: P 0 istenen numarası ekranın türüne bağlıdır. Örnek için Adım 3&#39;e bakın. P 0 solucanlar ışık tedavisi hemen sonra koordinasyonsuzluk görünebilir ve zamanla iyileşir. </li><li> bir kuluçka veya karanlık folyo kullanarak kapalı solucanlar tutun; ve onların döl 2,10 arasında istenen fenotipleri tarama başlamak için standart prosedürü izleyin. </li></ol> Bir İleri Genetik Ekran 3. Örnek Not:. Bu LED ve zorlanma çalışma olup olmadığını kontrol etmek 120 haploid genomlar ile klonal ekranın bir örneği Şekil 2 prosedürün şemasını göstermektedir. <ol><li> [Day1] mavi ışık tedavisinden sonra, P 0 olarak OP50 ile NGM plaka üzerine beş sağlıklı görünümlü solucanlar almak 20 ° C inkübatör tutmak ve bunları 1 için yumurtalarını izind ( &#39;DAY1&#39; plaka). </li><li> [Day2] Yeni numaralı seribaşı plaka üzerine Transferi P 0 ( &#39;DAY2&#39; plaka). </li><li> [Day3] seçin ve yakarak &#39;DAY2&#39; plaka üzerinde P 0 öldürmek. </li><li> [Day4] &#39;DAY1&#39; plaka 30 gebe genç erişkin F 1 Pick up ve bireysel plakalar ( &#39;DAY1&#39; için 30 F 1 tabak) üzerine aktarabilirsiniz. </li><li> &#39;DAY2&#39; plaka için [Day5] Adımı tekrarlayın 3.4 ( &#39;Day2&#39; için 30 F 1 tabak). </li><li> Onlar yetişkin olunca [Day7-9] standart stereomikroskop altında anormal morfolojiye ve / veya WT suşu (Şekil 3A) ile karşılaştırıldığında F 2 solucanlar davranışını kontrol edin. NOT: yüksek penetrans ile resesif F 2 arasında aynı fenotipe sahip solucanlar yaklaşık dörtte bir kalıtsal mutasyon görüntülenir. Bununla birlikte, bazı mutantlar yavaş büyür ve / veya kısmi bir penetrans gösterebilir. Nedenle, mutantların az dörtte bir bulunabilir olması mümkündürplaka. </li><li> ayrı ayrı görünür fenotipleri ile solucanlar almak ve gelecek nesil fenotip kalıtım kontrol edin. </li></ol> bir kromozom dışı transgen entegrasyon 4. Örnek <ol><li> Bir sahip CZ20310 juSi164 ilgi konusu DNA&#39;yı enjekte [Pmex-5-His-72 :: miniSOG-3&#39;UTR (TBB-2) + CB-unc-119 (+)] unc-119 (ED3) hazırlamak transgenik solucanlar standart bir enjeksiyon protokolü 12,13 aşağıdaki düşük iletim hızı (% 10-30). NOT: Alternatif olarak, kurulan ekstrakromozomal diziler juSi164 suşları içine geçilebilir. JuSi164 için genotipleme önlemek için, juSi164 heterozigot etkinliği azalmış olabilir, ancak P 0 olarak kullanılabilir. JuSi164 için genotipleme yöntemi Bölüm 5&#39;te tarif edilmektedir. </li><li> Aşama 2&#39;de tarif edildiği gibi [Day1] ~, mavi ışık ile P 0 20 olarak transgenik hayvanların tedavi edin. NOT: Daha fazla P 0 solucanlar tr bağlı olarak gerekli olan<html> ansmission oranı. Transgenlerin fenotipleri veya ko-enjeksiyon işaretleyici 12,13 ile tespit edilebilir. </li><li> Bireysel plakalar üzerine 10 sağlıklı görünümlü P 0 pick 20 ° C inkübatör tutmak ve onları bir kaç gün (P 0 plakaları) için yumurtalarını izin </li><li> [Day4] Tek: 20 transgenik F 1 bireysel plakalar üzerine her P 0 plaka solucanlar ve onları yumurtalarını edelim. (20 F 1 x 10 P 0 tabak = toplam 200 F 1 tabak.) </li><li> [Day7] F&gt;% 75 F 2 olan transgenlerin 1 tabak bulun. </li><li> F&gt;% 75 iletim hızı 1 tabak (F 2 tabak), beş transgenik hayvanlar seçin. </li><li> [Day10] F potansiyel entegre çizgiler olarak% 100 iletim hızı ile 2 tabak tutun. </li><li> Mendel ayrımını kontrol etmek ve potansiyel arka plan mutasyonları kaldırmak için standart bir prosedür 10 kullanılarak potansiyel entegre çizgiler outcross. </li></ol>le &quot;&gt; 5. Genotiplendirme <ol><li> Lyse 20 standart bir protokol 14 kullanılarak lizis tamponu F 2 solucanlar outcrossed. </li><li> Standart bir protokol 14 Aşağıdaki 58 ° C&#39;lik bir tavlama sıcaklığı ile MosSCI yerleştirilmesi 9 juSi164 (Tablo 1) primerleri kullanılarak PCR gerçekleştirin. NOT: unc-119 (ED3) Aşağıdaki Adımlar 5.4 ve 5.5 tahlisiye transgen (juSi164) çıkardıktan sonra koordinasyonsuzluk davranış tarafından tespit edilebilir, çünkü unc-119 (ED3) genotipinin gereksizdir. </li><li> Agaroz jel elektroforezi 15 çalıştırın ve juSi164 için WT bulmak için bant boyutları (Tablo 1) inceleyin. </li><li> F 2 döl nedeniyle unc-119 (ED3) varlığına solucanlar felç olup olmadığını inceleyin. </li><li> koordinasyonsuzluk solucanlar adım 5.4 bulunursa, tek birkaç nonuncoordinated solucanlar nesil tüm nonuncoordinated solucanlar elde etmek. </li></ol>

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The authors have nothing to disclose.

Burada, Optogenetic mutagenez ayrıntılı bir prosedür His-MSOG germline ifade ile ve küçük ölçekli bir ileri genetik ekranının bir örneğini sağlar tarif eder. standart kimyasal mutajenezi ile karşılaştırıldığında, bu Optogenetic mutajenez yöntemi, bir çok avantaja sahiptir. Öncelikle, bu sayede uzak gibi EMS, TRK gibi toksik kimyasal mutajen gelen laboratuar personelinin tutmak ve ultraviyole ışık (TMP / UV) ile Trimethylpsoralen, zehirsizdir. İkinci olarak, mutagenez deney prosedürü, p <su...

Ileri genetik ekranlar yaygın olarak Caenorhabditis elegans (C. elegans: 1) gibi model organizmaların çeşitli biyolojik süreçleri bozarak genetik mutantlarını üretmek için kullanılmıştır. Bu tür mutantlar analizleri işlevsel olarak önemli genlerin keşfi yol ve sinyalizasyon 1,2 yolaklarıyla. Geleneksel olarak, C. mutagenez elegans mutajenik kimyasallar, radyasyon, veya transpozonlar 2 kullanılarak elde edilir. Örneğin etil metansülfonat (EMS) ve N-etil-N -nitrosourea (TRK) insanlar için toksik olduğu gibi kimyasalları; gama ışını ya da ultraviyole (UV) radyasyon mutagenez özel ekipman gerektirir; ve Mutator suşları 3 gibi transpozon aktif suşları, bakım sırasında gereksiz mutasyonlara neden olabilir. Biz genetik olarak kodlanmış foto 4 kullanılarak kalıtsal mutasyonlar ikna etmek için basit bir yaklaşım geliştirdik. Reaktif Oksijen Türleri (ROS) DNA 5 zarar verebilir.Mini Singlet Oksijen Jeneratörü (miniSOG) DL (Işık, Oksijen ve Voltaj) için tasarlanmış olan 106 amino asit Arabidopsis phototropin 2 6 etki bir yeşil floresan protein. Mavi ışık (~ 450 nm), miniSOG maruz kalma üzerine ROS bütün hücrelerde mevcut olan bir kofaktör 6-8 olarak fl avin mononükleotid singlet oksijen de dahil olmak üzere oluşturur. Bu histon-72, bir C, C-terminaline miniSOG etiketleyerek bir His-MSOG füzyon proteinini inşa Histon 3. elegans varyantı Bunun C tohum çizgisinden Onun-MSOG ifade etmek için bir tek kopya transgen 9 oluşturulan elegans. karanlıkta normal kültür koşulları altında, His-MSOG transgenik solucanlar, normal kuluçka büyüklüğü ve ömrü 4 var. Mavi LED ışığına maruz kalma üzerine, His-MSOG solucanlar soylarına 4 arasında kalıtsal mutasyonlara üretir. T: C: A ve G: C C: G ve küçük chromoso indüklenen mutasyon spektrumu, G gibi nükleotid değişiklikleri içerirBana 4 silmeleri. Bu mutagenez prosedürü gerçekleştirmek için basit ve minimal laboratuvar güvenliği kurulum gerektirir. Burada, optogenetic mutagenez için LED aydınlatma kurulum ve prosedürler açıklanmaktadır.

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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.

Biz 120 Mutagenezli haploid genomlar karşılık gelen 60 F 1 tabak arasında Bölüm 2 ve 3&#39;te açıklanan standart protokol izlenerek 30 dakika ışığa maruz kullanarak CZ20310 juSi164 unc-119 (ED3) ile bir ileri genetik ekran gerçekleştirilen biz 8 F 1 tabak bulundu F vücut büyüklüğü defekt (Şekil 3B), koordinasyonsuzluk hareketi (Şekil 3C), ve yetişkin öldürücülüğü (Şekil 3D) ol...

Watch this Scientific Journal Video about Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans at JoVE.com

C. elegans'ta Histone-miniSOG Kullanılarak Optogenetik Rastgele Mutajenez | Genetik | Video

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetik olarak kodlanmış histon miniSOG mavi ışığa bağımlı bir şekilde genom kalıtsal mutasyonlara neden olur. Bu mutajenez yöntemi, hızlı, basit toksik kimyasalların serbest ve ileri genetik tarama ve transgen entegrasyonu için çok uygundur.

Histon-miniSOG Kullanılan optogenetic rastgele mutagenez<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

Classical and Modern Genetics

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9.0K Görüntüleme.  University of California in San Diego and Howard Hughes Medical Institute.  Genetik olarak kodlanmış histon miniSOG mavi ışığa bağımlı bir şekilde genom kalıtsal mutasyonlara neden olur. Bu mutajenez yöntemi, hızlı, basit toksik kimyasalların serbest ve ileri genetik tarama ve transgen entegrasyonu için çok uygundur. 

Video: Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

Hedeflenen<em&gt; In situ</emBudding Maya&gt; Mutajenezis Histon ve Genler

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Genetik

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

arasında Transkriptomik Analizi<em&gt; C</em&gt;.<em&gt; elegans</emGalaxy Projesi Smokin Suite sayesinde&gt; RNA Sıralama Veri

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Kromatin İmmunopresipitasyon Yöntemi Kullanılarak c-KIT Ligand Promotör Analizi

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites &#8211; the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) &#8211; flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short &#34;GGATCC&#34; scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.

CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

Cpf1&#39;i Kullanarak C-Brick DNA Standart Montajı İçin Protokoller

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on animal physiology and behavior. Recently, we showed that modulation of longevity in the nematode Caenorhabditis elegans by food abundance is more complex than previously recognized. The responsiveness of the lifespan to changes in food level is determined by specific genes that act by controlling information processing within a neural circuit. Our framework combines genetic analysis, high-throughput quantitative imaging and information theory. Here, we describe how these techniques can be used to characterize any gene that has a physiological relevance to broad-range dietary restriction. Specifically, this workflow is designed to reveal how a gene of interest regulates lifespan under broad-range dietary restriction; then to establish how the expression of the gene varies with food level; and finally, to provide an unbiased quantification of the amount of information conveyed by gene expression about food abundance in the environment. When several genes are examined simultaneously under the context of a neural circuit, this workflow can uncover the coding strategy employed by the circuit.

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Here, we present a framework to relate broad-range dietary restriction to gene expression and lifespan. We describe protocols for broad-range dietary restriction and for quantitative imaging of gene expression under this paradigm. We further outline computational analyses to reveal underlying information processing features of the genetic circuits involved in food-sensing.

C. elegans gen ifade seviyeleri sırasında ömrü modülasyon altında geniş menzilli diyet kısıtlama tarafından kodlanmış bilginin bir miktar

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

Caenorhabditis elegans Ploitlik manipülasyon

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Fisyon mayası Heterochromatin kararsız hale Proteasome mutantlar seçmek için rasgele Mutagenesis Gene hedefli

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

Bir hızlı ve genomik noktası mutantlar C. elegans kullanarak CRISPR/Cas9 Ribonucleoproteins üreten Facile boru hattı

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

Sitogenetik teknikle Bemisia tabaci yumurta döllenme oranı belirleme

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

Nanoakışkan Teknoloji Kullanarak Tek ve Toplu C. elegans Örneklerinde Yüksek Verimli Nicel RT-PCR

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

Histon-miniSOG Kullanılan optogenetic rastgele mutagenez

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