formaldehyde crosslinking of the f1 generation of caenorhabditis elegans for chip

Caenorhabditis elegans'ta Epigenetik Kalıtım ve ChIP

Epigenetic inheritance allows the transmission of gene regulatory information across generations and can therefore allow the environment or experiences of parents to affect their progeny. In C. elegans, germline gene silencing initiated by exogenous double-stranded RNA (dsRNA) can be inherited for multiple generations in progeny not exposed to the original trigger1,2,3,4. This process, termed RNA interference (RNAi) inheritance, is one of several related epigenetic silencing phenomena in C. elegans, including piRNA-initiated multigenerational silencing2,5, paramutation/RNAe (RNA-induced epigenetic silencing)6,7,8, and multicopy array-initiated silencing9, which have overlapping but distinct requirements for small RNA and chromatin regulation machinery. In C. elegans exogenous RNAi, dsRNA is processed into small interfering RNAs (siRNAs) that act in a complex with primary Argonaute proteins to recognize their target mRNA. This targeting leads to the amplification of secondary siRNAs that associate with secondary Argonautes to silence target mRNA through both cytoplasmic and nuclear silencing pathways. For germline-expressed RNAi targets, the nuclear secondary Argonaute HRDE-1 and additional nuclear RNAi factors target nascent transcripts, resulting in transcriptional repression and recruitment of histone methyltransferases to deposit repressive chromatin marks, including H3K9me310. Histone H3K9me3 promotes the establishment of heritable silencing of germline-expressed green fluorescent protein (GFP) transgenes by RNAi inheritance11,12.
The goal of this protocol is to use a transgene expressing a GFP-histone fusion protein in the germline as a reporter for RNAi inheritance and to assay changes in histone modifications at the reporter transgene locus using chromatin immunoprecipitation and quantitative polymerase chain reaction (ChIP-qPCR). This protocol describes a standardized plate-based RNAi feeding approach to initiate reporter silencing. It also provides a detailed timeline for passaging animals between generations by isolating in utero embryos from gravid adults by alkaline hypochlorite treatment (&#39;bleaching&#39;). Methods and representative data for monitoring the frequency of GFP silencing in a subset of the population by fluorescence microscopy and for histone H3K9me3 ChIP-qPCR are also described. Reporter-based RNAi inheritance assays provide a highly tractable system to functionally dissect the roles of genetic and environmental factors in epigenetic regulation13,14, and genetic screens using such reporters have identified both genes that are required for2,3,15 and genes that negatively regulate16,17 the duration of transgenerational epigenetic inheritance.

Yazar Spotlight: C. elegans'ta RNAi Kalıtım ve ChIP 

Nesiller Arası Epigenetik Kalıtımın Analizi C. elegans Floresan Raportör ve Kromatin İmmünopresipitasyon (ChIP) Kullanılarak

Watch this Scientific Journal Video about Formaldehyde Crosslinking of the F1 Generation of Caenorhabditis elegans for ChIP at JoVE.com

Formaldehyde Crosslinking of the F1 Generation of Caenorhabditis elegans for ChIP  

ChIP için F1 Nesil Caenorhabditis elegans'ın formaldehit çapraz bağlanması

Solucanları içeren tüpe% 1.8'lik bir son konsantrasyona formaldehit ekleyerek ve tüpü oda sıcaklığında altı dakika döndürerek başlayın. Daha sonra tüpleri hemen sıvı nitrojen içinde dondurun ve numuneleri 80 santigrat derecede saklayın. Devam etmek için formaldehit çapraz bağ numunesini oda sıcaklığındaki bir su banyosunda üç dakika çözdürün ve numuneleri oda sıcaklığında 16 dakika döndürün.1.25 molar glisinde 125 milimolar son konsantrasyona kadar ve oda sıcaklığında beş dakika döndürün. Bu adımı takiben, numuneleri buz üzerinde veya dört derecede tutun ve manyetik boncuk elüsyonuna kadar buz gibi soğuk tamponlar kullanın. Numuneyi 1000 g'da üç dakika santrifüjledikten sonra, bir mililitre PBSTX ile üç kez yıkayın.Ardından bir mililitre Resüspansiyon Tamponu ile iki kez yıkayın. Son yıkamadan sonra, toplam hacmin pelet hacminin en az üç katı ve minimum 100 mikrolitre olmasını sağlamak için yeterli Tampon bırakın.

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Research

JoVE Journal

Genetics

Formaldehyde Crosslinking of the F1 Generation of Caenorhabditis elegans for ChIP

360 Görüntüleme. Solucanları içeren tüpe% 1.8'lik bir son konsantrasyona formaldehit ekleyerek ve tüpü oda sıcaklığında altı dakika döndürerek başlayın. Daha sonra tüpleri hemen sıvı nitrojen içinde dondurun ve numuneleri 80 santigrat derecede saklayın. Devam etmek için formaldehit çapraz bağ numunesini oda sıcaklığındaki bir su banyosunda üç dakika çözdürün ve numuneleri oda sıcaklığında 16 dakika döndürün.1.25 molar glisinde 125 milimolar son konsantrasyona kada...

Video: Formaldehyde Crosslinking of the F1 Generation of Caenorhabditis elegans for ChIP  

Analysis of Transgenerational Epigenetic Inheritance in C. elegans Using a Fluorescent Reporter and Chromatin Immunoprecipitation (ChIP)

Transgenerational epigenetic inheritance (TEI) allows the transmission of information through the germline without changing the genome sequence, through factors such as non-coding RNAs and chromatin modifications. The phenomenon of RNA interference (RNAi) inheritance in the nematode Caenorhabditis elegans is an effective model to investigate TEI that takes advantage of this model organism's short life cycle, self-propagation, and transparency. In RNAi inheritance, exposure of animals to RNAi leads to gene silencing and altered chromatin signatures at the target locus that persist for multiple generations in the absence of the initial trigger. This protocol describes the analysis of RNAi inheritance in C. elegans using a germline-expressed nuclear green fluorescent protein (GFP) reporter. Reporter silencing is initiated by feeding the animals bacteria expressing double-stranded RNA targeting GFP. At each generation, animals are passaged to maintain synchronized development, and reporter gene silencing is determined by microscopy. At select generations, populations are collected and processed for chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) to measure histone modification enrichment at the GFP reporter locus. This protocol for studying RNAi inheritance can be easily modified and combined with other analyses to further investigate TEI factors in small RNA and chromatin pathways.

This protocol describes an RNA interference and ChIP assay to study the epigenetic inheritance of RNAi-induced silencing and associated chromatin modifications in C. elegans.

Transgenerational epigenetic inheritance (TEI) allows the transmission of information through the germline without changing the genome sequence, through ...