Name: Collection of F1 Generation Caenorhabditis elegans for Chromatin Immunoprecipitation (ChIP)
Uploaded: 2023-05-05T12:10:00
Duration: 59 s
Description: Watch this Scientific Journal Video about Collection of F1 Generation Caenorhabditis elegans for Chromatin Immunoprecipitation ChIP at JoVE.com

collection of f1 generation caenorhabditis elegans for chromatin immunoprecipitation (chip)

Epigenetisk arv og ChIP i Caenorhabditis elegans

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.

Forfatter Spotlight: RNAi-arv og ChIP i C. elegans 

Analyse af transgenerationel epigenetisk arv i C. elegans ved hjælp af en fluorescerende reporter og kromatinimmunudfældning (ChIP)

Research in our lab examines how chromatin-associated proteins, histone modifications, and small RNA pathway factors work together in the context of development and transgenerational epigenetic inheritance. The RNA inheritance assay, using a GFP reporter, has become a very powerful tool. This protocol describes how to standardize the preparation, scoring, and passaging of worms, and the preparation of ChIP samples, which can help to generate reproducible results.One of the experimental challenges when studying germline-expressed transgenes is isolating the effects in the animal's germline. We're currently using whole animals for ChIP, but in the future we would like to develop assays to examine chromatin, specifically in the germline. Our laboratory is interested in histone methylation and proteins that recognize these marks.Going forward, we would like to investigate the interplay between these chromatin readers and small RNA pathways in the context of RNAi inheritance at transgenes and the regulation of endogenous sequences.

Watch this Scientific Journal Video about Collection of F1 Generation Caenorhabditis elegans for Chromatin Immunoprecipitation ChIP at JoVE.com

Collection of F1 Generation Caenorhabditis elegans for Chromatin Immunoprecipitation (ChIP)  

Indsamling af F1 Generation Caenorhabditis elegans til kromatin immunudfældning (ChIP)

Begynd med at plating ca. 3.500 C.elgans F1 generation embryoner på 14 plader og lad dem vokse til den unge voksne fase i tre dage ved 21 grader Celsius. Vask og saml C.elgans i 1,5 milliliter rør ved hjælp af PBS / TX. Centrifuger ved 1000 G i to minutter, aspirer og træk ormene fra en stamme ind i et rør.Gentag tre vaske med PBS/TX. Derefter suges supernatanten og efterlader en milliliter i røret og inverteres for at blande. Efter at have talt antallet af C.elgans-orme i tre mikroliter tre gange, beregnes koncentrationen af orme og overføres et volumen svarende til 3000 orme til et nyt rør til formaldehydtværbinding.

Research

JoVE Journal

Genetics

Collection of F1 Generation Caenorhabditis elegans for Chromatin Immunoprecipitation (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 ...

Bleaching and Plating Caenorhabditis elegans P0 Generation Embryos onto RNAi-NGM plates

Bleaching and Plating Caenorhabditis elegans P0 Generation Embryos onto RNAi-NGM plates  

Blegning og plettering Caenorhabditis elegans P0 Generation Embryos på RNAi-NGM plader

Passaging and Scoring the Caenorhabditis elegans P0 Generation for Germline GFP Expression

Passaging and Scoring the Caenorhabditis elegans P0 Generation for Germline GFP Expression  

Passaging og scoring af Caenorhabditis elegans P0-generationen til GFP-ekspression af kimlinjen

Formaldehyde Crosslinking of the F1 Generation of Caenorhabditis elegans for ChIP

Formaldehyde Crosslinking of the F1 Generation of Caenorhabditis elegans for ChIP  

Formaldehydtværbinding af F1-generationen af Caenorhabditis elegans til ChIP

Sonication of Formaldehyde-Crosslinked Caenorhabditis elegans and Chromatin Immunoprecipitation

Sonication of Formaldehyde-Crosslinked Caenorhabditis elegans and Chromatin Immunoprecipitation  

Sonikering af formaldehyd-tværbundet Caenorhabditis elegans og kromatin immunudfældning