Name: Musehjernedissektion til mikrogliaisolering
Uploaded: 2023-09-15T13:20:00
Duration: 1 min 1 s
Description: Watch this Scientific Journal Video about Mouse Brain Dissection for Microglia Isolation at JoVE.com

mouse brain dissection for microglia isolation

Kvantificering af histonmodifikation af musehjerne ved hjælp af flowcytometri

Epigenetics is the study of the mechanisms that regulate gene expression without altering the underlying DNA sequence. Epigenetic regulation of gene expression is dynamic within cells and can allow for rapid and coordinated responses to various environmental stimuli. The dynamic regulation occurs in part due to changes in the chromatin structure at the level of the nucleosome, which is comprised of histone proteins (H2A, H2B, H3, H4) assembled into an octamer core tightly wound by DNA1. The interactions between the histone proteins and the DNA can control the accessibility of DNA to transcription machinery, which can ultimately control gene expression and other aspects of chromatin biology2. Histone proteins have unstructured tails which feature positively charged residues that form electrostatic interactions with the negatively charged DNA backbone. These interactions result in tight packing of the DNA and reduced DNA accessibility. Covalent modifications to the histone tails, termed histone post-translational modifications (HPTMs), can regulate these interactions3,4. Some of the most well-characterized HPTMs include histone tail acetylation and methylation, which can change the affinity of electrostatic interactions between the histone tails and DNA, resulting in differential accessibility to the underlying DNA and recruitment of transcription factors which recognize these HPTMs at specific sites. HPTMs are regulated by three important classes of enzymes termed readers- which recognize, writers- which deposit, and erasers- which remove HPTMs. Thus, the recruitment or dissolution of reader, writer, or eraser enzymes can ultimately change the landscape of HPTMs and govern the structure and function of chromatin, making their regulation and readout essential for understanding cellular biology and function3,4.
The cells in the central nervous system (CNS) are epigenetically flexible as they change their transcriptome to adapt to environmental stimuli. Accumulating evidence suggests that changes in the epigenome, such as DNA methylation, non-coding RNAs, and HPTMs, play an essential role in memory formation and synaptic function5. Disrupting HPTM dynamics through manipulation of the relevant readers, writers, or erasers can block or enhance associative learning and long-term potentiation6,7,8. Microglia, the resident immune cell of the CNS, rapidly regulate their transcriptome in response to immune stimulation through dynamic changes in their epigenome9,10,11. This high level of adaptation to their local brain environment makes them difficult to examine in an isolated context as studies have shown that the epigenome and transcriptome of microglia becomes altered after only a few hours in culture media after removal from the brain environment11. In addition, as microglia only make up 10% of the brain&#39;s cells, measures examining changes at the whole tissue level lack sensitivity and specificity12,13. As a result, microglia need to be rapidly isolated to examine the epigenetic changes such as HPTM levels, ex vivo.
The methods commonly used to examine HPTMs include chromatin-immunoprecipitation sequencing (ChIP-seq) and cleavage under targets and tagmentation sequencing (CUT&#38;Tag-seq)4. While these techniques are highly specific to an individual HPTM and can inform the presence of HPTMs within a specific genomic context, they can only examine one of the many possible HPTMs within a single experiment11,14 Therefore, before proceeding with such experiments, which require a significant time and money investment, it is highly valuable to narrow down the list of potentially interesting HPTMs for further investigation by first examining changes in global levels of HPTMs. The two main approaches for examining global HPTM levels are immunohistochemistry and western blot analysis, but both approaches are only semi-quantitative, low-throughput, and require large numbers of tissue sections or isolated cells15,16. Thus, we aimed to develop a highly sensitive, quantitative method that could be used to examine the global HPTM levels rapidly and at the single cell level.
The protocol presented enables detection of global HPTM levels rapidly using intranuclear flow cytometry. Previous studies in cancer cells have justified the importance of examining global levels from a clinical perspective17,18. It is also common for studies to use global levels as a screening method prior to assessing genomic location of specific HPTMs of interest19,20. For microglia, assessing global levels following isolation is challenging due to the low cell yield; Pan et al present global HPTM levels from isolated microglia, in which microglia from three animals was pooled to enable protein level detection by western blot19. Using our protocol, we are able to detect global changes with much lower cell inputs, enabling screening of multiple marks per animal and eliminating the need to pool samples.
Here, we describe a protocol to detect HPTM levels rapidly via quantitative intranuclear flow cytometry in isolated microglia. While we focus specifically on HPTM quantification for the sake of brevity, this protocol can be used in the same way to quantify global levels of reader, writer, and eraser enzymes. The protocol is delivered in two parts: first, the isolation method for microglia and, second, the flow cytometry-based method for determining HPTM levels. The isolation method yields cells that can be used for both RNA isolation and HPTM level assessment which allows for the evaluation of gene expression and HPTM levels from the same sample. In addition, the method for HPTM assessment can be used on other cell types as indicated in the protocol.

Forfatter Spotlight: Forbedringer i forskning i regulering af genekspression

Kvantificering af globale histonposttranslationelle modifikationer ved hjælp af intranukleær flowcytometri i isoleret musehjernemikroglia

Our research aims to understand the fundamental regulatory mechanisms controlling gene expression in the brain, and we're particularly interested in understanding how disruption of these mechanisms contributes to neurodevelopmental and neuropsychiatric conditions. We focus on understanding how genetic and environmental risk factors combined to alter cellular function and produce behavioral impairments. This protocol includes methods for high-throughput screening of histone modifications in isolated microglia, allowing us to screen through dozens of different epigenetic modifications before committing to high-throughput sequencing, and functional follow-up studies.Our protocol provides measures of global histo modifications similar to Western plot. However, our technique is both faster, requires fewer input cells, is more quantitative, and can be multiplexed.

Watch this Scientific Journal Video about Mouse Brain Dissection for Microglia Isolation at JoVE.com

Musehjernedissektion til mikrogliaisolering

Begynd med at placere det isolerede musehjernevæv i 1 milliliter fordøjelsesbuffer pr. hjerne i separate petriskåle på is. Hak hjernevævene grundigt i små stykker ved hjælp af et rent skalpelblad. Brug en plastoverførselspipette med spidsen afskåret til forsigtigt at overføre hakkede hjerner til individuelle brønde inde i en 24-brønds plade på is.Dæk pladen med et stort stykke gennemsigtig, fleksibel film, luk låget og inkuber på is i 30 minutter. Overfør derefter den fordøjede hjerneopløsning til individuelle 7 ml isglas, dounce homogenisatorer på is indeholdende 5 ml kold FACS-buffer. Forsigtigt dounce hver hjerne med en løs støder, indtil der opnås en enkeltcellesuspension.Dounce blødt med den stramme støder tre til fire gange for at sikre en enkeltcellesuspension, før den overføres til 15 ml polypropylenrør.

Research

JoVE Journal

Neuroscience

Mouse Brain Dissection for Microglia Isolation

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...