Rapid Dissection and Dissociation of the Mouse Olfactory Epithelium for Single-Nucleus Suspensions

Benjamin  D. W. Belfort; Anthony M. Insalaco; Claude C. Chew; Johnathan D. Jia; Julia Younis; Benjamin R. Arenkiel

doi:10.3791/68472

Dissecção rápida e dissociação do epitélio olfatório de camundongo para suspensões de núcleo único

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Neuroscience

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JoVE Journal Neuroscience

Rapid Dissection and Dissociation of the Mouse Olfactory Epithelium for Single-Nucleus Suspensions

Dissecção rápida e dissociação do epitélio olfatório de camundongo para suspensões de núcleo único

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03:42 min

August 1, 2025

DOI: 10.3791/68472-v

Benjamin D. W. Belfort^1,2,3,4, Anthony M. Insalaco^2,3, Claude C. Chew⁵, Johnathan D. Jia³, Julia Younis², Benjamin R. Arenkiel^2,3

¹Genetics and Genomics Graduate Program,Baylor College of Medicine, ²Department of Molecular and Human Genetics,Baylor College of Medicine, ³Jan and Dan Duncan Neurological Research Institute,Texas Children's Hospital, ⁴Medical Scientist Training Program,Baylor College of Medicine, ⁵Cytometry and Cell Sorting Core,Baylor College of Medicine

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Overview

This article outlines a protocol for the rapid dissection and dissociation of murine olfactory epithelium aimed at facilitating single-nucleus RNA sequencing. The method prioritizes speed and ease of execution, making it suitable for applications where tissue homogenization is necessary.

Key Study Components

Area of Science

Neuroscience
Genomics
Olfactory System Research

Background

Single-cell and single-nucleus RNA sequencing are crucial for understanding cellular diversity.
Previous dissection methods emphasized anatomical integrity, which may not be ideal for certain applications.
The olfactory epithelium plays a fundamental role in olfactory sensory transduction.
Enhancing dissection techniques can improve the efficiency of genetic studies related to olfaction.

Purpose of Study

To provide a user-friendly protocol for olfactory epithelium dissection.
To facilitate rapid tissue processing for single-cell sequencing investigations.
To promote research on the genetic mechanisms of the olfactory system.

Methods Used

The method employs dissection of olfactory epithelium from murine models.
This biological model involves the mouse olfactory system, emphasizing the need for rapid extraction.
Bone removal and careful manipulation ensure integrity of the main olfactory epithelium.
Key steps include incisions in the cranial vault and extraction of the olfactory structures.
Quality controls were implemented to maintain consistent sequencing quality.

Main Results

Over half of collected events were identified as nuclei, indicating successful isolation.
High circularity of nuclei suggests quality preservation during dissection.
Quality control demonstrated uniform gene and mitochondrial distributions.
Marker genes aligned with established benchmarks for cell types.

Conclusions

The study presents a rapid methodological advancement for dissecting the olfactory epithelium.
By enabling efficient single-nucleus sequencing, it contributes to understanding genetic factors in olfaction.
This protocol could enhance the analysis of neuronal mechanisms and plasticity in sensory systems.

Frequently Asked Questions

What are the advantages of this dissection protocol?

This protocol emphasizes rapid execution, allowing for efficient processing of olfactory tissue without losing critical cellular details necessary for sequencing.

How is the olfactory epithelium obtained?

The olfactory epithelium is achieved through a series of precise incisions in the mouse skull, leading to careful extraction of the nasal structures.

What type of data can be obtained from this method?

This method yields single-nucleus RNA sequencing data, enabling insights into gene expression and cellular diversity in the olfactory system.

Can this technique be adapted for other tissue types?

While primarily designed for olfactory epithelium, the principles of rapid dissection could potentially be adapted to other neural tissues with similar anatomical features.

Are there any limitations to this study?

One limitation may be the focus on speed which could affect the anatomical integrity of the extracted tissues, potentially influencing certain types of analyses.

How does this contribute to understanding olfactory function?

By fostering genetic studies through effective dissection, it provides a clearer picture of the molecular underpinnings of olfactory perception and function.

Este artigo fornece um protocolo simplificado para a dissecção e dissociação do epitélio olfatório murino para fins de sequenciamento de RNA de núcleo único.

Aqui, fornecemos um protocolo projetado para auxiliar na dissecção rápida do epitélio olfatório para fins de sequenciamento de célula única ou núcleo único. Nosso protocolo prioriza a velocidade e a facilidade de aprendizado e é ideal para aplicações de célula única em que o tecido é homogeneizado e a dissecção rápida é mais importante do que preservar a integridade anatômica, ao contrário dos métodos anteriores. Nosso protocolo permite a dissecção rápida e fácil do epitélio olfatório, com o objetivo de facilitar estudos de sequenciamento de células únicas e promover pesquisas sobre a base genética do sistema olfatório.

Para começar, obtenha a cabeça sem pele de um rato. Use uma tesoura fina e de ponta afiada para fazer duas incisões para quebrar os arcos zigomáticos do osso esquamosal bilateralmente, colocando uma ponta de tesoura na órbita posterior e a outra adjacente ao canal auditivo. Faça uma incisão superficial de órbita em órbita ao longo da face mais anterior do osso frontal, incisando apenas a superfície dorsal do osso frontal a uma profundidade de aproximadamente um a dois milímetros.

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