Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

Sarah R. Oca&#241;as; Jos&#233;  V. V. Isola; Tatiana D. Saccon; Kevin D. Pham; Ana J. Chucair-Elliott; Augusto Schneider; Willard M. Freeman; Michael B. Stout

doi:10.3791/64765

Interrogation par appariement spécifique à la cellule de l’épigénome ovarien et du transcriptome ...

JoVE Journal
Biology

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Biology

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

Interrogation par appariement spécifique à la cellule de l’épigénome ovarien et du transcriptome de la souris

Full Text

1,392 Views

12:25 min

February 24, 2023

DOI: 10.3791/64765-v

Sarah R. Ocañas*^1,2,3, José V. V. Isola*^1,3, Tatiana D. Saccon¹, Kevin D. Pham², Ana J. Chucair-Elliott², Augusto Schneider⁴, Willard M. Freeman^2,3, Michael B. Stout^1,3

¹Aging and Metabolism Research Program,Oklahoma Medical Research Foundation, ²Genes & Human Disease Research Program,Oklahoma Medical Research Foundation, ³Oklahoma City Veterans Affairs Medical Center, ⁴Nutrition College,Federal University of Pelotas

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study optimizes the translating ribosome affinity purification (TRAP) and isolation of nuclei tagged in specific cell types (INTACT) methods for paired interrogation of the ovarian transcriptome and epigenome in a mouse model. The approach enables analysis of specific ovarian cell populations without cell sorting, facilitating insights into cell type-specific changes associated with aging.

Key Study Components

Research Area

Cell type-specific analysis
Ovarian aging
Transcriptomic and epigenomic investigation

Background

Understanding ovarian aging is crucial for addressing female infertility.
Current methods often require cell sorting, which can limit throughput.
This technique offers a streamlined way to analyze ovaries from the same mouse.

Methods Used

Optimized TRAP and INTACT methods
NuTRAP mouse model crossed with Cyp17a1-Cre line
Single ovary collection and specific protocols for nuclear isolation

Main Results

Enabled paired analysis of transcriptomic and epigenomic data.
Uncovered cell type-specific changes in the ovaries related to aging.
Validated the potential of this method for broader application across various cell types.

Conclusions

The method demonstrates effectiveness in isolating nucleic acids from specific ovarian cell populations.
This approach has significant implications for studies focused on female reproductive health.

Frequently Asked Questions

What are the advantages of this method?

It increases throughput, reduces costs, and eliminates the need for specialized cell sorting equipment.

Can this method be applied to other cell types?

Yes, as long as a specific Cre driver is available for the cell type of interest.

What biological effects were observed in the study?

The study identified specific changes in ovarian cells associated with aging, relevant for understanding reproductive senescence.

Why is cell specificity important?

It ensures that the analysis accurately reflects changes within the targeted cell populations, which is crucial for understanding their biological roles.

What preliminary steps are involved in this method?

The method begins with precise collection and homogenization of the ovary, followed by centrifugation to isolate nuclei.

How significant are the findings regarding female infertility?

The findings enhance the understanding of ovarian aging, which is essential for developing treatments for female infertility.

Dans ce protocole, la méthode de purification par affinité des ribosomes de traduction (TRAP) et la méthode d’isolement des noyaux marqués dans des types cellulaires spécifiques (INTACT) ont été optimisées pour l’interrogation par appariement du transcriptome ovarien et de l’épigénome spécifiques à la cellule à l’aide du modèle murin NuTRAP croisé vers une lignée de souris Cyp17a1-Cre.

Cette méthode fournit une technique pour isoler les acides nucléiques de populations de cellules ovariennes spécifiques de la même souris sans avoir besoin de tri cellulaire. Cette technique peut aider à identifier comment des populations cellulaires spécifiques changent dans diverses conditions. Par exemple, avec le vieillissement.

Le principal avantage de cette technique est qu’elle permet une analyse épigénomique et transcriptomique appariée à partir d’un type spécifique de cellules ovariennes sans avoir besoin de tri cellulaire. Cela augmente considérablement le débit, diminue le coût et élimine le besoin d’équipement spécialisé lors de l’analyse spécifique du type cellulaire à partir du tissu ovarien. Ici, notre objectif est d’identifier les changements spécifiques au type cellulaire qui se produisent avec le vieillissement ovarien avant la sénescence reproductive, ce qui a des implications dans le traitement de l’infertilité féminine.

View the full transcript and gain access to thousands of scientific videos