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Biology

Получение 2-dGuo обработанных Thymus культур Органный

doi: 10.3791/906 Published: August 28, 2008

ERRATUM NOTICE

Summary

В этом видеоролике показано вскрытие и удаление вилочковой железы плода, а также подготовку бывших культур естественных условиях от 2-dGuo обработанных тимуса.

Abstract

В вилочковой железе, незрелых CD4 +8 + тимоцитов выражения случайно переставить Т-клеточного рецептора α-и бета-цепи генов претерпевают позитивной и негативной селекции событий основано на их способности распознавать self-peptide/major комплекса гистосовместимости (MHC) молекулы выраженное тимуса стромальных клеток. В естественных условиях анализ роли тимуса стромальных клеток при intrathymic выбор затрудняется сотовой сложности тимуса микроокружения в стационарном тимуса взрослого, и из-за отсутствия соответствующей ориентации стратегии манипулировать экспрессии генов, в частности, тимуса стромальных отсеков. Мы показали, что вилочковая микроокружения можно легко манипулировать в пробирке с помощью reaggregate тимуса культур орган, который позволит подготовки трехмерных тимуса долях от определенных стромальных и лимфоидных клеток. Хотя другие в системах пробирке поддержку некоторых аспектов Т-клеточного развития, reaggregate тимуса органной культуры остается только в пробирке система в состоянии поддерживать эффективную MHC класса I и II-опосредованной тимоцитов события выбора, и поэтому может быть использован как эффективный инструмент для изучения клеточной и молекулярной регуляции позитивной и негативной селекции в тимусе.

Protocol

Пожалуйста, посетите Springer протоколы для получения дополнительной информации о подготовке бывших культур естественных тимуса органа.

Erratum

Formal Correction: Erratum: Preparation of 2-dGuo-Treated Thymus Organ Cultures
Posted by JoVE Editors on 04/01/2012. Citeable Link.

A correction was made to: Preparation of 2-dGuo-Treated Thymus Organ Cultures. A revised abstract was republished due to a publisher error. The abstract was corrected to:

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

from

In the thymus, immature CD4+8+ thymocytes expressing randomly rearranged T-cell receptor α- and b-chain genes undergo positive and negative selection events based on their ability to recognize self-peptide/major histocompatibility complex (MHC) molecules expressed by thymic stromal cells. In vivo analysis of the role of thymic stromal cells during intrathymic selection is made difficult by the cellular complexity of the thymic microenvironment in the steady-state adult thymus, and by the lack of appropriate targeting strategies to manipulate gene expression in particular thymic stromal compartments. We have shown that the thymic microenvironment can be readily manipulated in vitro through the use of reaggregate thymus organ cultures, which allow the preparation of three-dimensional thymus lobes from defined stromal and lymphoid cells. Although other in vitro systems support some aspects of T-cell development, reaggregate thymus organ culture remains the only in vitro system able to support efficient MHC class I and II-mediated thymocyte selection events, and so can be used as an effective tool to study the cellular and molecular regulation of positive and negative selection in the thymus.

Получение 2-dGuo обработанных Thymus культур Органный
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Cite this Article

Jenkinson, W., Jenkinson, E., Anderson, G. Preparation of 2-dGuo-Treated Thymus Organ Cultures. J. Vis. Exp. (18), e906, doi:10.3791/906 (2008).More

Jenkinson, W., Jenkinson, E., Anderson, G. Preparation of 2-dGuo-Treated Thymus Organ Cultures. J. Vis. Exp. (18), e906, doi:10.3791/906 (2008).

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