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Biology

2 dGuo - 치료 Thymus 오르간 문화의 준비

doi: 10.3791/906 Published: August 28, 2008

ERRATUM NOTICE

Summary

이 동영상은 절개와 태아 thymus의 제거뿐만 아니라 2 dGuo - 처리 thymus의 전 생체내 문화의 준비를 보여줍니다.

Abstract

thymus, 미숙 CD4 8 + 무작위로 재배열 T - 세포 수용체를 표현 thymocytes에서 α - 및 B - 체인 유전자 self-peptide/major의 histocompatibility 복합 (MHC) thymic로 표현 분자를 인식하는 능력에 따라 긍정적이고 부정적인 선택 이벤트를 받다 stromal 세포. intrathymic 선택하는 동안 thymic stromal 세포의 역할의 생체내 분석에 정상 상태 성인 thymus에서 thymic microenvironment의 세포 복잡하여 어렵게 만든, 적절한 타겟팅 전략의 부족에 의해 특정 thymic stromal 구획의 유전자 발현을 조작할 수 있습니다. 우리는 thymic microenvironment가 쉽게 정의 stromal과 림프 세포의 입체 thymus 엽 (叶)의 준비를 허용 thymus 장기 문화를 reaggregate의 사용을 통해 체외로 조작할 수있는 것으로 나타났습니다. 체외 시스템의 다른 T 세포 개발의 일부 측면을 지원하지만, thymus 장기 문화 I 및 II - 중재 thymocyte 선택 이벤트, 그래서이 연구는 효과적인 도구로 사용할 수있는 유일한 체외 시스템의 효율적인 MHC 클래스를 지원할 수 남아 reaggregate thymus에서 긍정적이고 부정적인 선택의 세포 및 분자 규제.

Protocol

방문하십시오 스프링거 프로토콜 예 생체내 thymus 기관 문화의 준비에 대한 자세한 내용은합니다.

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.

2 dGuo - 치료 Thymus 오르간 문화의 준비
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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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