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2 - dGuo इलाज thymus अंग संस्कृतियों की तैयारी

doi: 10.3791/906 Published: August 28, 2008



यह वीडियो विच्छेदन और भ्रूण thymus को हटाने के रूप में अच्छी तरह से 2 - dGuo इलाज thymus के पूर्व vivo संस्कृतियों की तैयारी को दर्शाता है.


Thymus, अपरिपक्व 8 CD4 + बेतरतीब ढंग से पुनर्व्यवस्थित टी सेल रिसेप्टर व्यक्त thymocytes में α और ख श्रृंखला जीन सकारात्मक और नकारात्मक चयन उनके self-peptide/major उतक अनुरूपता (MHC) जटिल thymic द्वारा व्यक्त अणुओं की पहचान करने की क्षमता के आधार पर की घटनाओं से गुजरना stromal कोशिकाओं. Intrathymic चयन के दौरान thymic stromal कोशिकाओं की भूमिका के vivo विश्लेषण में स्थिर राज्य वयस्क thymus में thymic microenvironment के सेलुलर जटिलता से मुश्किल बना है, और उपयुक्त लक्ष्यीकरण रणनीतियों की कमी से विशेष thymic stromal डिब्बों में जीन की अभिव्यक्ति में हेरफेर करने के लिए. हम पता चला है कि thymic microenvironment reaggregate thymus अंग संस्कृतियों, जो परिभाषित stromal और lymphoid कोशिकाओं से तीन आयामी thymus lobes के तैयारी की अनुमति के उपयोग के माध्यम से आसानी से किया जा सकता है इन विट्रो में हेरफेर. हालांकि इन विट्रो प्रणालियों में अन्य टी सेल विकास के कुछ पहलुओं का समर्थन reaggregate, thymus अंग संस्कृति इन विट्रो प्रणाली में ही रहता है कुशल MHC वर्ग का समर्थन करने में सक्षम मैं और द्वितीय की मध्यस्थता thymocyte चयन घटनाओं, और इसलिए अध्ययन करने के लिए एक प्रभावी उपकरण के रूप में इस्तेमाल किया जा सकता है thymus में सकारात्मक और नकारात्मक चयन के सेलुलर और आणविक विनियमन.


कृपया यात्रा स्प्रिंगर प्रोटोकॉल पूर्व vivo thymus अंग संस्कृतियों की तैयारी के बारे में अधिक जानकारी के लिए.


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).


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 इलाज thymus अंग संस्कृतियों की तैयारी
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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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