Intrathymic Delivery of MHC Genes Using Recombinant Adenoviruses
Advances in the study of thymic selection have relied heavily on animals that are genetically manipulated either by transgenesis and/or by knock out technology. Besides being costly and time consuming, these methods suffer inherent limitations (reviewed in refs. 1, 2, 3). In an effort to first, complement the existing panoply of methodologies used in the study of thymocyte selection and second, circumvent some of the problems inherent to these methods, we have developed an adenovirus-based vector system to deliver MHC class II genes to the thymic stroma. When injected into MHC class II-deficient animals (IIo) (4), these vectors punctually restore the expression of MHC class II molecules on the surface of transduced thymic stromal cells (5). In turn, the block in T-cell development associated with the absence of MHC class II molecules in these thymi is relieved and significant numbers of mature CD4 single positive thymocytes appear (5). Alternatively, this gene delivery system has also been used to look at positive selection of T cell receptor (TCR) transgenic thymocytes by a known peptide engineered in the invariant chain gene (6). Although variability is seen in the reconstitution of injected animals, this method is a reliable delivery tool for punctual expression of genes in the thymic stroma. The experiments we have carried out indicate that the cortex of the thymus is the main target for MHC class II expression. Here, we will first describe the procedures to make, characterize, and produce high titer stocks of recombinant viruses, followed by the surgical procedure allowing intrathymic delivery of the virus.
KeywordsSucrose Microwave Phenol Agar EDTA
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