Research objectives and content
Recent studies in the mouse have shown that double negative-inmature thymocytes can assemble a new TCR isoform that is devoid of TCRa chain and is referred to us the pre-TCR complex. The assembly and inducing functions of this complex are likely to be coupled to the achievement of productive TCRBeta chain gene rearrangement. In this way, the CD3 subunits associated with the pre-TCR trigger a cascade of signals wich drive the inmature double negative cells to the more differentiated double positive stage: e.g., thymocytes from mice deficient in one chain of the CD3 complex (# -chain) are unable to assemble a pre-TCR and blocked at the double negative stage. In our project the main objective is the identification of gene products acting downstream of the pre-TCR/CD3 complex. To this end, we will try to develope a genetic screen that combines the production high-titer helper-free retroviruses by transient transfection and the subsequent infection of early thymocytes in reaggregate organ culture. Such kind of approach, combined with the use of thymuses originated from various mutant mice whose T cell development is arrested at different stages, should permit us to analyse any candidate genes implicated in T cell development in a brief period (2-3 weeks for each). In this context, the CD3-E deficient mice developed at the host institution constitute a particularly appropriate gene transfer recipient to study the regulation of TCR gene rearrangement since their V(D)J recombination machinery is intact. However, my long term objective is to identifie new genes implicated in the development of thymocytes. To this end, I plan to construct a cDNA library and introduce it into CD3-E-deficient thymocytes; the resulting double positive cells will be isolated and the cDNA expressed by the integrated proviruses identified by PCR.
Training content (objective. benefit and expected impact)
In the development of the research project, I will receive training on different techniques of Molecular Biology (transient transfection with retroviruses, construction of DNA libraries, etc.) and cell culture techniques, with many applications in other fields of research. Indeed, the host institution has major facilities for flow cytometry and confocal microscopy. The development of this research project will permit us to understand how the pre-TCR complex controls the expression of different new genes essential for T cell maturation and other cellular processes. Links with industry / industrial relevance (22)
The CIML has several programs of collaboration with industry (including the group headed by Dr Malissen). Depending on the results obtained in the project is possible to try to establish a contract with industry to comercialize the new genes cloned and identified implicated in T cell development.