Genetics

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This core aims to provide access, support and training for three new and emerging technologies for the projects described in this program. In addition, the core seeks to continue to develop and improve of the application of the three technologies described for the specific needs of the projects described in this program. The specific technologies include: a) the development of a comprehensive and technically advanced resource for the design, generation and production of viral-based shRNA vectors as well as retroviral approaches to manipulate ectopic gene expression in primary cells and cell lines of interest, b) the use of genetically modified mice for the efficient immortalization of autoreactive B-cells, and c) the use of novel technologies to conditionally immortalize long term hematopoietic stem cells in order to generate cell lines from transgenic mice of interest for these projects. The three specific aims of this core are: To develop a suite of viral vectors that will be useful for genetic manipulation of primary lymphoid cells. The use of viral mediated delivery of shRNA will be the primary tool for loss-of-function studies whereas the use of retroviral driven cDNA expression will be the primary means of gain-of-function studies in mice. 1. To use mice that overexpress MYC in an inducible, and B-cell specific manner to immortalize autoreactive B-cells (AN-1 cells). This approach will be important for Dr. Cambier's project and their attempts to determine the nature and specificity of the B-cell antigen receptors on normal AN1/T3 cells. 2. To define the hematopoietic pluripotency of conditionally immortalized long-term hematopoietic stem cell clones. The genetic pathways we will focus on will be of broad interest and application to the other projects in this program, and we will train and assist those investigators in this process as needed. This core will be important in allowing the different projects described in this program to define the molecular basis by which antigen receptor derived signals are able to regulate lymphocyte tolerance, homeostasis and survival. Depending on the specific circumstances, the antigen receptor can anergize, tolerize or kill lymphoid cells with the ultimate goal of generating an innocuous but useful repertoire. The projects described in this project will focus initially on the role of members of the Bcl-2 family in these functions of the antigen receptors.