Core - Vector 2 - Retrovirus

Sadelain, Michel

Abstract

Recombinant retroviruses are powerful tools for gene transfer that are used in experimental as well as clinical applications. Retroviral-mediated gene transfer leads to stable proviral integration in the target-cell genome, a key feature when foreign genetic material must be reliably transmitted to the progeny of transduced progenitor cells or clonogenic cells. The projects that make up this Program Project rely in part or exclusively on retroviral-mediated gene transfer to modify primary cells prepared from heart, lung, blood, bone marrow, and embryonic stem cells. The construction of a retroviral vector and the generation of a stable high-titer producer cell line are elaborate procedures with well- defined Checkpoints that are best handled in a highly specialized core laboratory. Centralized generation of high-quality producer cell lines and retroviral stocks will be cost effective, permit standardization of procedures, and facilitate the exchange of information and expertise between the different projects.
The specific aims of the Retrovirus Core are: 1. construction of the recombinant retroviral vectors; 2. transfection of vector DNA in packaging cells and selection of producer cell lines; 3. characterization of vector transmission in standard cell lines by Southern blot analysis; 4. expansion of independent clones and identification of the best clone for specific target cells;5. titration of cell-free retroviral stocks; 6. detection of replication-competent retrovirus in cell-free viral stocks. The Retrovirus Core is directed by M. Sadelain, M.D., Ph.D., who is assisted by a senior research technician with considerable experience in cell culture and molecular techniques.