Research Project
3455592
Somatic mutation of immuoglobulin (Ig) variable (V) genes plays an important role in the generation of antibody diversity. The goal of this project is to develop an assay to detect and isolate cells undergoing somatic mutation in vivo and in vitro, by making transgenic mice in which a somatic mutation in an Ig transgene results in expression of a reporter gene, lacZ, that is easily detected. The transgene to be used encodes a fusion protein that pairs a rearranged VDJ, containing a stop codon, with a marker gene, lacZ. The VDJ is 5' of the lacZ; its stop codon therefore prevents translation of the lacZ "exon". If the stop codon is reverted by somatic mutation, the entire fusion protein will be translated, and lacZ expressed. LacZ expression can be readily detected by histochemical or fluorescence assays. Cells that have undergone somatic mutation will turn blue when the chromogenic substrate (X-gal) is used, or fluorescence if the fluorogenic substrate FDG is added. Somatic mutation can be studied in animals by sectioning organs, or even entire embryos, and staining with X-gal; sites of mutation will turn blue. The amount of mutation can be quantitated with FDG and analysis on the Fluorescence Activated Cell Sorter (FACS). Positive cells can be isolated using the FACS and further characterized as to phenotypic and functional properties. The initial step is to make this construct and an appropriate control construct (with a translatable VDJ), transfect them into cell lines to ensure that they are expressed, and then to make transgenic mice. These mice can then be used to directly test many of the current hypotheses regarding somatic mutation, including: 1) somatic mutation occurs in a burst of activity after immunization; 2) somatically mutating B cells are found in germinal centers; 3) such cells are, or are destined to become, memory cells; 4) Lyl+ B cells do not somatically mutate; 5) T cells do not somatically mutate; and 6) somatic mutation is not triggered in responses to thymus independent antigens. The transgene can also be bred onto mice with various immune dysfunctions, such as autoimmune strain MRL/lpr, the autoimmune and immunodeficient motheaten strain, and the immunodeficient scid mouse strain. Somatic mutation in these mice can be analyzed and compared to the normal situation.
