Analysis of endogenous immunoglobulin gene rearrangement in the SCID mouse

grantor: University of TorontoThe critical event in lymphoid development is the assembly of germline variable gene segments forming the variable exon of the antigen receptors. This somatic assembly occurs through a process called V(D)J recombination. Mice with severe combined immune deficiency (SCID) have a profound deficiency in lymphoid development. The scid mutation results in a defect in the ability to repair double strand DNA damage, and consequently affects normal V(D)J rearrangement. Early studies showed that endogenous rearrangements in transformed SCID cell lines exhibited large deletions in the recombined genetic elements. Indeed, the estimated frequency of normal coding joint resolution in SCID cells is $10\sp2$- to $10\sp3$-fold lower than wild type. However, the scid mutation is not absolute as older mice become "leaky", exhibiting serum immunoglobulin (Ig) and/or T cell activity. During the course of my studies, the SCID gene product was identified as the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs); a molecule implicated in ds DNA damage repair, V(D)J recombination and transcription regulation. The goal of my thesis is to acquire greater understanding of the process of V(D)J recombination and its role in B cell development. To achieve this objective, I have examined the extent of assembly of Ig variable genes in non-leaky SCID mice. In Chapter III, I show that DJ scH rearrangements are found at 1-10% the frequency observed in C.B-17 whole bone marrow. Qualitative analysis of these joins demonstrated that these junctions are indistinguishable from wild type with respect to the number of deletions. Collectively however, the SCID joins exhibited a profoundly restricted repertoire characterized by a general absence of N addition and an increase in homology mediated joining. Chapter IV provides a detailed analysis of the extent to which VDJ scH and $\kappa$ rearrangements are found in SCID B lineage. I show that SCID mice undergo normal VDJH and kappa rearrangement at an appreciable frequency (0.1-1%), and many of these rearrangements are functional (86%, 44% respectively). About 40% of the rearrangements could have arisen by homology mediated ligation, suggesting that homology is used in the resolution of coding ends in the absence of functional SCID protein. Despite the relatively high level of Ig gene rearrangement, SCID mice do not have detectable pre-B cells. Moreover, SCID mice transgenic for either fully rearranged heavy and light chain Ig do not exhibit increased leakiness. My data provides evidence which suggests that the scid defect has a profound and lasting effect on B cell development beyond deficiencies in V(D)J recombination.Ph.D