CRISPR EDITING OF CHICKEN DF-1 CELL LINE TO EXPRESS DUCK RIG-I
Kathy Magor, University of Alberta
Aradana Muthupandian, Doaa Waly, Harrison Anzinger, Tera Burris, Brad Magor
Dept. Biological Sciences, University of Alberta, Edmonton, AB
Germinal centres, the histologically distinct sites of antibody affinity maturation, have long been thought to be an evolutionary invention of the homeotherms. Indeed the question of if, or the degree to which, poikilothermic vertebrates affinity mature their antibody repertoires has remained contentious. We have previously demonstrated that the mediator of antibody (Ab) affinity maturation, Aicda, is expressed in cells within catfish melanomacrophage clusters (MMCs). We recently reported that we could isolate these clusters from spleen and kidney of zebrafish, to allow for analyses of Ab VDJ repertoires using BRILIA software. The conclusion of these analyses was that 1 – 3 B-cells nucleate a MMC and clonally expand while accumulating somatic mutations in their VDJ exons. This is akin to antibody affinity modification in germinal centres. These observations have since been verified using Alakazam clonal lineage reconstruction. To assess whether there is a mechanism for selection of these Ab affinity modified B-cells we vaccinated and then boosted goldfish and zebrafish with Alexa 647 conjugated BSA or KLH. Subsequent FACS analysis revealed that all Alexa 647+ spleen and kidney leukocytes also had the autofluorescence profiles of melanomacrophages. Confocal imaging revealed that the ‘trapped’ antigen was on the melanomacrophage cell surface. Furthermore magnetic beads coated with monoclonal anti-BSA could capture melanomacrophages from BSA vaccinated fish only, indicating that the Ag was retained intact on the cell surface. Collectively these results support the argument that MMCs are proto-germinal centres in the spleen and kidney of fishes.
Funded through NSERC Canada.