The role of signaling through mIgM in chicken B cell development /

We and others have hypothesize that, in the developing chick, signaling through the membrane bound form IgM (mIgM) expressed on bursal lymphocytes plays a critical role in the progression of normal B cell development. In order to specifically evaluate the role of bursal mIg, we want to introduce by retrovirally mediated gene transfer a transgenic mIg construct which should be expressed on the B cell surface independent of endogenous Ig gene rearrangement. We therefore cloned the transmembrane (TM) form of the chicken $ mu$ heavy chain from a bursal cDNA library. Sequence analysis revealed 80% homology of the TM/cytoplasmic regions of chicken $ mu$ to the mouse homologue, in contrast to the much lower ($ sim$30%) overall homology when comparing $ mu$ extracellular domains. We have cloned a truncated form of the $ mu$TM (tr. $ mu$TM) lacking the VDJ-C$ mu$1 domains into the retroviral vector RCAS BP(B). In vitro transfection of chicken embryo fibroblasts (CEFs) was used to assay for $ mu$ protein expression. Strikingly, the CEFs transfected with the $ mu$ transgene expressed the tr. $ mu$TM transgene on the cell surface, as determined by flow cytometry following viable cell staining with several anti-chicken $ mu$ mAbs. At this time, in vivo detection of the tr. $ mu$TM transgene has been unsuccessful, possibly due to rapid degradation of the transgenic protein. The successful expression of this molecule into the RCAS vector system will allow us to further determine whether expression of the tr. $ mu$TM will affect the progression of normal B cell development