Abstract
Our lab has generated transgenic mice for studying somatic mutation of antibody genes. The transgene encodes a mouse/human chimeric heavy chain with a stop codon in the variable region (HUG-X). If somatic mutation reverts the stop codon, the HUG heavy chain is expressed. HUG-X mice were immunized to promote somatic mutation. B-cells from these mice were then immortalized, creating hybridomas. The HUG-X hybridomas were then subcloned to isolate possible mutants. While hybridomas are maintained in tissue culture, they secrete antibodies into the culture medium. This medium can be drawn off and tested by Enzyme-Linked Immunosorbent Assay (ELISA). Previously in our lab, HUG-X hybridomas expressing HUG (presumably due to somatic mutation) stopped expressing HUG as they were maintained in culture. We have now altered the procedure previously used to limit loss of HUG expression. We freeze a cell line down after getting positive HUG results. When this putative HUG positive line is thawed, we immediately subclone the hybridomas again. This separates hybridomas that have stopped expressing the HUG heavy chain from those who continue to express it. After testing for HUG by ELISAs, the hybridomas are frozen down again to minimize loss of HUG expression. Subclones of the hybridoma lines HyXX5, HyXX6, HyXX7, and HyXX8 have been assayed for HUG expression through ELISAs. Initial results suggest that somatic mutation resulting in stable HUG expression has occurred in all lines except HyXX6, indicating that this procedure is a successful method of testing for somatic mutation.
