Testing Changes in Avidity and Somatic Mutation of the Human Gamma 1 Transgene.
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In the bone marrow, as B-cells mature, they undergo rearrangement of their gene sequence to produce an antibody with an affinity for one specific antigen. Once these antibodies, which are attached to the membranes of their B-cells, bind with the antigen they have an affinity for, a series of point and frame shift mutations of the gene sequence that codes for their variable region occurs. These somatic mutations results in higher or lower affinity for their antigen. If affinity is increased, the B-cell will proliferate into memory and plasma cell. To prove this theory we are working with transgenic mice that express the Human Gamma 1 transgene. This transgene encodes an antibody heavy chain with a human constant region and a mouse variable region that binds to the antigen dansyl. Using hybridomas generated from transgenic mice, experiments such as avidity testing and DNA sequencing can be used to see if the HuG 1 antibodies, that have an affinity for dansyl, would demonstrate higher avidity through somatic mutation after repeated exposures to this antigen. Reverse transcriptase-polymerase chain reaction (RT-PCR), to amplify the variable region of the heavy chain, and dansyl-bovine serum albumin (BSA) conjugation were performed in preparation for these procedures. The dansyl-BSA conjugation was successful, as was, RNA isolation and RT-PCR. Jessica Padilla completed TA cloning, and future work on DNA sequencing is pending. Also pending is an avidity assay with our quantified dansyl and HuG 1 antibodies.