Analysis of Somatic Mutation in Transgenic Mice Using the HUG-X Reporter Gene.
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Our lab uses a gene construct called HUG-X to study somatic mutation of immunoglobulin genes.HUG-X is a mouse/human chimeric heavy chain composed of a human g1 constant region and a mouse variable region containing a stop codon.If somatic mutation reverts the stop codon, the HUG heavy chain is expressed.Mice containing the transgene (HUG-X mice) were immunized to promote somatic mutation. After immunization, B cells from these mice were immortalized, creating hybridomas.Those hybridomas that produce the HUG heavy chain eventually stop doing so as they are kept in tissue culture.The aim of this project is to successfully isolate monoclonal HUG+ hybridoma lines, then determine the sequence of the (presumably) mutated HUG-X gene.Currently, eight such lines have been isolated and RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) is being done on the extracted RNA.The RT-PCR will create cDNA that can then be sequenced. By comparing these sequences to the known sequence of the original HUG-X gene, any somatic mutation can be observed and analyzed.