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Analysis
of Somatic Mutation in Transgenic Mice Using the HUG-X Reporter Gene.
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. |
