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Date of Award
James D. Tucker
Mutations in the human superoxide dismutase gene (hSOD1) cause Amyotrophic Lateral Sclerosis (ALS), a paralytic disorder that leads to degeneration of the motor neurons in the brain and spinal cord and generally causes death within five years of onset. This work focused on characterizing the site of insertion of the human SOD1 G93A mutant transgene in a mouse model for ALS. These transgenic mice exhibit a phenotype similar to that of human ALS patients, although the exact number of gene copies and the precise location of the transgene have not been determined. Since the number and location of the transgene may affect the expression of nearby loci, which in turn may alter the phenotype of these animals, it is important to map precisely the site of insertion with respect to possible deletions or additions in DNA sequences and to determine the exact number of copies present. Previous experiments have suggested that there are approximately 25 copies of the transgene and that they are present on mouse chromosome 12, band E. We confirmed the chromosomal location by fluorescence in situ hybridization, but our real-time PCR analyses showed the copy number to be 102 +/- 5 (mean +/- S.E.). Although the mouse flanking sequence has not been determined, our results indicate that all the concatamerized transgene monomers are present in the head-to-tail orientation, and that the individual hSOD1 gene monomers appear to be adjacent to each other with perhaps only a few bases added or deleted at the junctions. Our results also indicate the presence of (a) ~3.3 kb of the pBR322 plasmid, which was used to create the transgenic mouse, at the 5' end of the transgene complex, (b) plasmid SVneo sequence at the 3' end of the transgene complex, (c) at least two SVneo regions in tail-to-tail orientation at locations that remain to be determined, and (d) DNA sequences in the transgene complex from vector(s) that were supposedly not used in making these transgenic mice. The presence of plasmid sequences at the 5' and the 3' ends of the transgene complex was unexpected, and raises the possibility that such sequences may also exist in between at least some of the hSOD1 monomers. This observation as well as the presence of sequences from other vectors, significantly complicates the efforts to identify the flanking mouse sequences.
Loganathan, Ramya Malarini, "Characterization Of The G93a Hsod1 Transgene Complex In The Mouse Model Of Amyotrophic Lateral Sclerosis" (2010). Wayne State University Theses. 6.