Wagner, Nicholas:

A mouse model for the overexpression of methyltransferases

Duisburg, Essen (2010), VI, 114 S.
Dissertation / Fach: Biologie
ehem. Fakultät für Biologie und Geografie
Horsthemke, Bernhard (Doktorvater, Betreuerin)
Ehrenhofer-Murray, Ann (GutachterIn)
Dissertation
Abstract:
One of the epigenetic mechanisms by which gene expression is regulated is the methylation of DNA. Erroneous DNA methylation can cause a vast spectrum of diseases. A mouse model was created in this study to investigate whether the overexpression of the somatic form of Dnmt1, Dnmt1s, is sufficient to cause erroneous methylation and disease. A CAG promoter-driven transgene containing the Dnmt1s DNA methyltransferase was constructed. Transgene functionality was tested in 3T3 murine fibroblasts in cell culture. Because the ubiquitous overexpression of Dnmt1 has been reported to be embryonically lethal, a conditional transgene was constructed using the Cre-LoxP system. This technique allows the initial expression of the eGFP marker protein only. Upon cross-ins with Cre-recombinant mouse lines, the eGFP sequence is cut out and degraded. This leads to the expression of the Dnmt1s under control of the CAG promoter. To easily distinguish between endogenous and transgenic methyltransferase, a RGS-His-tag was added to the N-terminal end of the Dnmt1s transgene. Pronucleus injections with the Dnmt1s transgene resulted in six founder lines verified by PCR, Southern blot and eGFP fluorescence. Cross-ins of these founders with CMV-Cre mouse lines yielded viable offspring. The ubiquitous overexpression of Dnmt1s was investigated in cross-ins that carry the recombined version of the Dnmt1s transgene and express RGS-His-tagged Dnmt1 as verified by RT-PCR. TaqMan analyses of total Dnmt1 expression of the line with the strongest overexpression showed a ~ 3-fold (3.17 ± 0.65) overexpression in liver, a ~ 3.5-fold (3.47 ± 0.15) overexpression in testis, a ~ 5.5-fold (5.44 ± 0.39) overexpression in brain and a ~ 6-fold (6.24 ± 1.48) overexpression in kidney, but no overexpression in the spleen of CMV-Cre recombined transgenic mice in comparison to sibling wild type controls. Dnmt1 overexpression was confirmed on the protein level by quantitative western blots, which detected ~ 3-fold Dnmt1 levels in brain and ~ 6-fold Dnmt1 levels in testis of transgenic mice in comparison to sibling wild type controls. Upcoming analyses will test the in vivo functionality of the transgenic protein and the phenotypic consequences of Dnmt1s overexpression.

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