TY  - THES
T1  - Conditional RNA interference, altered nuclear transfer and genome-wide DNA methylation analysis
A1  - Meissner,Alexander
Y1  - 2006/07/18
N2  - The goal of the studies described here was to establish a set of methods aimed at ultimately enhancing the efficiency of epigenetic reprogramming. The use of RNA interference (RNAi) for studying and influencing gene function has become an essential part of biology. In this work, we have described two lentivirus-based vectors used for conditional, Cre-lox regulated RNAi in cells and in mice. One vector triggers Cre-dependent activation (pSico) and the other Cre-dependent termination (pSicoR) of shRNA expression. These vectors were used to conditionally and reversibly knock-down p53, Npm, and Dnmt1 expression in ES cells and in MEFs. As a proof of principle, pSico was used to generate conditional and tissue-specific knock-down mice. As outlined in Chapter 1 conditional depletion of various gene products by RNAi will provide better understanding of the factors involved in epigenetic reprogramming. This knowledge should ultimately lead to enhancing the efficiency of successful reprogramming. The pSicoR system was applied in later experiments to temporally suppress Cdx2 function in donor nuclei prior to nuclear transfer, a modification of the current procedure, termed altered nuclear transfer (ANT). Finally, deciphering the epigenome of different cell types is critical for understanding the regulation of both normal development and disease states. In the last part of this work we have devised a new strategy that permits high-resolution comparative DNA methylation analysis. The system was tested in ES cells depleted of DNA methylation by elimination of the DNA methyltransferases Dnmt1, Dnmt3a and 3b. Dnmt1 was knocked down in Dnmt3a and 3b double knockout ES cells using a pSicoR-Dnmt1 vector.
KW  - Kerntransplantation
KW  - DNS-Methyltransferase
KW  - RNS-Interferenz
KW  - Embryonalzelle
CY  - Saarbrücken
PB  - Universitäts- und Landesbibliothek
AD  - Postfach 151141, 66041 Saarbrücken
UR  - http://scidok.sulb.uni-saarland.de/volltexte/2006/626
ER  -