Towards conditional gene inactivation in the mouse colon and functional analysis of genes influencing colorectal carcinogenesis

• Main Author: Read, Jason Tyler
• Published: University of Edinburgh 2001
• Subjects: 616.994
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.660977

Dysregulation of the Wnt signalling pathway is implicated in the carcinogenic transformation of normal cells in a number of human tissues. Mutations in Wnt pathway signalling molecules appear to be particularly important in the development of colorectal neoplasia. This thesis investigates the effects of modulating levels of Wnt pathway components on downstream molecules <i>in vivo</i> and <i>in vitro</i>. To examine the effects of loss of Wnt pathway components on the biology of the mouse large intestine work was undertaken to assemble a tissue specific, inducible Cre (CreER^tm) transgene construct. This formed part of a group effort using various lengths of upstream sequence from the mouse homeobox gene Cdx1 to drive CreER^tm. In order to ensure that sufficient Cdxl promoter sequence to achieve adequate expression levels and localization a recently described methodology based on the Red a-b-g recombination system of bacteriophage Lambda was used in an effort to insert the CreER^tm coding sequence into a Bacterial Artificial Chromosome (BAC). The BAC used in the construction of the transgene was determined to contain previously uncharacterised upstream Cdx1 promoter elements, which may play a role in determining correct tissue expression pattern. In order to further characterise the newly isolated Cdxl promoter elements, portions of the BAC derived sequence were used in the construction of reporter plasmids encoding the Enhanced Green Fluorescent Protein (EGFP). The EGFP based reporter plasmids, which contained various fragments of the Cdx1 promoter containing a number of transcriptional control elements, were microinjected and liposome transfected into murine embryonic stem cells, colorectal cell lines and primary murine colonocytes in order to determine the contribution of the transcriptional elements to levels of expression. The work presented in this thesis contributes to the development of a transgenic mouse line bearing a conditional, inducible intestinal epithelial specific Cre recombinase and to the understanding of regulation of the Wnt signalling pathway in response to genotoxic damage as well as expression of the murine homeobox gene Cdxl.