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Title: Design of binary system for the identification and targeted expression of mouse genes
Author: Brown, S. L.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2003
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This work describes the design and validation of a binary system in a mouse that will enable (i) identification and functional analysis of novel genes, and (ii) study of the function of previously-cloned genes by their targeted expression. It utilises the yeast transcriptional activator GAL4 and extends to the mouse, a method devised to study the development of Drosophila melanogaster. Mouse embryonic (ES) cells were used since these are pluripotent and can be manipulated in vitro to production of the transgenic mouse. First it was demonstrated, using two transactivator (PGKGAL4 and PGKGAL4236) and three target (UASb-gal, UASb-geo and UAShygro) constructs, that GAL4 can transactivate in mouse ES cells. This was confirmed using two other cell lines - mouse NIH 3T3 fibroblasts and human 293T cells. In all cases, transactivation of a UAS-reporter was observed, demonstrating that GAL4 in functional in mouse ES cells. Next, three GAL4 gene traps were prepared, pSAGAL4IRESb-geopA, pSAGAL4pA and pSAGAL4neo. It was shown that the former can act as a gene trap in mouse ES cells and that b-geo expression, hence GAL4 also, can be placed under the control of an endogenous promoter. This demonstrates the principle underlying the binary system is valid. For the other constructs, ES cells with UASb-geo or UASb-gal integrated into the genome are needed. It proved impossible to generate such cell lines. The final part of the study involved a more elegant solution, design of a GAL4 gene trap system which could potentially trap any gene without the need for specific target ES cell lines. This was achieved by placing GAL4 under transational control of an IRES, such that translation of GAL4 occurs independently of upstream endogenous exons. The STOP-IRES-GAL4 cassette was shown to synthesise GAL4 via the IRES.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available