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Title: Long range analysis of the mammalian casein locus
Author: Tomlinson, Anthony M.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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Current notions of genome organisation hold that chromatin is structurally and functionally partitioned into domains delimited by specialised chromatin elements. In transgenic studies a variety of chromatin domains and functional elements isolated from them have been shown to direct physiological levels of gene expression independent of the integration position of the transgene within the genome. The principal aim of this project was to assay for position independent, copy number dependent and tissue- and developmental stage-specific gene expression from the human and murine casein gene loci in transgenic mice. Two overlapping YACs covering the murine casein gene locus were restriction mapped, generating the first reported physical map of this locus. The order of the five casein genes is α-β-γ-ε-κ; as in other mammals the κ casein gene, though evolutionarily unrelated to the other members of the locus, is closely linked, which may imply that casein gene expression is under locus control. The YACs are collinear within the region of overlap, suggesting that neither is rearranged. One of these YACs was manipulated with the intention of inserting a reporter gene under the control of an ovine β-lactoglobulin promoter downstream of the murine β casein polyadenylation site, so that gene expression from the YAC could be detected against a murine background in cell culture or transgenic mice. In parallel, a YAC bearing the human casein locus was restriction mapped and used to establish transgenic mice. Unexpectedly, none expressed detectable levels of human casein RNA when analysed by northern blot, although four produced human κ casein RNA that was detectable by reverse transcriptase polymerase chain reaction, indicating basal levels of transcription of the gene. The reasons for this are unknown, though initial characterisation of genomic DNA from the transgenic animals suggests that the YAC is not intact in any of the eight lines. The human casein YAC was also transfected into HC11 murine mammary epithelial cells with a view to assessing the level of human casein gene expression from the YAC in vitro, but unfortunately no stably transfected cell lines were established. These results and their implications for the use of large DNA constructs in transgenic mammals are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available