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Title: In vitro culture of chicken and mouse embryo-derived cells
Author: Lodge, Peter Graham
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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Mouse embryonic stem (mES) cells can be maintained in vitro without loss of pluripotency in the presence of leukaemia inhibitory factor (LIF). Germline competent mES cells can be genetically modified in vitro and used to make transgenic mice via chimaeras. Germline competent chicken ES (chES) cells would be a powerful tool for the production of transgenic chickens. ES cells have been isolated from primates and mice but attempts from other species have been unsuccessful. Novel ES cell isolation strategies have been tested using inbred mouse strains. Using standard techniques, mES cells cannot be isolated from CBA strain embryos whereas they can be isolated from 129Sv strain embryos. A vital function of LIF in mES cells is activation of signal transducer and activator of transcription 3 (STAT3). LIF also activates the mitgoen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathway that appears to promote differentiation. LIF is a member of the interleukin-6 (IL-6) family of cytokines that includes ciliary neurotropic factor (CNTF). Manipulation of IL-6 family signals is a new approach to mES cell isolation that may be applied to development of methods of chES cell isolation. Prior to investigation of new approaches, standard mES cell isolation was performed. chES cells were not isolated in conditions adapted from standard mES cell isolation. A new approach involving the manipulation of LIF-mediated signals were evaluated in mES cell isolation experiments. The drug PD98059 inhibits the MEK/ERK pathway by preventing phosphorylation of MEK I. ES cell isolation frequency from strain 129Sv embryos increased significantly in the presence of 25μM PD89059 and 500 U/ml LIF (p<0.1) however, CBA ES cells could be isolated in the same conditions. The drug U0126 blocks and MEK/ERK pathway by directly inhibiting both phosphorylated MEK I and MEK II. CBA ES cells were isolated at a frequency of 22.3% in medium containing 2μM U0126 and 2x103 U/ml LIF.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available