Conditional mutagenesis allows the introduction of tissue specific mutations in the mouse and is of crucial importance in converting genome sequence information into functional data for biomedical research. Mice expressing the Cre recombinase in a spatially controlled manner are essential in creating such conditional knock-outs. A wide variety of Cre mice have been generated, but there is a distinct lack of models expressing the recombinase faithfully and at high levels in cells of the innate immune system. To address this need, three target genes, Itgb2l, Marco and Msr1, were chosen to create novel neutrophil and macrophage specific knock-in models harbouring iCre2, a recombinase engineered for increased expression levels. Two strategies were employed. Initially gene specific bacterial artificial chromosomes in which the iCre2 fragment replaced the endogenous translation start codon were created by Red/ET recombineering. Utilization of these BAC vectors for embryonic stem cell targeting successfully created knock-ins but the identification of homologous recombinants was complicated by the vectors’ large size. As the discovery of mutations impeding iCre2 functionality in the knock-in lines necessitated repeating the vector creation process, novel shorter vectors were designed. These vectors achieved targeting frequencies of around 10% and facilitated the isolation and verification of 9 Itgb2l and Marco specific iCre2 knock-in murine embryonic stem cell lines on the 129 genetic background. To determine tissue specific iCre2 expression before generating mouse models, an in vitro haematopoietic differentiation system, utilising three-dimensional embryoid body formation and selective expansion of progenitors in the presence of IL-3 and MCSF, was adapted. Embryonic stem cells were successfully differentiated into macrophages as assessed by CD11b and F4/80 marker expression. Collectively, this work has established the foundations for obtaining viable myeloid specific Cre producer mouse strains and discusses the potential of their future application in elucidating the role of macrophages and neutrophils in innate immune function.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:564992 |
| Date | January 2010 |
| Creators | Könitzer, J. D. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/20230/ |
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