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Investigation of annexin function by targeted gene disruption

Hawkins, Timothy Edward; (1998) Investigation of annexin function by targeted gene disruption. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Members of the Annexin family of calcium-binding proteins are generally expressed in the cytosol of most eukaryotic cells from plants to humans. In vertebrates there are at least ten members of the family of which any one cell type expresses a selection. Annexins are characterised by the ability to bind calcium-dependently to negatively charged phospholipids, and by the presence of a 70 aa motif usually repeated four times. Numerous functions have been attributed to the annexins including calcium channel activity, regulation of exocytosis, phospholipase A2 inhibition and involvement in endocytosis. None of these postulated roles has been proved conclusively. This thesis describes work designed to define more rigorously the roles of members of this gene family in vivo by targeted disruption of the loci encoding the genes in the chick pre-B-cell DT40 lineage and mice. As a first step, targeting constructs were generated by a novel PCR-based method. Mice were then generated that have lost expression of Annexin VI, a unique member of the family in that it contains two instead of one set of four 70 aa repeats. One allele was disrupted in embryonic stem (ES) cells using the now well-documented method of homologous recombination, and genetically modified ES cells, identified by PCR, were then injected into blastocysts and reimplanted into recipient females. Chimaeric offspring that gave germline transmission were then bred to produce hetero- and finally homozygous null-mutant mice. Mice lacking Annexin VI are viable and fertile, and show no gross morphological defects. While no defects in B and T-cell development, or heart function were identified, adult male annexin VI knock-out mice gain weight at about half the rate of their wild-type counterparts, suggesting a novel role for annexin VI in animal physiology. In addition, clones of DT40 cells were generated with disruptions at both alleles of annexins II and V. The chick pre-B-cell DT40 lineage expresses several annexins including annexins I, II, V and VI. Targeted disruption is facilitated in this cell line by the expression of high levels of recombinase during IgG chain rearrangement. Loss of annexin II or V is also not lethal, but does lead to complex but distinct phenotypes involving calcium signalling, apoptosis and cell clumping. Detailed analysis of knock-out DT40 cells shows that these genes have non-redundant functions and play important roles in the cell. This work provides the experimental systems and information necessary to make significant progress in the understanding of the true roles of Annexins in cellular physiology.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Investigation of annexin function by targeted gene disruption
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Annexin function
URI: https://discovery.ucl.ac.uk/id/eprint/10101968
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