Gomez, Charles Keith Rizleigh; (2007) Demonstration of Tissue Factor expression using a Tissue Factor – Green Fluorescent Protein Reporter Model. Doctoral thesis (Ph.D), Imperial College, University of London.

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Abstract

Blood coagulation is initiated when circulating factor (F) VII comes into contact with membrane bound tissue factor (TF). Subsequently a series of enzymatic cleavages results in the formation of insoluble fibrin. The coagulation network is of fundamental importance in maintaining the integrity of the circulation and is closely integrated with other processes such as inflammation. Both under- and over-activity of the network have significant pathological consequences and thus the network is tightly regulated. The first point of regulation is in the formation of the TF•FVIIa initiating complex. Circulating FVII is kept separate from membrane-bound TF by an intact vascular wall. The initiation of coagulation is thus dependant on the site of TF expression. In addition abnormal TF expression has been shown to be important in various pathological processes such as atherosclerosis and cancer metastasis. The accurate demonstration of TF activity is of critical importance in the understanding of these processes. This thesis describes the development of a TF-Enhanced Green Fluorescent Protein (EGFP) reporter model for analysis of TF expression. A targeting vector containing the cDNA for EGFP in-frame with and flanked by regions homologous to the mouse TF gene was generated. The vector was designed such that EGFP would be knocked in to the TF locus by homologous recombination to produce a fusion protein with EGFP tagged onto the intact cytoplasmic domain of TF. Two strategies were used for generating transgenic mice. A targeting vector was electroporated into mouse embryonic stem (ES) cells and correctly targeted ES cells were then used in blastocyst injections. Alternatively a bacterial artificial chromosome (BAC) containing the mouse TF gene and flanking regions was targeted in a bacterial host using a similar vector. The targeted BAC was used in pro-nuclear injections of fertilized mouse ova resulting in random integration into the mouse genome. The function of the TF-EGFP reporter model was investigated by analysis of transgene expression in whole tissues and specific cell types taken from transgenic mice.

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