Kenny, Raechel; (1992) Towards a mouse model for haemophilia. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Haemophilias A and B are X-linked recessive bleeding disorders caused by deficiencies of coagulation factors VIII and IX respectively. Present treatment for the disease, by replacement therapy with products extracted from large volumes of pooled blood, carries a high risk of infection. The aim of this project is to develop a mouse model for haemophilia, in which proposed gene therapy protocols can be studied. To do this I have proposed to use homologous recombination to disrupt the endogenous factor VIII and IX genes in murine embryonic stem cells. Correctly targeted cells would be introduced into a developing mouse embryo where they would contribute to the germline and subsequently to a line of transgenic, haemophilia mice. As a first step towards this model, genomic bacteriophage libraries have been screened for the murine factor VIII and IX genes. Five bacteriophage clones were isolated and shown to contain the mouse factor IX gene. This locus has subsequently been characterised by dideoxy-sequencing and bacteriophage mapping methods. The mouse factor IX gene spans more than 53kb and shares, in the coding regions, 86% sequence identity with the human gene. The structure of mouse factor IX is discussed. No factor Vlll-containing clones have been isolated, although sequences thought to be part of the murine factor VIII gene have been amplified by reverse transcription/PCR. Targeting constructs have been produced which contain part of the mouse factor IX gene disrupted by the selectable Neo cassette, and the HSV-tk gene which will allow the use of positive/negative selection procedures. These constructs have been introduced into the Embryonic Stem Cell line, E14, by electroporation, but no targeting events have yet been identified.

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