Phylactides, Marios Stellou; (1998) Generation and analysis of granulocyte elastase-deficient Cre-transgenic mice. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Neutrophils form a major proportion of the phagocytic population in the body canying out the first line of defence against bacterial and fungal infections. The ingested microbes are killed by a combination of reactive oxygen radicals and antimicrobial proteases in the phagocytic vacuole. Discharge of reactive oxygen radicals and proteases by activated phagocytes into the extracellular space is thought to play a major role in inflammatory and other immunopathological reactions (e.g. emphysema, rheumatoid arthritis, inflammatory bowel disease). To determine the physiologic and pathologic function of granulocyte elastase (GE), one of the major proteases in neutrophils. we have cloned and inactivated the gene encoding GE by homologous recombination in mouse embryonic stem cells. Correctly targeted clones were introduced into blastocysts for the generation of chimeric mice used to establish a GE-deficient mouse line The GE-deficient mice were shown by Northern blot analysis to have no detectable GE mRNA Western blot analysis further showed absence of GE protein The GE-deficient mice appear to be phenotypically normal under the conditions encountered in the animal house facilities. Neutrophil numbers and neutrophil migration into the peritoneum following a sterile inflammatory stimulus are normal as is neutrophil degranulation. GE-deficient mice showed no increase in mortality rate when challenged with Staphylococcus aureus (S. aureus), a gram positive bacterial pathogen. Tissue clearance of the S. aureus infection by wild type and mutant mice was comparable with both types of mice developing acute, but not fatal, progressive pyelonephritis five days post-infection. Concomitant with GE inactivation, the P1 bacteriophage cre recombinase gene was introduced into the mouse genome 5' of GE so that its start of translation site directly replaced that of GE. The aim was to establish a granulocyte specific knockout program based on the Cre-loxp site-specific recombination process. Cre recombinase activity was shown to be prominent in granulocyte fractions and at much reduced levels in other tissues examined, probably resulting from contaminating granulocytes.

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