To obtain a better understanding of the relationships between atherogenesis and genetically controlled molecular variability, I propose to make specific alterations in several lipid-metabolism- related genes of otherwise normal experimental animals. Modification of each gene will be achieved in mouse embryonic stem cells by targeted homologous recombination between the endogenous gene and exogenous DNA. Cells with the specifically modified gene will be identified, clonally propagated, and reintroduced into recipient blastocysts for the production of chimeric mice. Such chimeric mice can transmit the modified gene to their offspring. The resulting animals, having fully characterized single-gene changes, should be very valuable (i) for dissecting molecular aspects of atherogenesis, (ii) as models of human atherosclerosis and (iii) for the development of (new) drug treatments. Specifically, (I) I will modify the apolipoprotein B (apo B) gene so as to replace the low density lipoprotein (LDL) receptor binding sites of apo B by an equal number of amino acids from an unrelated protein. Because each LDL particle contains only a single apo B molecule, which functions as a ligand for the clearance of the particle, this type of mutation in apo B will have a co-dominant effect. The resulting mice are expected to show elevated levels of LDL ln their plasma, and to be susceptible to developing atherosclerotic lesions at an early stage in their lives. (II) I will inactivate the apolipoprotein E (apo E) gene by inserting the bacterial B-galactosidase gene into one of its exons. This mutation should behave in a recessive fashion, and homozygotes for the inactive gene are expected to show phenotypes similar to type III hyperlipoproteinemia in humans. (III) Golden hamsters may provide better animal models of human atherosclerosis than mice because the overall balance of the lipid metabolic pathways in golden hamsters is much closer to that in humans than is the case in mice. I plan to set up procedures allowing me to generate transgenic hamsters from embryonic stem cells in order to accomplish comparable modifications in golden hamster apolipoprotein genes.
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