Apolipoprotein (apo) E, a constituent of several lipoproteins, is a ligand for the low density lipoprotein receptor, and this interaction is important for maintaining cholesterol and triglyceride homeostasis. We have used a gene replacement strategy to generate mice that express the human apoE3 isoform in place of the mouse protein. The levels of apoE mRNA in various tissues are virtually the same in the human apoE3 homozygous (3/3) mice and their littermates having the wild type mouse allele (+/+). Total cholesterol and triglyceride levels in fasted plasma from the 3/3 mice were not different from those in the +/+ mice, when maintained on a normal (low fat) chow diet. We found, however, notable differences in the distribution of plasma lipoproteins and apolipoprotein E between the two groups: β-migrating lipoproteins and plasma apoB100 levels are decreased in the 3/3 mice, and the apoE distribution is shifted from high density lipoproteins to larger lipoprotein particles. In addition, the fractional catabolic rate of exogenously administered remnant particles without apoE was 6-fold slower in the 3/3 mice compared with the +/+ mice. When the 3/3 and +/+ animals were fed a high fat/high cholesterol diet, the 3/3 animals responded with a dramatic increase (5-fold) in total cholesterol compared with the +/+ mice (1.5-fold), and after 12 weeks on this same diet the 3/3 animals developed significantly (at least 13-fold) larger atherosclerotic plaques in the aortic sinus area than the +/+ animals. Thus the structural differences between human APOE3 and mouse ApoE proteins are sufficient to cause an increased susceptibility to dietary-induced hypercholesterolemia and atherosclerosis in the 3/3 mice. Apolipoprotein (apo) E, a constituent of several lipoproteins, is a ligand for the low density lipoprotein receptor, and this interaction is important for maintaining cholesterol and triglyceride homeostasis. We have used a gene replacement strategy to generate mice that express the human apoE3 isoform in place of the mouse protein. The levels of apoE mRNA in various tissues are virtually the same in the human apoE3 homozygous (3/3) mice and their littermates having the wild type mouse allele (+/+). Total cholesterol and triglyceride levels in fasted plasma from the 3/3 mice were not different from those in the +/+ mice, when maintained on a normal (low fat) chow diet. We found, however, notable differences in the distribution of plasma lipoproteins and apolipoprotein E between the two groups: β-migrating lipoproteins and plasma apoB100 levels are decreased in the 3/3 mice, and the apoE distribution is shifted from high density lipoproteins to larger lipoprotein particles. In addition, the fractional catabolic rate of exogenously administered remnant particles without apoE was 6-fold slower in the 3/3 mice compared with the +/+ mice. When the 3/3 and +/+ animals were fed a high fat/high cholesterol diet, the 3/3 animals responded with a dramatic increase (5-fold) in total cholesterol compared with the +/+ mice (1.5-fold), and after 12 weeks on this same diet the 3/3 animals developed significantly (at least 13-fold) larger atherosclerotic plaques in the aortic sinus area than the +/+ animals. Thus the structural differences between human APOE3 and mouse ApoE proteins are sufficient to cause an increased susceptibility to dietary-induced hypercholesterolemia and atherosclerosis in the 3/3 mice. Apolipoprotein (apo) 1The abbreviations used are: apo, apolipoprotein; LDLR, low density lipoprotein receptor; VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; R, receptor; HDL, high density lipoprotein; HSPG, heparan sulfate proteoglycans; 3/3, mice homozygous for human apoE3 allele; 3/+, mice heterozygous for human apoE3; +/+, mice homozygous for wild type mouse apoE allele; kb, kilobase; ES cell, embryonic stem cell; TC, total cholesterol; ELISA, enzyme-linked immunosorbent assay; hu, human; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: apo, apolipoprotein; LDLR, low density lipoprotein receptor; VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; R, receptor; HDL, high density lipoprotein; HSPG, heparan sulfate proteoglycans; 3/3, mice homozygous for human apoE3 allele; 3/+, mice heterozygous for human apoE3; +/+, mice homozygous for wild type mouse apoE allele; kb, kilobase; ES cell, embryonic stem cell; TC, total cholesterol; ELISA, enzyme-linked immunosorbent assay; hu, human; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis. E is important for the transport of cholesterol and triglyceride throughout the body. It is an amphipathic protein that stabilizes the structure of lipoprotein particles via its ability to bind lipid, and it functions as a ligand for lipoprotein receptors, such as the low density lipoprotein receptor (LDLR) and the low density lipoprotein receptor-related protein (1Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, 2Herz J. Hamann U. Rogne S. Myklebost O. Gausepohl H. Stanley K.K. EMBO J. 1988; 7: 4119-4127Crossref PubMed Scopus (736) Google Scholar, 3Mahley R.W. Innerarity T.L. J. Biol. Chem. 1977; 252: 3980-3986Abstract Full Text PDF PubMed Google Scholar). ApoE is a major protein component of chylomicron remnants, very low density lipoproteins (VLDL), and intermediate density lipoproteins (IDL), but it is not present on low density lipoproteins (LDL). ApoE is also enriched in a subclass of high density lipoproteins (HDL) and functions as an effective ligand for their binding to the LDLR (4Mahley R.W. Dietschy J.M. Gotto Jr., A.M. Ontko J.A. Disturbances in Lipid and Lipoprotein Metabolism. American Physiological Society, Bethesda1978: 181-197Google Scholar).The human APOE gene is located on chromosome 19 (5Olaisen B. Teisberg P. Gedde-Dahl Jr., T. Hum. Genet. 1982; 62: 233Crossref PubMed Scopus (110) Google Scholar) as part of an apolipoprotein cluster that also includes the genes encodingAPOCI, APOCII, and APOCIV (6Kardassis D. Laccotripe M. Talianidis I. Zannis V. Hypertension. 1996; 27: 980-1008Crossref PubMed Scopus (34) Google Scholar). Multiple tissue-specific enhancers and negative elements have been identified in the region proximal to the gene (7Paik Y.-K. Chang D.J. Reardon C.A. Walker M.D. Taxman E. Taylor J.M. J. Biol. Chem. 1988; 263: 13340-13349Abstract Full Text PDF PubMed Google Scholar). A distal enhancer, the hepatic control region, that is required for liver expression is located 15 kb downstream of the gene (8Smith J.D. Melian A. Leff T. Breslow J.L. J. Biol. Chem. 1988; 263: 8300-8308Abstract Full Text PDF PubMed Google Scholar, 9Simonet W.S. Bucay N. Lauer S.J. Taylor J.M. J. Biol. Chem. 1993; 268: 8221-8229Abstract Full Text PDF PubMed Google Scholar). The liver synthesizes approximately 70% apoE found in the body and 20% is found in the brain, and the remainder is synthesized in several tissues including the spleen, lung, heart, ovary, testis, kidney, and skin (10Blue M.L. Williams D.L. Zucker S. Khan S.A. Blum C.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 283-287Crossref PubMed Scopus (138) Google Scholar, 11Basu S.K. Ho Y.K. Brown M.S. Bilheimer D.W. Anderson R.G.W. Goldstein J.L. J. Biol. Chem. 1982; 257: 9788-9795Abstract Full Text PDF PubMed Google Scholar).Three major APOE alleles, ε2, ε3, and ε4, occur in humans at frequencies of 7.3, 78.3, and 14.3%, respectively (12Hallman D.M. Boerwinkle E. Saha N. Sandholzer C. Menzel H.J. Csázár A. Utermann G. Am. J. Hum. Genet. 1991; 49: 338-349PubMed Google Scholar). These three alleles are distinguished by coding differences at positions 112 and 158 (13Rall Jr., S.C. Weisgraber K.H. Mahley R.W. J. Biol. Chem. 1982; 257: 4171-4178Abstract Full Text PDF PubMed Google Scholar). The most common isoform, apoE3, has a cysteine at position 112 and an arginine at position 158; the apoE2 isoform has a cysteine at both positions, whereas the apoE4 isoform has an arginine at both positions. Functionally, the three isoforms differ in their affinity for the LDLR, with apoE3 and E4 exhibiting 100% binding and apoE2 displaying only 1% normal binding affinity (14Weisgraber K.H. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1982; 257: 2518-2521Abstract Full Text PDF PubMed Google Scholar). Despite the lower affinity of apoE2 for the LDLR, individuals homozygous for ε2 typically have lower than normal plasma cholesterol levels, except for the fraction of homozygotes (5–10%) who develop type III hyperlipoproteinemia (15Utermann G. La Ricerca Clin. Lab. 1982; 12: 23-33PubMed Google Scholar). Individuals homozygous for ε4 have higher total plasma cholesterol and LDL cholesterol compared with ε3 homozygotes and are at increased risk for developing coronary artery disease (16Boerwinkle E. Visvikis S. Welsh D. Steinmetz J. Hanash S.M. Sing C.F. Am. J. Hum. Genet. 1987; 27: 567-582Crossref Scopus (163) Google Scholar). The ε4 allele is also associated with the development of Alzheimer's disease (17Schmechel D.E. Saunders A.M. Strittmatter W.J. Crain B.J. Hulette C.M. Joo S.H. Pericak-Vance M.A. Goldgaber D. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9649-9653Crossref PubMed Scopus (1336) Google Scholar, 18Saunders A.M. Strittmatter W.J. Schmechel D. George-Hyslop S.T. Pericak-Vance M.A. Joo S.H. Rosi B.L. Gusella J.F. Crapper-MacLachlan D.R. Albert M.J. Hulette C. Crain B. Goldgaber D. Roses A.D. Neurology. 1993; 43: 1467-1472Crossref PubMed Google Scholar).ApoE isoform-specific effects are important in the etiology of atherosclerosis and other diseases, but appropriate animal models to rigorously investigate these effects are currently lacking. To date, transgenic animals made by pronuclear injection of human DNA have been used to study these effects, but this method produces mice with varying levels of transgene expression due to differences in chromosomal location and copy number, and expression of the endogenous mouse apoE complicates the interpretation of the data (19Smith J.D. Plump A.S. Hayek T. Walsh A. Breslow J.L. J. Biol. Chem. 1990; 265: 14709-14712Abstract Full Text PDF PubMed Google Scholar, 20Simonet W.S. Bucay N. Lauer S.J. Wirak D.O. Stevens M.E. Weisgraber K.H. Pitas R.E. Taylor J.M. J. Biol. Chem. 1990; 265: 10809-10812Abstract Full Text PDF PubMed Google Scholar, 21Fazio S. Horie Y. Simonet W.S. Weisgraber K.H. Taylor J.M. Rall Jr., S.C. J. Lipid Res. 1994; 35: 408-416Abstract Full Text PDF PubMed Google Scholar, 22Mortimer B.-C. Redgrave T.G. Spangler E.A. Verstuyft J.G. Rubin E.M. Arterioscler. Thromb. 1994; 14: 1542-1552Crossref PubMed Google Scholar, 23van den Maagdenberg A.M.J.M. Hofker M.H. Krimpenfort P.J.A. de Bruijn I. van Vlijmen B. van der Boom H. Havekes L.M. Frants R.R. J. Biol. Chem. 1993; 268: 10540-10545Abstract Full Text PDF PubMed Google Scholar, 24Xu P.-T. Schmechel D. Rothrock-Christian T. Burkhart D.S. Qiu H-L. Popko B. Sullivan P.M. Maeda N. Saunders A.M. Roses A.D. Gilbert J.R. Neurobiol. Dis. 1996; 3: 229-245Crossref PubMed Scopus (159) Google Scholar, 47van Vlijmen B.J.M. van Dijk K.W. van't Hof H.B. van Gorp P.J.J. van der Zee A. van der Boom H. Breuer M.L. Hofker M.H. Havekes L.M. J. Biol. Chem. 1996; 271: 30595-30602Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). To overcome these difficulties we have used targeted gene replacement to generate mice that express only the human apoE3 isoform at levels that are in the physiological range. In these mice the coding sequences for mouseapoE were replaced with sequences coding for humanAPOE3 without disturbing any of the known regulatory sequences. This replacement results in animals with expression of human apoE mRNA, identical in tissue distribution and levels, to that of mouse apoE mRNA in wild type animals.We here describe the essentially normal cholesterol and triglyceride levels of mice homozygous for human apoE3 (3/3) when maintained on a normal (low fat) mouse chow diet but show that they are markedly more susceptible to diet-induced atherosclerosis than their wild type littermates (+/+).DISCUSSIONWe have successfully replaced the coding sequence of the mouseapoE gene with the equivalent human APOE3 coding region without altering any known endogenous regulatory sequences. Expression of the human apoE mRNA in the liver, brain, and other tissues parallels that found normally in wild type mice. Thus, the human APOE3 protein is functional and is expressed at physiological levels in these modified mice, thereby providing a model for studying the function of human APOE3 in vivo.To date, pronuclear injection of DNA has been used to generate transgenic animals expressing various forms of human APOE to study its role in lipid metabolism (19Smith J.D. Plump A.S. Hayek T. Walsh A. Breslow J.L. J. Biol. Chem. 1990; 265: 14709-14712Abstract Full Text PDF PubMed Google Scholar, 20Simonet W.S. Bucay N. Lauer S.J. Wirak D.O. Stevens M.E. Weisgraber K.H. Pitas R.E. Taylor J.M. J. Biol. Chem. 1990; 265: 10809-10812Abstract Full Text PDF PubMed Google Scholar, 21Fazio S. Horie Y. Simonet W.S. Weisgraber K.H. Taylor J.M. Rall Jr., S.C. J. Lipid Res. 1994; 35: 408-416Abstract Full Text PDF PubMed Google Scholar, 22Mortimer B.-C. Redgrave T.G. Spangler E.A. Verstuyft J.G. Rubin E.M. Arterioscler. Thromb. 1994; 14: 1542-1552Crossref PubMed Google Scholar, 23van den Maagdenberg A.M.J.M. Hofker M.H. Krimpenfort P.J.A. de Bruijn I. van Vlijmen B. van der Boom H. Havekes L.M. Frants R.R. J. Biol. Chem. 1993; 268: 10540-10545Abstract Full Text PDF PubMed Google Scholar, 24Xu P.-T. Schmechel D. Rothrock-Christian T. Burkhart D.S. Qiu H-L. Popko B. Sullivan P.M. Maeda N. Saunders A.M. Roses A.D. Gilbert J.R. Neurobiol. Dis. 1996; 3: 229-245Crossref PubMed Scopus (159) Google Scholar, 47van Vlijmen B.J.M. van Dijk K.W. van't Hof H.B. van Gorp P.J.J. van der Zee A. van der Boom H. Breuer M.L. Hofker M.H. Havekes L.M. J. Biol. Chem. 1996; 271: 30595-30602Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). There are three severe limitations to this approach as follows: the number of copies of the transgene can vary over wide ranges; there are marked and uncontrolled differences in the expression of transgenes at different chromosomal locations; and the presence of the endogenous mouse protein complicates the interpretation of the results. Most importantly, rigorous comparisons between different isoforms are not possible because of the variable expression inherent in conventional transgenic animals.Mice made by replacement of the endogenous mouse coding region with the equivalent region of the human gene retain the natural chromosomal context at the apoE locus. Thus, locus-specific sequences and enhancers are not altered except for any possible intragenic control sequences that differ between mice and humans. Therefore, it was expected that tissue-specific expression of the human replacement APOE3gene would closely parallel that of the endogenous mouse apoE gene. Our mRNA studies show that this expectation was indeed fulfilled, with the exception of intestinal apoE mRNA which was slightly reduced in the 3/3 mice compared with the +/+ mice. The significance of this reduction in intestinal expression is not clear, considering that intestinal apoE expression is very low compared with other tissues and that chylomicrons originating from the small intestine are known to contain very little apoE (34Robinson S.F. Quarfordt S.H. Biochim. Biophys. Acta. 1978; 541: 492-503Crossref PubMed Scopus (13) Google Scholar, 35Imaizumi K. Fainaru M. Havel R.J. J. Lipid Res. 1978; 19: 712-722Abstract Full Text PDF PubMed Google Scholar). Differences between the 3/3 and +/+ mice consequently are highly informative as far as function of the apolipoprotein itself are concerned. Thus, gene replacement demonstrates the advantages over pronuclear injection in generating transgenic animals.The lipid profiles of the 3/3 and +/+ mice are very similar when the mice are maintained on a chow (low fat) diet, and the fasting levels of total cholesterol, triglyceride, and HDL cholesterol are essentially identical in the two animals. However, when the same mice were fed a high fat diet, the 3/3 animals responded with a 5-fold increase in total cholesterol compared with a 1.5-fold increase in the +/+ animals. Second, after 3 months on a high fat diet, the atherosclerotic plaques seen in all the 3/3 animals were 13-fold larger than those observed in the wild type controls. Third, there was a 6-fold slower clearance rate of remnant lipoprotein particles in the 3/3 animals. The accumulation of cholesterol and the delayed clearance of remnant particles observed in the 3/3 animals are likely causes of the increased susceptibility to lesion development.The dramatic delay in clearance of remnant particles in the 3/3 mice was unexpected since previous studies using transgenic mice expressing human APOE have revealed normal or enhanced clearance kinetics (22Mortimer B.-C. Redgrave T.G. Spangler E.A. Verstuyft J.G. Rubin E.M. Arterioscler. Thromb. 1994; 14: 1542-1552Crossref PubMed Google Scholar,36de Silva H.V. Lauer S.J. Wang J. Simonet W.S. Weisgraber K.H. Mahley R.W. Taylor J.M. J. Biol. Chem. 1994; 269: 2324-2335Abstract Full Text PDF PubMed Google Scholar). One possible explanation for the observed delay in clearance is that the human APOE3 protein has a lower affinity to mouse LDLR and/or other receptors than mouse ApoE because of species differences in their structures. Mouse and human apoE differ in 30% of their amino acid sequences with most of the differences being at the amino and carboxyl termini (1Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). The receptor binding domain (residues 136–160) is highly conserved between mouse and human apoE since 21 of 25 residues are identical in this region including seven residues designated as critical for receptor binding (37Yang Y.-W. Chan L. Li W.-H. J. Mol. Evol. 1991; 32: 469-475Crossref PubMed Scopus (17) Google Scholar, 38Wardell M.R. Brennan S.O. James E.D. Fraser R. Carrell R.W. J. Clin. Invest. 1987; 80: 483-490Crossref PubMed Scopus (96) Google Scholar). The residues that differ have conservative substitutions (e.g. methionine for leucine) that do not affect the net charge or hydrophobicity of the protein and would not be predicted to have any substantial effects on the protein conformation (39Lalazar A. Weisgraber K.H. Rall Jr., S.C. Giladi H. Innerarity T.L. Levanon A.Z. Boyles J.K. Amit B. Gorecki M. Mahley R.W. Vogel T. J. Biol. Chem. 1988; 263: 3542-3545Abstract Full Text PDF PubMed Google Scholar). Thus, it must be ascribed to differences outside the receptor binding domain if the affinity for the receptor differs between the mouse and human apoE proteins. Species differences in the receptors that interact with apoE, such as the LDLR, and low density lipoprotein receptor-related protein, could also contribute to a lower affinity.A dysfunctional interaction between human APOE3 and mouse heparan sulfate proteoglycans (HSPG) may also contribute to the delay in clearance. It has been postulated that HSPG's are involved in the initial sequestration process that enhances the uptake of apoE containing lipoproteins (40Landis B.A. Rotolo F.S. Meyers W.C. Clark A.B. Quarfordt S.H. Am. J. Physiol. 1987; 252: C805-C810PubMed Google Scholar, 41Mahley R.W. Hussain M.M. Curr. Opin. Lipidol. 1991; 2: 170-176Crossref Scopus (104) Google Scholar). The interaction of apoE with HSPG's is thought to occur mainly via electrostatic attraction (42Mahley R.W. Weisgraber K.H. Innerarity T.L. Biochim. Biophys. Acta. 1979; 575: 81-91Crossref PubMed Scopus (102) Google Scholar). Human APOE3 has a cysteine at position 112 where the mouse protein has an arginine at the equivalent position (mouse apoE residue 104). This charge difference could be significant, even though residue 112 is outside the known heparin-binding domain.The delayed clearance could also be due to protein-protein interactions not involving receptors. Several proteins are known to interact with apoE and play a role in lipid metabolism. For example, the interaction of apoE with hepatic and lipoprotein lipases mediates/or enhances the catabolism of lipoproteins (41Mahley R.W. Hussain M.M. Curr. Opin. Lipidol. 1991; 2: 170-176Crossref Scopus (104) Google Scholar). Lipoprotein particles containing human APOE3 may be less efficient in this type of interaction than mouse ApoE. This would have important consequences in the remodeling and processing phase of lipoprotein catabolism.Another consideration in the clearance experiment is that the exogenously introduced remnant particles have to acquire apoE prior to their removal by receptors. In +/+ mice most of the plasma apoE is associated with HDL particles that are presumably the source of apoE used to clear the exogenously added remnants. In the 3/3 animals, however, the majority of plasma apoE3 is associated with large lipoproteins and may not transfer as easily to exogenously added particles. In support of this, we and others (27de Silva H.V. Más-Oliva J. Taylor J.M. Mahley R.W. J. Lipid Res. 1994; 35: 1297-1310Abstract Full Text PDF PubMed Google Scholar) have noted that the distributions of apoE among mouse plasma lipoproteins analyzed by FPLC and by ultracentrifugation do not always agree and that apoE associated with HDL are more readily lost by ultracentrifugation. Although the capacity for apoE3 to exchange between different classes of lipoproteins has not been documented, the decreased ability of apoE3 to transfer from large lipoproteins may have important implications.The association of apoE3 with larger lipoproteins in mice (Fig. 5,A and C) is different from observations made in humans that demonstrate a preference of apoE3 for HDL (1Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, 43Weisgraber K.H. J. Lipid Res. 1990; 31: 1503-1511Abstract Full Text PDF PubMed Google Scholar). It is very unlikely that this difference is the result of a mutation unexpectedly introduced into the APOE3 gene during its manipulation because in our work 2P. M. Sullivan and N. Maeda, unpublished observations. we observed that transgenic apoE3 mice, in an endogenous apoE (−/−) background, had a virtually identical apoE3 distribution pattern as seen in the 3/3 replacement mice. These mice were made by pronuclear injection of a human cosmid clone isolated from a different individual from the one we used to make our mice (24Xu P.-T. Schmechel D. Rothrock-Christian T. Burkhart D.S. Qiu H-L. Popko B. Sullivan P.M. Maeda N. Saunders A.M. Roses A.D. Gilbert J.R. Neurobiol. Dis. 1996; 3: 229-245Crossref PubMed Scopus (159) Google Scholar). Similar findings were also reported by Taylor et al. (44Taylor J.M. Simonet W.S. Bucay N. Lauer S.J. de Silva H.V. Curr. Opin. Lipidol. 1991; 2: 73-80Crossref Scopus (16) Google Scholar) in transgenic apoE3 mice (that also express mouse apoE) which showed that the majority of human APOE3 resided in larger lipoprotein particles using SDS-PAGE immunoblot analysis of fractions separated by sequential density ultracentrifugation. In this work the authors also found that mouse ApoE and human APOE co-distributed among lipoproteins. This is different from the results of our FPLC analysis of 3/+ heterozygote plasma which shows that approximately half of the mouse ApoE and only a minor fraction of human APOE3 was associated with HDL (data not shown). It is not known whether the observed differences between our work and Taylor et al. are the result of different techniques used to fractionate the plasma lipoproteins or the result of different levels of human APOE and mouse ApoE expression in the two types of mice. Further characterization of these large apoE3-rich lipoprotein particles is required to understand the observed delay in clearance in the 3/3 animals.The reduced clearance rate of remnant particles in the 3/3 mice, whatever its cause eventually proves to be, is not sufficient to cause any alteration in fasted plasma cholesterol levels in these animals relative to their +/+ littermates when the animals are on a normal chow diet. The 3/3 mice are, however, much more susceptible to diet-induced hyperlipidemia and atherosclerosis than their +/+ littermates. Possibly, on the high fat diet a postprandial accumulation of β-VLDL's occurs that leads to the development of atherosclerosis, as suggested by Zilversmit (45Zilversmit D.B. Circulation. 1979; 60: 473-485Crossref PubMed Scopus (1424) Google Scholar). In human APOE2 homozygous individuals with type III hyperlipoproteinemia, there is an abnormal accumulation of remnant particles that has been suggested to cause accelerated coronary and peripheral vascular disease (46Slyper A.H. Lancet. 1992; 340: 289-291Abstract PubMed Scopus (54) Google Scholar). Even small changes in lipid metabolism that decrease the clearance efficiency of lipoproteins are likely to have dramatic effects on the development of atherosclerosis, and thus the 3/3 animals give us an opportunity to investigate what changes are important.In conclusion, we have demonstrated the use of targeted gene replacement to generate mice expressing the most common human isoform, APOE3, at physiological levels. These mice allow us to document the behavior of the human APOE3 isoform in vivo in mice without any co-expression of mouse apoE. Future experiments comparing these mice with mice expressing the human apoE2 and E4 isoforms created in an identical manner should prove invaluable for studying diseases related to the different human apoE isoforms. Apolipoprotein (apo) 1The abbreviations used are: apo, apolipoprotein; LDLR, low density lipoprotein receptor; VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; R, receptor; HDL, high density lipoprotein; HSPG, heparan sulfate proteoglycans; 3/3, mice homozygous for human apoE3 allele; 3/+, mice heterozygous for human apoE3; +/+, mice homozygous for wild type mouse apoE allele; kb, kilobase; ES cell, embryonic stem cell; TC, total cholesterol; ELISA, enzyme-linked immunosorbent assay; hu, human; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: apo, apolipoprotein; LDLR, low density lipoprotein receptor; VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; R, receptor; HDL, high density lipoprotein; HSPG, heparan sulfate proteoglycans; 3/3, mice homozygous for human apoE3 allele; 3/+, mice heterozygous for human apoE3; +/+, mice homozygous for wild type mouse apoE allele; kb, kilobase; ES cell, embryonic stem cell; TC, total cholesterol; ELISA, enzyme-linked immunosorbent assay; hu, human; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis. E is important for the transport of cholesterol and triglyceride throughout the body. It is an amphipathic protein that stabilizes the structure of lipoprotein particles via its ability to bind lipid, and it functions as a ligand for lipoprotein receptors, such as the low density lipoprotein receptor (LDLR) and the low density lipoprotein receptor-related protein (1Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, 2Herz J. Hamann U. Rogne S. Myklebost O. Gausepohl H. Stanley K.K. EMBO J. 1988; 7: 4119-4127Crossref PubMed Scopus (736) Google Scholar, 3Mahley R.W. Innerarity T.L. J. Biol. Chem. 1977; 252: 3980-3986Abstract Full Text PDF PubMed Google Scholar). ApoE is a major protein component of chylomicron remnants, very low density lipoproteins (VLDL), and intermediate density lipoproteins (IDL), but it is not present on low density lipoproteins (LDL). ApoE is also enriched in a subclass of high density lipoproteins (HDL) and functions as an effective ligand for their binding to the LDLR (4Mahley R.W. Dietschy J.M. Gotto Jr., A.M. Ontko J.A. Disturbances in Lipid and Lipoprotein Metabolism. American Physiological Society, Bethesda1978: 181-197Google Scholar). The human APOE gene is located on chromosome 19 (5Olaisen B. Teisberg P. Gedde-Dahl Jr., T. Hum. Genet. 1982; 62: 233Crossref PubMed Scopus (110) Google Scholar) as part of an apolipoprotein cluster that also includes the genes encodingAPOCI, APOCII, and APOCIV (6Kardassis D. Laccotripe M. Talianidis I. Zannis V. Hypertension. 1996; 27: 980-1008Crossref PubMed Scopus (34) Google Scholar). Multiple tissue-specific enhancers and negative elements have been identified in the region proximal to the gene (7Paik Y.-K. Chang D.J. Reardon C.A. Walker M.D. Taxman E. Taylor J.M. J. Biol. Chem. 1988; 263: 13340-13349Abstract Full Text PDF PubMed Google Scholar). A distal enhancer, the hepatic control region, that is required for liver expression is located 15 kb downstream of the gene (8Smith J.D. Melian A. Leff T. Breslow J.L. J. Biol. Chem. 1988; 263: 8300-8308Abstract Full Text PDF PubMed Google Scholar, 9Simonet W.S. Bucay N. Lauer S.J. Taylor J.M. J. Biol. Chem. 1993; 268: 8221-8229Abstract Full Text PDF PubMed Google Scholar). The liver synthesizes approximately 70% apoE found in the body and 20% is found in the brain, and the remainder is synthesized in several tissues including the spleen, lung, heart, ovary, testis, kidney, and skin (10Blue M.L. Williams D.L. Zucker S. Khan S.A. Blum C.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 283-287Crossref PubMed Scopus (138) Google Scholar, 11Basu S.K. Ho Y.K. Brown M.S. Bilheimer D.W. Anderson R.G.W. Goldstein J.L. J. Biol. Chem. 1982; 257: 9788-9795Abstract Full Text PDF PubMed Google Scholar). Three major APOE alleles, ε2, ε3, and ε4, occur in humans at frequencies of 7.3, 78.3, and 14.3%, respect