Proc. Natl. Acad. Sci. USAVol. 91, pp. 2130-2134, March 1994Genetics

Expression of human apolipoprotein B and assembly oflipoprotein(a) in transgenic miceMATTHEW J. CALLOW*, Lowi J. STOLTZFUS*, RICHARD M. LAWNt, AND EDWARD M. RUBIN*I*Life Sciences Division, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720; and tFalk Cardiovascular Research Center, StanfordUniversity, Stanford, CA 94305

Communicated by Charles V. Shank, November 5, 1993

ABSTRACT The atherogenic macromolecule lipopro-tein(a) [Lp(a)] has resisted in vvo analyses partly because it isfound in a limited number of experimental animals. Althoughtransgenic mice expressing human apolipoprotein (a) [apo(a)]have previously been described, they faied to assemble Lp(a)particles because of the inability of human apo(a) to associatewith mouse apolipoprotein B (apoB). We isolated a 90-kilobaseP1 paemid containing the human apoB gene and with thisDNA generated 13 lines of tnsgec mice of which 11 ex-pressed human apoB. The human apoB transcript was ex-pressed and edited in the liver of the transgenic mice. Plasmaconcentrations of human apoB, as well as low density lipopro-tein (LDL), were related to transgene copy number; thetransgnic line with the most copies of human apoB had a>44old Increase in LDL cholesterol compared with nontrasgenics and a lipoprotein profile similar to that of humans.When human apoB and apo(a) transgenic mice were bredtogether, plasma apo(a) in mice expressing both human pro-teins was tightly aciated with lipoproteins in theLDL densityregion. These studies dem ate the successful expression ofhuman apoB and the efficient assembly of Lp(a) in mice.

Numerous studies have demonstrated that an elevatedplasma level of lipoprotein (a) [Lp(a)] is a major independentrisk factor for atherosclerosis (1-3). The Lp(a) moleculeconsists of a highly polymorphic glycoprotein, apolipopro-tein(a) [apo(a)], linked to a low density lipoprotein (LDL)molecule (1-5). The mechanism by which high plasma levelsof Lp(a) lead to increased atherosclerosis is unknown. Theextensive sequence homology between apo(a) and plasmin-ogen (6) has raised the possibility that Lp(a) may competewith the normal function of plasminogen. Although some ofthe properties ofLp(a) have been studied in vitro, the fact thatthis lipoprotein is absent in most species other than higherprimates has hindered in vivo analysis ofthe biology of Lp(a).

In humans, the association between apo(a) and LDL toform the Lp(a) particle is the result of a disulfide linkagebetween apo(a) and apolipoprotein B (apoB) ofLDL (4, 5, 7).Transgenic mice that express human apo(a) have been engi-neered, but, unlike in humans, the apo(a) in these animalswas found in the lipoprotein-free plasma fraction and was notassociated with mouse LDL (8, 9). Infusion ofisolated humanLDL, but not mouse LDL, into apo(a) transgenic animalsresults in the appearance of Lp(a) particles (8). This suggeststhat the failure to form Lp(a) in the apo(a) transgenic mice iscaused by the inability of mouse apoB-containing LDL toproperly interact with human apo(a). Thus, the developmentof mice to examine the biological properties of Lp(a) in vivorequires the creation of animals that express both humanapoB and apo(a) transgenes to facilitate assembly of Lp(a).ApoB is one ofthe largest known proteins, with 4536 amino

acids (550 kDa) (10). In addition to the full-length molecule,

a truncated version of apoB (B48) is produced from editedapoB mRNA transcripts exclusively in the intestines ofnonrodent mammals and in the intestines plus the livers ofrodents (11). The large size of the apoB gene, >43 kb, hasmade it difficult to clone an intact apoB genomic fragment inA phage or cosmid vectors for introduction into the mousegenome.A recently developed cloning system using P1 phagemids

allows the cloning of 80- to 90-kb DNA inserts (12, 13). Wehave isolated a clone containing the entire human apoB geneplus extensive 5' and 3' regions from a human P1 library. ThisDNA was introduced into fertilized mouse embryos, andnearly all of the resulting transgenic mice had significantplasma levels of human apoB and increased plasma levels ofLDL-cholesterol. Mice containing both human apo(a) andhuman apoB transgenes efficiently assembled Lp(a) parti-cles.

MATERIALS AND METHODSIsolin of Hum apoB Genomic DNA and Creation of

Trangenic Mice. A human genomic library contained in thebacteriophage P1 vector (DuPont/NEN) was screened byPCR using primers to exon 18 of the apoB gene (PCR 1:5'-TGTGGAGTTTGTGACAAATATGG-3'; PCR 2: 5'-CAGCTTGACTGGTCTCTTTGG-3'). Pi-phagemid DNAwas isolated by alkaline lysis ofthe bacterial cells followed byphenol extraction and ethanol precipitation of the DNA. TheDNA was gently treated with GeneClean (Bio 101) beforeinjection into inbred FVB zygotes as described (14, 15).Transgenic mice were screened with primers to the 5' pro-moter region of human apoB (PCR 3: 5'-AGAAGGTTCCA-GATGTCTATGAGG-3'; PCR 4: 5'-TCCAAGTATCT-GTCTTCAAGAAACC-3'). The apo(a) transgenic mice usedin this study have been described (8, 9). All animals were feda Purina Rodent laboratory chow 5001 diet.

Southern Blot Analysis. Mouse genomic DNA was isolatedfrom mouse tails as described (15). Mouse DNA was restric-tion enzyme-digested, electrophoresed in 1% agarose, andvacuum-blotted to a Duralon membrane (Stratagene). TheDNA was UV-crosslinked to the membrane and probed witha 32P-labeled 2.7-kb HindIII fragment ofexon 26 ofthe humanapoB gene (provided by R. Farese, Gladstone FoundationLaboratories, San Francisco), according to standard proce-dures (16). The transgene copy number was estimated bydetermining the emission by phosphor-imaging analysis (Mo-lecular Dynamics) relative to a human genomic sample (twogene copies per genome) and then rounding off to the nearestwhole number.RNA Analysis. Total RNA was isolated from Hep G2 cells

and from various mouse tissues using the RNAStat6O reagent

Abbreviations: Lp(a), lipoprotein(a); apoB and apo(a), apolipopro-teins B and (a); HDL, high density lipoprotein; LDL, low densitylipoprotein; VLDL, very low density lipoprotein; mAb, monoclonalantibody.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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(TelTestB, Friendswood, TX) according to the supplier'sinstructions. RNA (15 ,ug per lane) was subjected to electro-phoresis on a 1% agarose/0.6 M formaldehyde gel andtransferred to a Duralon membrane. Filters were hybridizedwith either a 2.7-kb HindIII fragment from exon 26 of humanapoB or a 168-bp PCR product corresponding to part of the5' untranslated region and exon 1 of mouse apoB that wassynthesized by amplifying mouse genomicDNA with primers5'-CAAGTACCTGCGTGAGCTCC-3' and 5'-AAGAA-CAGTAGCAGGAACAGCAG-3'. Probes were 32P-labeledby random priming (MegaPrime Kit; Amersham).Immunoblot Analysis. Plasma was obtained from blood

collected by tail bleeds and supplemented with 0.4 ,uMaprotonin, 1 mM benzamidine, 0.005% gentamicin sulfate,and 0.015% phenylmethylsulfonyl fluoride (PMSF). An ali-quot of plasma or lipoprotein fraction (15 /1) was combinedwith 20 td of "buffer A" and 50 of loading buffer and was

boiled for 5 mmi as described (8). Dithiothreitol and 2-mer-captoethanol were excluded for nonreducing conditions. Aportion was electrophoresed on precast SDS/polyacrylamidegels (6% acrylamide; Schleicher & Schuell). Proteins weretransferred to nitrocellulose at 200 mA for 2 hr, and themembrane was blocked with 3% gelatin in TBS (20 mMTris/500 mM NaCl, pH 7.5) before incubation with theappropriate biotinylated antibody in TBS with 1% gelatin and0.05% Tween 20. Monoclonal antibodies (mAbs) were usedfor the detection of apo(a) (mAb 4F3, Cappel Laboratories)and human apoB (provided by E. Krul, Washington Univer-sity, St. Louis). The primary antibody was detected with anExtravidin-alkaline phosphatase conjugate (Sigma), andcolor was developed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagents in 0.1 M Tris buffer (pH9.5).

Lipoprotein and Apolipoprotein Measurement. Total andhigh density lipoprotein (HDL)-cholesterol were determinedby using commercially available assay kits (BoehringerMannheim) adapted for use with a microtiter plate reader(17). Lipoproteins from the p < 1.21 g/ml fraction of plasmawere analyzed by agarose gel electrophoresis (18) and by2-16% nondenaturing gradient PAGE (19). Human apoBplasma levels were determined by ELISA with a human-specific antibody (International Immunology, Murrieta, CA).Mouse apoB plasma levels were determined by quantitativeimmunoblot analysis using a rabbit polyclonal antibody di-rected against mouse LDL (provided by E. Krul) withpreadsorption of human-reactive components by incubationwith p < 1.063 g/ml lipoproteins from human plasma; apo(a)plasma levels were determined by quantitative immunoblotanalysis and by ELISA.

Density Gradient Ultracentrifugation and Separation of To-tal Lipoprotein Fractions. Plasma was pooled from two mice(200 p1) and adjusted to a volume of 500 ,u and a density of1.31 g/ml with NaBr. The plasma was sequentially overlayedwith NaBr solutions of 1.21 g/ml, 1.063 g/ml, 1.019 g/ml, and1.006 g/ml (20). The tubes were centrifuged at 40,000 rpm, for26 hr at 15°C in the SW 50.1 rotor (Beckman). Ten fractionsof 520 Au were removed, and a portion of each fraction wasdesalted, concentrated in a Centricon-10 microconcentrator(Amicon), mixed with sample buffer, and subjected to elec-trophoresis on SDS/PAGE gels before immunoblot analysis.Mean densities were determined by centrifuging tubes inwhich plasma was replaced with a solution of NaBr (1.31g/ml) and measuring fraction densities with a Mettler PaarDMA 46 density meter. To isolate total lipoproteins, plasma(25 A4) was adjusted to a volume of 230 ul and to a density of1.21 g/ml with NaBr before centrifugation at 40,000 rpm for18 hr at 10TC in the type 42.2 rotor. A volume of 25 wasremoved from the tops of the tubes (p < 1.21 g/ml) and alsofrom the bottoms (p > 1.21 g/ml).

RESULTSapoB-Containing P1 Clone and apoB Transgenic Mice.

Digestion of the apoB-containing P1 clone (DMPC-HFF#1-0261G) with Not I, Cla I, Sal I, or Nru I followed bypulsed-field gel electrophoresis revealed the presence of -19kb of genomic sequence 5' to the translational start site forapoB and -14 kb of genomic DNA 3' to the transcriptiontermination site. Restriction enzyme analysis of the P1 clonealso confirmed that no gross rearrangement of the apoB genehad occurred during cloning and replication of the P1 ph-agemid. Microinjection of supercoiled or linearized P1 DNAinto fertilized embryos resulted in 46 offspring, of which 13were positive for human apoB sequences by PCR withhuman-specific primers PCR 3 and PCR 4. Southern blotanalysis of these PCR-positive mice showed the presence ofmultiple copies of the gene when compared with the humangenomic DNA. As expected, DNA from nontransgenic con-trol mice did not hybridize with the human apoB probe.Transgene copy number was roughly correlated with plasmalevels ofhuman apoB in the founder animals, with the highestplasma levels of human apoB seen in the high-copy numberanimals (Table 1).

Tissue Specificity ofHuman apoB Expression. Northern blotanalysis revealed that human apoB mRNA was expressed inthe livers of transgenic mice from both a low-expressing line(transgenic line 1) and high-expressing line (transgenic line11) (Fig. 1). Low levels of the transcript were also detectedin the heart of the high-expressing animal. However, nohuman apoB transcripts were detected in the small intestineof mice from either line. Editing of human apoB mRNA wasassessed by primer extension performed essentially as de-scribed by Driscoll et al. (21) (data not shown). The predictedhuman apoB48 extension product was observed by usingRNA derived from the liver ofthe apoB transgenic mouse andfrom human small intestine but was absent when using RNAfrom nontransgenic mice and Hep G2 cells, a cell line knownnot to edit human apoB transcripts (22).The amount of mouse apoB mRNA in liver and intestine

was examined in both a low-expressing (data not shown) andhigh-expressing human apoB transgenic mouse (lines 1 and11, respectively) and compared with that in a nontransgeniclittermate (Fig. 1 Right). In the high-expressing animal, a

Table 1. Effect of human apoB transgene on plasma humanapoB and cholesterol concentrations

Cholesterol,Human apoB mg/dl

In plasma, Copy non-Mouse mg/dl no. HDL HDL Sex

Tg 1 1.6 1 31 130 MTg 2 6.1 2 33 160 FTg 3 6.5 1 39 125 MTg 4 9.3 2 23 133 MTg 5 12.6 6 44 104 FTg6. 13.2 5 30 99 FTg7 16.4 12 42 135 MTg 8 20.8 ND 79 91 FTg 9 23.2 10 56 112 FTg 10 26.6 ND 46 123 MTg 11 71.5 15 98 73 FNon-Tg 1 0.00 0 23 159 FNon-Tg 2 0.00 0 25 101 F

Eleven founder mice (6-10 weeks old) expressing human apoBidentified by immunoblot analysis were examined. Transgene copynumber, apoB concentration, and cholesterol levels were determinedas described in text. Single determinations of apoB concentrationsand cholesterol were performed in duplicate. ND, not determined;Tg, transgenic; M, male; F, female.

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AB100 -;- 4

B48 P "

1 2 3 4 5 6 7 8 9 10Fraction

C

FIG. 2. Distribution of human apoB and lipoproteins within theplasma of mice expressing human apoB. (A) Plasma from twotransgenic mice derived from transgenic founder mouse 4 wassubfractionated by density-gradient ultracentrifugation as describedin text and electrophoresed under nonreducing conditions beforetransfer to nitrocellulose and immunoblotting with a human apoB-specific mAb. The equivalent of 72 A4 of each density fraction wasloaded onto the gel. The mean densities of each fraction are asfollows: 1.024 g/ml (lane 1), 1.030 Qane 2), 1.040 (lane 3), 1.053 (lane4), 1.071 (lane 5), 1.092 (lane 6), 1.115 (lane 7), 1.139 (lane 8), 1.161(lane 9), and 1.178 (lane 10). (B) Equivalent amounts of the p < 1.21g/ml plasma fraction from high expressor transgenic mouse 11 anda nontransgenic mouse were separated on 2-16% nondenaturingpolyacrylamide gels as described in text, stained for protein, andscanned densitometrically. A typical human profile is presented forcomparison of particle size. The LDL region encompasses a sizerange of 23-28 nm. (C) Equivalent amounts of plasma from high-expressor transgenic mouse 11 and a nontransgenic mouse wereseparated by agarose gel electrophoresis, stained for lipid, andscanned as above. H-apoB, human apoB; IDL, intermediate densitylipoprotein.

examined under nonreducing and reducing conditions (Fig.3A). Apo(a) in the apo(a) transgenic mice was only present inthe lipoprotein-free fraction and migrated as a monomer(r500 kDa) under both reducing and nonreducing conditions.In contrast, apo(a) in the apoB/apo(a) mice was foundprimarily in the lipoprotein fraction. Furthermore, undernonreducing conditions, apo(a) existed as a much largermolecular weight form, which upon reduction was replacedby the 500-kDa band. To further define the lipoproteinfractions containing apo(a), plasma from apoB/apo(a) trans-genic animals was separated by density-gradient ultracentrif-

B

MouseapoB probe

FIG. 1. Northern blot analysis ofRNA from an apoB transgenicmouse and a nontransgenic (n.t.) littermate. Total RNA derived fromvarious mouse tissues (transgenic line 11) was subjected to electro-phoresis in the indicated lanes. Filters were hybridized with a humanapoB probe (Left) or a mouse apoB probe (Right) as described intext. s., Small.

slight reduction was observed in mouse apoB mRNA com-pared with that in the control mouse, while in the low-expressing animal, the mouse apoB mRNA levels wereapproximately equal to those of the nontransgenic mouse.

Distribution of Human apoB Among Lipoproteins and theLipoprotein Profile in apoB Transgenic Mice. Animals withhigh transgene copy number had increased plasma levels ofhuman apoB and non-HDL cholesterol (Table 1). Lipopro-tein fractions from the apoB transgenic mice were isolated bydensity-gradient ultracentrifugation followed by electropho-resis and immunoblotting with a human-specific apoB mAb.Human apoB was found exclusively in the LDL and very lowdensity lipoprotein (VLDL) plasma fractions (Fig. 2A). Asignificant percentage of the human apoB immunoreactivematerial in the VLDL density range migrated as apoB48,while the vast majority of immunoreactive material in theLDL density range migrated as apoB100.LDL was a minor component of plasma lipoproteins in

nontransgenic mice (Fig. 2B). In contrast, a high-expressinghuman apoB transgenic mouse (transgenic line 11) containedLDL as the major lipoprotein component, even on a MouseChow diet (Fig. 2B). When plasma lipoproteins were sepa-rated by agarose gel electrophoresis (Fig. 2C), P-migratinglipoproteins were considerably increased, and a-migratinglipoproteins were decreased, in the high-expressing trans-genic mouse compared with a nontransgenic mouse. Thus,the overall lipoprotein profile of the human apoB transgenicanimal roughly resembled that of the human despite themouse's low fat, low cholesterol diet.The effect of high plasma levels of human apoB on mouse

apoB levels was assessed by immunoblotting with an anti-mouse LDL polyclonal antibody. The preadsorption withhuman lipoproteins eliminated detectable crossreactivitywith human apoB. Plasma levels of mouse apoB appearedslightly increased in human apoB transgenic mice (data notshown).

apo(a) in Mice Expresing Both Human apo(a) and HumanapoB. To examine the interaction between human apoB andapo(a), transgenic mice expressing apo(a) (8, 9) were bredwith the human apoB transgenic founder animal (transgenicline 9), and offspring containing one or both transgenes wereidentified by immunoblotting of plasma proteins. The li-poprotein-free fraction (p > 1.21 g/ml) and the lipoproteinfraction (p < 1.21 g/ml) were isolated from plasma, and themigration of human apo(a) immunoreactive material was

... j

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Beta Pre-Beta Alpha

2132 Genetics: Callow et al.

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A NON-REDUCED REDUCED

(a)/B (a) (a)/B (a)

500 kDa_

b t b t

B

500 kDa _-

Ai.:#0

:.:,.. 0:.:

b t b t

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1 2 3 4 5 6 7 8 9 10Fraction

FIG. 3. Immunoblot with anti-apo(a) antibody of mouse plasmafractions from mice expressing apo(a) or apo(a) + human apoB. (A)Lipoprotein fraction of plasma from the top of the tube (p < 1.21g/ml) (lanes t) or lipoprotein-deficient fraction from the bottom ofthetube (p > 1.21 g/ml) (lanes b) was collected as described in text. Ananti-apo(a) antibody was used for immunodetection of sampleselectrophoresed and transferred to nitrocellulose. (a)/B, apo(a) andhuman apoB transgenic mouse; (a), apo(a) transgenic mouse. (B)Plasma was subfractionated as described in text from mice express-ing apo(a) and human apoB and electrophoresed under reducingconditions before transfening to nitrocellulose and immunoblottingwith an anti-apo(a) antibody. The equivalent of 78 p1 of each densityfraction was loaded onto the gel lanes. Mean densities of eachfraction are described in Fig. 2.

ugation; apo(a) was primarily seen in the LDL and buoyantHDL density fractions (Fig. 3B), similar to that observed inhumans (1).A comparison of apo(a) levels in plasma from apo(a)

transgenic mice with apo(a)/apoB transgenic mice by ELISAshowed an increase from 6.3 mg/dl (n = 6 mice) to 9.6 mg/dl(n = 8 mice), respectively (P < 0.05, by the t test).

DISCUSSIONThese studies demonstrate efficient liver-specific expressionof human apoB when a large human genomic segment,including the apoB gene, is introduced into the mouse ge-nome. This, coupled with the general increase in apoBplasma levels associated with transgene copy number, sug-gests that the entire apoB domain participating in liver-specific expression is contained within the 75-kb clonedgenomic fragment. Similar findings of copy number-depen-dent expression were observed for the human ,B-globin genewhen distal 5' and 3' sequences defined to be important forefficient expression of this gene were included in transgeneconstructs (23).An unexpected finding from our analysis of the apoB

transgenic mice was the absence of human apoB expressionin the small intestine. This result suggests that specificelements necessary for intestinal expression may be lackingwithin the P1 clone or that necessary trans-acting factors donot recognize the human element. Separation of ci§-actingelements that determine liver and intestinal expression hasbeen seen for the apoAl gene (24). Alternatively, the P1vector sequences, present as part ofthe apoB transgene, maysomehow interfere with expression of human apoB in thesmall intestine. In humans, apoB mRNA is edited exclusivelyin the small intestine, while in rodents apoB mRNA is editedin both the small intestine and the liver (11). Editing ofhumanapoB transcripts occurred in the liver of the human apoBtransgenics. The ability of human apoB to be edited in thisheterologous system is consistent with studies in vitro (21, 25,

26) and in cultured cells (22, 27-29), confirming cross-speciescompatibility of trans-acting factors involved in editing.The marked increase in LDL in the high-expressing human

apoB transgenic mice was a notable feature of these animals.The explanation for this increase has yet to be determined. Itmay be due to an overall increased production of humanapoB100 in these animals and/or to reduced clearance ofhuman apoB-containing lipoproteins via mouse apoB recep-tors. Evidence supporting the latter comes from studiesshowing that uptake of human LDL was less efficient bymouse fibroblasts compared with human fibroblasts (30).Whatever the explanation for the increase of LDL in thehuman apoB transgenic mice, the highest expressing humanapoB transgenic line, line 11, has a lipoprotein profile notdissimilar to humans and should prove useful in the study ofhuman lipoprotein metabolism and atherosclerosis.The negligible effect of high plasma levels of human apoB

on mouse apoB plasma concentrations is surprising. Severalstudies have suggested that significantly more apoB is trans-lated than is secreted by hepatocytes (see ref. 31 for a review)possibly due to limiting lipid availability. The added synthesisof human apoB could have been expected to diminish therelative contribution of mouse apoB to total apoB secreted.The failure to observe such a reduction of mouse apoB intransgenic plasma suggests the possibility that inhibition ofclearance of mouse apoB due to high human apoB plasmaconcentrations may influence mouse apoB levels.The efficient assembly of Lp(a) particles in the apoB/

apo(a) transgenic animals is consistent with studies of infu-sion of human LDL into apo(a) transgenic mice (8). The highproportion of apo(a) associated with LDL in these animals,despite the fact that this interaction does not normally occurin mice, suggests a crucial role for the primary proteinsequence of human apoB and apo(a) in driving this interac-tion. The association ofapo(a) with LDL, rather than VLDL,in the apoB/apo(a) transgenic mice is consistent with humanstudies that show that the majority of apo(a) is found in thedensity range between LDL and HDL (1, 2). Even in miceexpressing low levels ofhuman apoB, a significant proportionof apo(a) was lipid-associated. This suggests that apoB andnot apo(a) is in excess in these animals, again similar to thesituation in humans. The =50% increase in plasma levels ofapo(a) in the apo(a)/apoB transgenic mice versus the apo(a)alone transgenic mice suggests greater stability or reducedclearance of apo(a) when bound to LDL rather than whenfree in plasma.Two classes of hypotheses have been presented to account

for the association of elevated Lp(a) plasma levels withatherosclerosis. In one case, apo(a) acts as a competitiveinhibitor of the activation of plasminogen, its close homo-logue (32, 33). In vitro, this leads to reduced fibrinolysis, areduction in the activation of transforming growth factor /,and a subsequent stimulation ofvascular smooth muscle cells(34). These processes might contribute to atherogenesis,even without the lipid association of apo(a). Alternatively,the lipid-bound form of apo(a) in the Lp(a) particle couldcontribute to cholesterol deposition and foam cell formationdue to its avid binding to cell receptor and extracellularmatrix sites. In this case, lipid association would be critical.Mice expressing apo(a) alone have been shown to haveincreased susceptibility to diet-induced fatty streak forma-tion (9). Thus, it will be instructive to compare the athero-genic response in mice that are transgenic for apo(a) alonewith those transgenic for both apo(a) and human apoB. Thefact that human apoB and apo(a) efficiently interact in theplasma of human apoB/apo(a) transgenic mice, resulting inthe assembly ofLp(a) particles, opens many avenues to studythe in vivo biology of Lp(a) in a manner not previouslyavailable.

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We thank P. Blanche for ELISA and lipoprotein assays, D.Muragesh for injection ofembryos, R. Krauss for critical comments,and Jeff Gingrich, Farideh Shadravan, and the Lawrence BerkeleyLaboratory Human Genome Project for screening of the P1 library.This work was supported by National Institutes of Health (NationalHeart, Lung, and Blood Institute) grants to E.M.R. (PPG HL18574)and a grant funded by the National Dairy Promotion and ResearchBoard and administered in cooperation with the National DairyCouncil. E.M.R. is an American Heart Association EstablishedInvestigator. L.J.S. was supported by National Research ServiceAward Postdoctoral Training Grant HL07279. M.J.C. was supportedby the American Heart Association. R.M.L. was supported byNational Institutes of Health Program Project Grant HL 48638-02.Research was conducted at the' Lawrence Berkeley Laboratory(Department of Energy Contract DE-AC0376SF00098), Universityof California, Berkeley.

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