Expression of the Human Apolipoprotein E Gene in Cultured Mammalian Cells*

The gene for human apolipoprotein (apo-) E was isolated from a human genomic library constructed in the cosmid shuttle vector pCV108. The transient expression of the apo-E gene was examined in cultured mammalian cells 48 h following calcium phosphate-mediated gene transfer. The expression of the cloned human apo-E gene, which contained between 0.7 and 29 kilobases of 5”flanking DNA, was not restricted to human cells or to cultured cells derived from tissues that have been shown to synthesize apo-E. Several independent mouse L cell stable transfectants with the human apo-E gene integrated into their genome were selected on the basis of G418 resistance, which is con- ferred by the selectable gene marker in the cosmid vector. The levels of human apo-E mRNA found in the stable transfected mouse L cells ranged from undetect-able to a level comparable to that found in the human liver. The size of the apo-E mRNA observed in the stable transfectants was identical to that found in the liver, indicating that the mouse L cells were capable of correctly processing the human apo-E gene transcripts. The integrated human apo-E genes had not undergone major rearrangements or deletions during transfer, and the level of apo-E mRNA found in the different stable transfectants correlated directly with the number of integrated copies of the human apo-E gene. The stable transfected L cells secreted authentic The medium was collected, concentrated 70- to 100-fold with an Amicon Ultrafiltration apparatus (Amicon) in the presence of 100 p~ phenylmethylsulfonyl fluoride and 1 pg of leupeptin/ml, dialyzed against 10 mM NH4HC03 containing 0.1% 2-mercaptoethanol, and applied to a heparin-Sepharose column. The bound proteins were eluted with a linear gradient between 0.01 and 0.70 M NH4HCOB. Elution of apo-E was monitored by quantitative radioimmunoassay. The partially purified apo-E was complexed with DMPC, and the complexes were purified on KBr gradients (50). Competitive binding assays were performed on human fibroblasts with Y-LDL as de- scribed

and lysine-rich region centered in the vicinity of residues 140-160 of the protein (6-8). Human apo-E has a complex isoelectric focusing pattern that is due to the presence of multiple alleles for apo-E at a single gene locus and to post-translational modifications involving the addition of sialic acid residues (9, 10). Variants of apo-E in which neutral amino acids substitute for arginine and lysine residues within the receptor binding region exhibit decreased receptor binding capacity and are associated with type 111 hyperlipoproteinemia (11)(12)(13)(14).
The liver is the major site of synthesis of apo-E (15). Mouse peritoneal macrophages (16) and human monocyte-derived macrophages (17) also have been shown to synthesize apo-E. Recently, apo-E mRNA has been detected in tissues of various peripheral organs, including the brain, adrenal, spleen, ovary, testis, and kidney (18)(19)(20). Immunocytochemical analysis has localized apo-E to astrocytic glial cells of the central nervous system and to nonmyelinating glial cells of the peripheral nervous system (21). It has been estimated that 10 to 20% of apo-E in the circulating plasma may originate from peripheral organs (18). The apo-E synthesized by the tissues in peripheral organs may be involved in the redistribution of cholesterol and other lipids among cells and tissues.
Human apo-E is a M , -34,000 protein composed of 299 amino acids of known sequence (22). It is initially synthesized with an 18-amino acid signal peptide that is removed cotranslationally (23,24). The nucleotide sequences of the mRNA for human (25,26) and rat (27) apo-E are known. Recently, the nucleotide sequence of the gene for human apo-E has been determined (28,29) and the gene has been mapped to chromosome 19 (29). The mRNA coding sequence is interrupted by three introns, which are located in the 5"nontranslated region of the mRNA, in the signal peptide region within the codon for Gly-,, and within the codon for Argel in the mature plasma protein region. The structure of the human apo-E gene with respect to the number and location of the introns is similar to that found in genes for human apo-A-I and apo-C-I11 (30)(31)(32), as well as for apo-A-11 (33).
Since the metabolism of cholesterol and other lipids may be affected by the expression of the apo-E gene, examination of the molecular basis of its regulation was begun. The human apo-E gene was isolated from a cosmid genomic DNA library, and the expression of the cloned gene was examined in cultured mammalian cells after both transient and stable transfections.

EXPERIMENTAL PROCEDURES
Materials-Radioisotopes and the plasmid pAT153 were purchased from Amersham Corp. EN3HANCE was obtained from New England Nuclear. All restriction endonucleases, T4 DNA ligase, and Klenow were purchased from Boehringer-Mannheim or New England Bio-Labs. Nitrocellulose paper for blots was obtained from Schleicher & Schuell, and HATF nitrocellulose filters were obtained from Millipore Corp. Cell culture media, trypsin EDTA solution, and G418, a neomycin analogue, were purchased from Gibco. Fetal bovine serum was obtained from Flow Laboratories. Serum-free medium (HB101) was obtained from Hana Biologicals (Berkeley, CA). Dimyristoylphosphatidylcholine (DMPC) was obtained from Avanti Polar Lipids (Birmingham, AL). Yeast RNA and phenylmethylsulfonyl fluoride were purchased from Sigma, and leupeptin was obtained from P-L Biochemicals.
Animals and CeU Cultures-Male Swiss-Webster mice were obtained from Charles River Breeding Laboratories (Wilmington, MA) and given free access to Purina rat chow. Cultured FQAH cells were maintained in modified Eagle's medium, and Chinese hamster ovary cells were maintained in Ham's F-12 medium. All other cells were grown in Dulbecco's modified Eagle's medium. All media were supplemented with 10% fetal bovine serum, 100 units of penicillin/ml, and 100 pg of streptomycin/ml.

Isolation of Human Apolipoprotein E Cosmid Clones-Construction
of the cosmid shuttle vector pCVlO8 and the preparation of the human genomic library have been described previously (34). The cosmid library was screened according to the high-density bacterial colony method of Hanahan and Meselson (35) using a 32P-iabeled (36) restriction endonuclease fragment derived from a full-length cloned human apo-E cDNA (25). Approximately 1 X 10' colonies were screened, and 16 positive clones were selected and isolated. A HindIII restriction fragment of pCHEGl containing the apo-E gene, described under "Results," was cloned into the HindIII site of the plasmid pAT153 (37). The recombinant plasmid was introduced into Escherichia coli RR1. Recombinant cosmids and plasmids were isolated by the alkaline-sodium dodecyl sulfate method (38), extracted with phenol/chloroform, and purified on CsCl gradients.
DNA Transfections-Cultured mammalian cells were transfected with the human apo-E-gene-containing cosmid DNAs by the calcium phosphate coprecipitation method of Graham and Van der Eb (39) as modified by Wigler et al. (40). To isolate stable transfectants, mouse L cells were seeded at 1 X l O a cells/100-mm dish and cultured for 20-24 h. Then, 5 pg of cosmid DNA in 1 ml of the DNA-calcium phosphate coprecipitate were added to the medium, and the cells were incubated for 4 h. The cells were shocked for 2 min with 15% glycerol in phosphate-buffered saline (140 mM NaCI, 8 mM NaH2P04, 1.5 mM KH2P04, and 3 mM KCl, at pH 7.5) (41), washed with phosphatebuffered saline, and allowed to grow for an additional 48 h. At this time the cells were subcultured and diluted 1:20 into medium supplemented with 400 pg of G418/ml (42). After 2 weeks of G418 selection, individual colonies were isolated and grown to mass culture. The cells were maintained in medium containing 200 pg of G418/ml. For transient expression, cells were seeded at 4 X 10' cells/75-cm2 flask, and the cells were transfected with 20 pg of DNA in 1 ml of DNAcalcium phosphate coprecipitate essentially as described above. Fortyeight hours after glycerol shock, total cellular RNA was prepared and analyzed as described below. Chloroquine was included at 100 p~ during the transfection of 5774.2 and P388D1 macrophage-like cell lines (43) to inhibit degradation of the transfected DNA.
Preparation and Analysis of RNA-Total cellular RNA was isolated from the cultured cells according to the guanidine thiocyanate procedure of Chirgwin et al. (44). Dot blot analysis was performed as described (18). Aliquots of total RNA (3.0, 2.0, 1.0, and 0.5 pg) were adjusted to 3 pg of RNA/sample with yeast RNA and denatured in 1.0 M formaldehyde, 0.9 M NaCl, and 0.09 M sodium citrate at 55 "C for 15 min. The samples were diluted into 20 volumes of 3 M NaCl containing 0.3 M sodium citrate and applied to nitrocellulose filters using a template manifold (Schleicher & Schuell). For Northern blot analysis (45), the RNA was denatured in 1% glyoxal, electrophoresed through a 1.1% agarose gel, transferred to nitrocellulose paper, and hybridized with cDNA probes.
Cosmid DNA Rescue and Apolipoprotein E Gene Copy Number-Total genomic DNA was extracted from the stable L cell transfectants and human leukocytes as described (46). The transfected cosmid DNA was excised from the genomic DNA and transfected into bacteria by in uitro packaging with extracts from lysogenic bacteriophages as described (34). The apo-E gene copy number was estimated by genomic DNA dot blots essentially as described by Palmiter et al. (47); human leukocyte DNA was used as the standard. Aliquots of genomic DNA (1-20 pg) were denatured in 0.1 N NaOH containing 2 M NaCl for 2 min at 100 "C, neutralized with 3 volumes of 1.0 M Tris (pH 8.0) containing 2 M NaCI, diluted with 15 volumes of 3 M NaCl containing 0.3 M sodium citrate, applied to nitrocellulose filters using a template manifold, and hybridized as described above. Blots were quantitated by densitometric scanning of several different exposures of the autoradiograms of the hybridized filters with a Beckman DU-8 spectrophotometer.
Protein Labeling-The cultured cells were grown in 75-cm2 tissue culture flasks in HBlOl serum-free medium until -75% confluent. The cells were labeled in Dulbecco's modified Eagle's medium containing 40 p~ unlabeled methionine and 100 pCi of ~-[~~S ] m e t h i onine/ml in the absence of serum for various lengths of time at 37 "C. The medium was adjusted to 100 pM phenylmethylsulfonyl fluoride and 1 pg of leupeptin/ml before immunoprecipitation with rabbit anti-human apo-E serum and was analyzed on 5.20% polyacry!amidesodium dodecyl sulfate gels (25).
Isolation of Macrophages-Peritoneal macrophages were harvested from thioglycolate-stimulated mice (48) and cultured for 24 h in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum. The macrophages were then incubated with 100 pg of acetoacetylated LDL protein/ml (17) for 24 h before being labeled with [35S]methionine as described above.
Lipoprotein Isolation and Potassium Bromide Density Gradient-To isolate total lipoproteins, cell culture medium was adjusted to a density of 1.215 g/ml with KBr and centrifuged at 55,000 rpm for 48 h. The d < 1.215 g/ml fraction was dialyzed against 25 mM NH4HC03, layered onto a linear 34-ml KBr gradient in the d = 1.006-1.21 g/ml range, and centrifuged at 27,000 rpm in a Beckman SW28 rotor at 4 "C for 60 h. Fractions (1 ml) were collected from the bottom of the tube and aliquots were examined for radioactivity. The density of each fraction was determined from the refractive index.
Receptor Binding Assay-To prepare apo-E for analysis, the apo-E secreted from the L cell transfectant L 1.1 into HBlOl serum-free medium was partially purified on a heparin-Sepharose column (49). The medium was collected, concentrated 70-to 100-fold with an Amicon Ultrafiltration apparatus (Amicon) in the presence of 100 p~ phenylmethylsulfonyl fluoride and 1 pg of leupeptin/ml, dialyzed against 10 mM NH4HC03 containing 0.1% 2-mercaptoethanol, and applied to a heparin-Sepharose column. The bound proteins were eluted with a linear gradient between 0.01 and 0.70 M NH4HCOB. Elution of apo-E was monitored by quantitative radioimmunoassay. The partially purified apo-E was complexed with DMPC, and the complexes were purified on KBr gradients (50). Competitive binding assays were performed on human fibroblasts with Y -L D L a s described (6).

Isolation of Human Apolipoprotein E Genomic Cosmid
Clones-Restriction endonuclease analysis and Southern blot hybridization revealed that the 16 positive apo-E-gene-containing cosmid clones selected and isolated were of two types.
A partial restriction endonuclease map showing the location of the apo-E gene in the genomic DNA insert of both types of cosmid clones is illustrated in Fig. 1. Both clones contained the entire apo-E gene. In pCHEG1, the apo-E gene was located at the 3' end of the DNA insert and contained approximately 29 kilobases (kb) of 5"flanking genomic DNA.
In pCHEG2, the apo-E gene was located at the 5' end of the

Expression of the Human Apolipoprotein E Gene
DNA insert and contained approximately 0.7 kb of 5"flanking genomic DNA. The partial restriction endonuclease map of the apo-E gene isolated from the cosmid genomic library was the same as that reported previously (28) for a human apo-E gene isolated from a X genomic library. Transient Expression of the Transfected Apolipoprotein E Gene in Cultured Mammalian Cells-To determine whether the apo-E gene in both cosmid clones was functional, the expression of the gene upon transfection of the cosmid DNA into cultured mammalian cells was examined. Several human and heterologous cultured cells (Table I) were transfected with both types of cosmid DNAs containing the apo-E gene by the calcium phosphate coprecipitation technique. Transient expression of the exogenous gene was determined 48 h after transfection. None of the cell lines contained detectable amounts of endogenous apo-E mRNA.
The transfected human apo-E gene was expressed in all of the cultured human cell lines, as well as in two mouse macrophage lines (5774.2 and P388DI), mouse fibroblasts (L cells), and African green monkey kidney cells COS^). It was not expressed in a rat hepatoma line (Fu5AH) or Chinese hamster ovary cells. Although the level of expression of the human apo-E gene in the different cell lines varied, the apo-E mRNA level for a given cell line was approximately the same for both types of apo-E-gene-containing cosmid DNAs. The apo-E mRNA transcribed in several of the transfected cell lines was examined on Northern blots and was found to be identical in size to the apo-E mRNA observed in the human liver (data not shown).
To characterize further the transcription unit of the human apo-E gene, a 10.5-kb Hind111 fragment of pCHEGl was cloned into the plasmid pAT153. This subclone contained the entire apo-E gene, with 5.2 kb of 5"flanking genome DNA and 1.6 kb of 3'-flanking genomic DNA. The same cultured cells were transfected with this subclone. The transcription pattern and level of expression observed using this clone were virtually identical to those observed for the larger pCHEGl and pCHEG2 cosmid clones. The results of the transient expression assays indicate that the regulatory sequences necessary for the transcription of the apo-E gene are located within 0.7 kb of 5"flanking sequence and 1.6 kb of 3"flanking sequence.
Stable Expression of the Transfected Human Apolipoprotein E Gene in Mouse L Cells-The human apo-E gene was introduced into mouse L cells to examine the expression of the transfected apo-E gene in greater detail. Mouse L cells were chosen because their endogenous apo-E gene is not expressed and because these cells can be readily adapted to grow in a defined serum-free medium, which would facilitate the characterization of the apo-E gene products.
Mouse L cells were transfected with both types of apo-Egene-containing cosmids. Since the pCV108 cosmid vector (34) contains the SV2-neomycin selectable marker gene (42), stable transfectants were selected using the neomycin analogue G418. Nine G418-resistant clones transfected with pCHEGl and six G418-resistant clones transfected with pCHEG2 were selected for analysis. Expression of the human apo-E gene in these clones was determined by examining the cellular RNA for the presence of human apo-E mRNA (Fig.  2). Eight of nine pCHEGl L cell transfectants expressed the human apo-E gene, with the level of apo-E mRNA observed in the transfectants varying from 1% (L 1.4) to 100% (L 1.1) of that found in the human liver, as determined from densitometric scans of the dot blots seen in Fig. 2. Cultured mouse cells transfected with pCHEG2 also yielded stable transfectants that expressed the human apo-E gene at levels comparable to those observed in pCHEGl stable transfectants, as illustrated by L 2.3 of Fig. 2. Therefore, it appears unlikely that distal 5"flanking sequences that are not present in the pCHEG2 human genomic insert are essential for apo-E gene expression.
Expression of the human apo-E gene in these clones was stable. The level of apo-E mRNA in the L cell transfectants remained constant even after the cells were maintained in selection medium for several months. In addition, maintaining the cells in a defined serum-free medium for several months had no significant effect on the level of apo-E mRNA in these cells. Transfection of the human apo-E gene and isolation of stable cell lines did not activate the endogenous mouse apo-E gene in any of the L cell transfectants, as determined by hybridization of RNA dot blots with a mouse apo-E cDNA probe (data not shown). The human apo-E gene transcripts appeared to be processed correctly by the L cell transfectants. Total RNA from human liver, control L cells, and L cell transfectants containing either pCHEGl or pCHEG2 were examined by Northern blot analysis (Fig. 3). The human apo-E mRNA in both L cell transfectants was identical in size to the apo-E mRNA found in the human liver. In addition, the apo-E mRNA from transfected cells was polyadenylated, since it could be isolated on a poly(U)-Sepharose column (data not shown).
Analysis of DNA in the L Cell Transfectants-The number of copies of the human apo-E gene in several L cell transfectants was determined by genomic DNA dot blots (Fig. 2). Assuming that there is only a single copy of the human apo-E gene per haploid genome (28), the apo-E gene copy number was found to vary from 1 to 11 in the L cell transfectants that were examined. In addition, there was a direct correlation between the number of copies of the human apo-E gene and the level of human apo-E mRNA in the different L cell transfectants.
Because several laboratories have reported that exogenous DNA sequences can be modified substantially following transfection into mammalian cells (51)(52)(53), the possibility that the transfected apo-E genes in the stable L cells were modified was investigated by cosmid DNA rescue (34). In this technique, when three or more transfected genomic cosmid DNA units are integrated tandemly into the cellular genome, the cosmid DNA can be recovered from the genomic DNA by in  vitro X bacteriophage packaging. After subsequent infection of a suitable bacterial host, the rescued cosmid DNA is recovered as a plasmid, and its restriction endonuclease pattern can then be directly compared with that of the original transfecting cosmid DNA for alterations that may affect gene expression.
Restriction endonuclease digestion and Southern blots of pCHEGl DNA and cosmid DNA rescued from pCHEGl L cell transfectants indicated that no major rearrangements or deletions of the human apo-E cosmid DNA had occurred during gene transfer (Fig. 4). The restriction pattern of the cosmid DNA rescued from the stable transfectants was identical to that of the original transfected cosmid DNA. Characterization of Human Apolipoprotein E Secreted from Stable Transfected Mouse L Cells-The L 1.1 transfectant was examined to determine whether the human apo-E mRNA that it contained was capable of functioning as a template for the synthesis of a secretable apo-E. As shown in Fig. 5, apo-E was immunoprecipitated from the medium of [35S]methioninelabeled L 1.1 cells, whereas no labeled proteins were immunoprecipitated from the medium of the control L cells. Human apo-E was a major secreted protein of the mouse L cell transfectant, representing -1 to 2% of total secreted proteins. The secreted human apo-E was found in two immunoprecipitated bands of M, -35,000 and 36,000 (Fig. 5). These forms often are found in highly sialylated apo-E. Immunoblots showed that the electrophoretic mobility of this protein was slightly slower than that of plasma apo-E (data not shown). The two-dimensional electrophoretic pattern (data not shown) of the human apo-E secreted by the transfected cells was consistent with the presence of multiple sialic acid residues on the secreted apo-E; the presence of these residues is characteristic of nascent apo-E (17, 54,55). Thus, the mouse L cells apparently are capable of post-translationally modifying the human apo-E that they synthesize.

C.
D.  (lanes 2 and 4 ) in 75-cm2 flasks were labeled with 100 pCi of [35S]methionine/ml for 6 h at 37 "C in the absence of serum. Aliquots of the medium containing 2 X 10' cpm of trichloroacetic acid-precipitable material were immunoprecipitated with anti-human-apo-E antibodies (25). Total medium, 2 X lo4 cpm/lane (lanes 1 and 2), and the immunoprecipitates (lanes 3 and 4 ) of the medium were electrophoresed on 5-20% polyacrylamide-sodium dodecyl sulfate gradient gels, and this fluorogram was prepared (25). fraction. As shown in Fig. 6, apo-E from both sources floated at a similar density, d -1.09. Analysis of aliquots of the peak fractions of both gradients by sodium dodecyl sulfate-polyacrylamide gels confirmed that apo-E was present in these fractions. No radiolabeled proteins were detected in any of the density fractions from medium of nontransfected control L cells.
To determine whether the apo-E secreted by the L cell transfectant possessed biological activity similar to that of authentic apo-E, the ability of the secreted apo-E to compete with Iz5I-LDL for binding to apo-B,E(LDL) receptors on human fibroblasts was examined. The human apo-E that was secreted from L 1.1 cells into serum-free medium was partially purified on a heparin-Sepharose column and complexed with DMPC. The apo-E secreted by the mouse L cells eluted from the column at the same ionic strength as that of plasma apo-E. As shown in Fig. 7, the apo-E secreted from the L cell transfectant effectively competed with LDL for binding to the receptor (50% displacement: L cell apo-E, 0.125 pg of protein/ ml; plasma apo-E3,0.062 pg of protein/ml).

DISCUSSION
We have isolated two genomic clones containing the human apo-E gene from a cosmid genomic library and characterized the expression of the gene after transfection into cultured mammalian cells. Although the two clones contain different 1.1 apo-E-DMPC to compete with human "'I-LDL for binding to normal fibroblasts. Purified plasma apo-E3 (0) and L 1.1 apo-E (0) partially purified on a heparin-Sepharose column were complexed with DMPC and then further purified on KBr gradients (50). The amount of apo-E in the isolated apo-EaDMPC complexes was determined by quantitative radioimmunoassay. Human fibroblasts were incubated in Dulbecco's modified Eagle's medium containing 10% human lipoprotein-deficient serum for 48 h before use. Each dish received 2 ml of the same medium with 2 pg of '251-LDL protein/ml and the indicated concentrations of apo-E.phospholipid complexes. After a 2-h incubation at 4 "C, the cells were washed extensively and the '=I-LDL that was specifically bound to the cells was determined. The 100% control value was 116 ng of '=I-LDL bound/mg of cellular protein.
amounts of 5'-and 3'-flanking genomic DNA, the apo-E gene in both clones was expressed with approximately equal efficiency in the transient expression assays to produce human apo-E mRNA that was identical in size to that found in the Expression of the Human Apolipoprotein E Gene 9863 human liver. This observation indicates that the regulatory sequences necessary for the expression of the apo-E gene are located in the gene itself or within -0.7 kb of 5"flanking sequence or 1.6 kb of 3'4anking sequence. Expression of the human apo-E gene was not restricted to human cell lines or to cultured cells derived from tissues that synthesize apo-E. This finding suggests that if tissue-specific transcription factors are involved in the regulation of apo-E gene expression, then these factors are present in more than one cell type. Different results might be expected for other apolipoproteins, which are synthesized in fewer tissues.
The inability of some cell lines to express the human apo-E gene does not result from any inability to introduce the exogenous DNA into these cells. For example, several independently selected stable transfectants of the Fu6AH cell line were prepared with the apo-E-gene-containing cosmid pCHEG1. These stable transfectants contained several copies of the human apo-E gene, as determined by genomic DNA dot blots, and they expressed the neomycin-resistant marker gene, but none of the clones expressed the apo-E gene. This finding suggests that even transient expression of the apo-E gene in the transfected DNA is suppressed by these cells or that these cells may lack an essential transcription factor required for apo-E gene expression.
The expression of the apo-E gene in several stable transfected mouse L cells that were selected on the basis of G418 resistance has also been characterized. The exogenous human gene was transcribed, and the mRNA was processed properly in the L cell transfectants to produce functional mRNA. The transfected cell apo-E mRNA comigrated with human liver apo-E mRNA during agarose gel electrophoresis, and it was translated into a protein that was secreted into the medium and interacted specifically with antibodies to human plasma apo-E. That this protein was authentic apo-E was demonstrated by its having an apparent molecular weight similar to that of plasma apo-E, by its ability to bind to a heparin-Sepharose affinity column and elute at the same ionic strength as plasma apo-E, and by its ability to compete with LDL for binding to the apo-B,E(LDL) receptor on human fibroblasts with an affinity similar to that of authentic human apo-E.
The stable L cell transfectants express the human apo-E gene at different levels. There are several possible explanations for the large variation in the level of human apo-E mRNA observed in these cells. In general, the expression of exogenous genes can be affected by rearrangements or deletions of the transfected DNA that can occur during gene transfer and stable selection, by the number of copies of the transfected DNA integrated into the genome of different transfected cells, and by the site of integration of the transfected DNA into the chromosomal DNA of the cell. It appears that no major rearrangements or deletions of the human apo-E gene occurred during gene transfer. The apo-E-gene-containing cosmid DNA that was rescued from the L cell transfectants had a restriction endonuclease pattern identical to that of the original transfected DNA. Furthermore, in the stable transfectants examined, apo-E gene expression did not appear to be influenced by the site of integration. Therefore, the differences in the level of human apo-E mRNA in the various L cell transfectants appear to be a direct function of the number of integrated copies of the human apo-E gene.
A cell line (mouse L cells) that does not normally secrete lipoproteins has been shown to be capable of secreting human apo-E associated with lipid upon transfection with the corresponding exogenous gene. Since the apo-E particles secreted from the transfected L cells have a flotation density identical to that of apo-E secreted from mouse peritoneal macrophages (17), it is likely that the apo-E secreted from the L cells is complexed with phospholipid as well. There are several possible sources of the lipid that is associated with the secreted apo-E. Nascent apo-E may become associated with lipid during intracellular transport and processing, or it may acquire lipid during or after secretion from either the cell membrane or from other lipid in the surrounding medium. The latter possibility seems unlikely, since iodinated delipidated human plasma apo-E incubated with L cells in serum-free medium did not become associated with lipid (data not shown). It will be of interest to determine whether other apolipoproteins secreted from L cells after gene transfer are also associated with lipid. In cells that secrete apolipoproteins, the association of the apolipoproteins with lipid may depend most on the properties of the protein moieties themselves and not on special assembly mechanisms of the cells. The ability to express efficiently the human apo-E gene in heterologous cultured mammalian cells will facilitate investigation of the regulatory elements associated with the expression of the human apo-E gene. Sequences upstream from the transcription initiation site, as well as sequences within introns, can be analyzed for their role in the expression of the apo-E gene.