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 conferred 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 undetectable 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 human apo-E into the medium. The secreted protein interacted specifically with antibodies to human plasma apo-E and had an apparent Mr = 35,000 to 36,000, which is slightly larger than that of plasma apo-E. The secreted human apo-E was associated with lipid (presumably phospholipids), floated at d approximately 1.09 g/ml, and bound with high affinity to the apo-B,E(LDL) receptor on fibroblasts.
D000818:Animals; D001057:Apolipoproteins E / Q000235:genetics*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004134:Dimyristoylphosphatidylcholine; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
D001057:Apolipoproteins E; D012333:RNA, Messenger; D004134:Dimyristoylphosphatidylcholine