Abstract
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 a apparent M(r) = 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 ~ 1.09 g/ml, and bound with high affinity to the apo-B,E(LDL) receptor on fibroblasts.
| Original language | English |
|---|---|
| Pages (from-to) | 9858-9864 |
| Number of pages | 7 |
| Journal | Journal of Biological Chemistry |
| Volume | 261 |
| Issue number | 21 |
| Publication status | Published - 1986 Dec 1 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
- Cell Biology
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Expression of the human apolipoprotein E gene in cultured mammalian cells. / Reardon, C. A.; Lau, Y. F.; Paik, Y. K.; Weisgraber, K. H.; Mahley, R. W.; Taylor, J. M.
In: Journal of Biological Chemistry, Vol. 261, No. 21, 01.12.1986, p. 9858-9864.Research output: Contribution to journal › Article
TY - JOUR
T1 - Expression of the human apolipoprotein E gene in cultured mammalian cells
AU - Reardon, C. A.
AU - Lau, Y. F.
AU - Paik, Y. K.
AU - Weisgraber, K. H.
AU - Mahley, R. W.
AU - Taylor, J. M.
PY - 1986/12/1
Y1 - 1986/12/1
N2 - 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 a apparent M(r) = 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 ~ 1.09 g/ml, and bound with high affinity to the apo-B,E(LDL) receptor on fibroblasts.
AB - 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 a apparent M(r) = 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 ~ 1.09 g/ml, and bound with high affinity to the apo-B,E(LDL) receptor on fibroblasts.
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UR - http://www.scopus.com/inward/citedby.url?scp=0023031929&partnerID=8YFLogxK
M3 - Article
C2 - 3733697
AN - SCOPUS:0023031929
VL - 261
SP - 9858
EP - 9864
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 21
ER -