Expression of human apolipoprotein B and assembly of lipoprotein(a) in transgenic mice.

Abstract

The atherogenic macromolecule lipoprotein(a) [Lp(a)] has resisted in vvo analyses partly because it is found in a limited number of experimental animals. Although transgenic 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 associate with mouse apolipoprotein B (apoB). We isolated a 90-kilobase P1 paemid containing the human apoB gene and with this DNA generated 13 lines of tnsgec mice of which 11 expressed human apoB. The human apoB transcript was expressed and edited in the liver of the transgenic mice. In humans, the association between apo(a) and LDL to form the Lp(a) particle is the result of a disulfide linkage between apo(a) and apolipoprotein B (apoB) of LDL (4, 5, 7). Transgenic mice that express human apo(a) have been engineered, but, unlike in humans, the apo(a) in these animals was found in the lipoprotein-free plasma fraction and was not associated with mouse LDL (8, 9). Infusion ofisolated human LDL, but not mouse LDL, into apo(a) transgenic animals results in the appearance of Lp(a) particles (8). This suggests that the failure to form Lp(a) in the apo(a) transgenic mice is caused by the inability of mouse apoB-containing LDL to properly interact with human apo(a). Thus, the development of mice to examine the biological properties of Lp(a) in vivo requires the creation of animals that express both human apoB 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 edited apoB mRNA transcripts exclusively in the intestines of nonrodent mammals and in the intestines plus the livers of rodents (11). The large size of the apoB gene, >43 kb, has made it difficult to clone an intact apoB genomic fragment in A phage or cosmid vectors for introduction into the mouse genome.A recently developed cloning system using P1 phagemids allows the cloning of 80-to 90-kb DNA inserts (12, 13). We have isolated a clone containing the entire human apoB gene plus extensive 5' and 3' regions from a human P1 library. This DNA was introduced into fertilized mouse embryos, and nearly all of the resulting transgenic mice had significant plasma levels of human apoB and increased plasma levels of LDL-cholesterol. Mice containing both human apo(a) and human apoB transgenes efficiently assembled Lp(a) particles.

Lipoprotein [a] is cleared from the plasma primarily by the liver in a process mediated by apolipoprotein [a]

“…Despite this limitation, studies using transgenic mice producing human Lp[a] have already contributed much to our understanding of Lp[a] synthesis (27)(28)(29)(30) and its role in atherosclerosis (31)(32)(33). Furthermore, we believe that mice may also offer an attractive model for the study of Lp[a] catabolism, because they are able to clear the lipoprotein in a way that appears to be similar to that in humans.…”

The assembly of lipoprotein Lp(a)

“…Following intravenous infusion of human LDL, but not of VLDL, into transgenic mice, Lp(a) that is indistinguishable from native Lp(a) found in humans is formed. Double transgenic mice expressing both human apo(a) and human apoB-100 synthesize bona fide Lp(a) directly [36,37]. It is interesting to note that the plasma apo(a) levels were higher in double transgenic mice than in those producing only apo(a), suggesting a faster removal rate of apo(a) that is not complexed to LDL.…”

A novel lipoprotein (a) lowering drug, D-47, decreases neointima thickening after vascular injury

“…Therefore, we have to use double transgenic mice with human apo (a) and human LDL to test the Lp(a) function. The previous studies showed that mice expressing the apo(a) gene developed atherosclerosis (16,17). Using this model animal, Kyutoku M et al (18) reported that DNA vaccine against apo(a) could significantly inhibit neointima formation in carotid artery ligation and concluded that the pro -atherosclerotic actions of Lp(a) could be prevented by reducing Lp(a) levels.…”