Transgenic mice expressing a human apolipoprotein[a] allele

Abstract

The most important determinant of plasma levels of Lp[a] are sequence differences at the highly polymorphic apolipoprotein[a] (apo[a]) locus. To define the sequences that mediate the regulation of apo[a] expression, we cloned a 370 kb DNA fragment that included a 130 kb apo[a] gene, and 40 kb 5 -and 200 kb 3 -flanking region from an individual with high plasma levels of Lp[a] using a YAC vector. This genomic clone was used to generate transgenic mice. In the YAC-apo[a] transgenic mouse, apo[a] was only expressed in the liver, as it is in humans. The mean serum level of apo[a] in 4-week-old YAC-apo[a] transgenic mice was 20 mg/dl. In the female mice the levels of apo[a] varied over a 1.5-fold range during the 4-day estrus cycle and the levels correlated directly with serum progesterone levels. The serum levels of apo[a] decreased to almost undetectable level in male mice after puberty and this decrease was reversed by castration. Ingestion of a high-fat diet resulted in a ϳ 100-fold fall in hepatic apo[a] mRNA levels and Ͼ 60fold decrease in serum apo[a] levels. To delimit the control elements that mediate tissue-specific and sex hormone-responsive apo[a] transcription, we derived a reporter YAC in which 40 kb of 5 flanking sequences from the cloned apo[a] allele were linked to a luciferase reporter gene. Analysis of four independent transgenic lines revealed no hepatic luciferase expression, suggesting that important cis -acting elements located outside the apo[a] 5 -flanking region are necessary for in vivo expression of apo[a].

Determinants of human apolipoprotein [a] secretion from mouse hepatocyte cultures

“…Human apo[a] promoter constructs show low activity in vitro (48,49). In addition, not all the gene regulatory regions have been identified (39) and some exist far upstream from the transcription start site (38,50,51). Primary human hepatocytes, which can now be purchased commercially, provide a potential model system and have been used successfully to study apo[a] secretion (26).…”

“…(35)(36)(37)]. Both transcriptional and posttranscriptional mechanisms may play roles in regulation of Lp[a] levels by these factors (38)(39)(40)(41)(42)(43)(44)(45)(46)(47).…”

“…Analysis of factors that influence Lp[a] production rate has been hampered by the limited tissue and species distribution of Lp[a], the low activity associated with apo[a] promoter fragments in vitro (48,49), and the presence of regulatory sequences Ͼ 20 kb upstream of the transcription start site (38,50,51) and in other regions of the apo[a] gene (39). The development of mice transgenic for a 17 K4 human apo[a] cDNA under the control of the mouse transferrin promoter (23,52), and subsequently of mice transgenic for both apo[a] and human apoB (Lp[a] transgenic mice) (53,54), has provided in vivo models to study some aspects of Lp[a] metabolism.…”

“…The more recent production of two strains of mice transgenic for yeast artificial chromosomes (YACs) encoding the entire human apo[a] gene in its own genomic context (39,40) has provided an additional opportunity to investigate factors that regulate apo[a] mRNA levels in vivo (39)(40)(41)(42). Hepatocytes cultured from YAC-apo[a] mice may also provide a valuable in vitro model system with which to study factors that regulate human apo[a] gene transcription.…”

“…Mice hemizygous for a 17 K4 human apo[a] cDNA under the control of the mouse transferrin promoter, and homozygous for full-length human apoB (Lp[a] transgenic mice), or hemizygous for a YAC encoding the entire gene for a 13 K4 human apo[a] isoform as well as substantial 5 Ј and 3 Ј sequence (YAC-apo[a] mice), were as described (39,53). Mice hemizygous for the 17 K4 human apo[a] cDNA in the absence of the human apoB transgene (apo[a] mice) were obtained by crossing Lp[a] mice with C57BL6/SJ wild-type mice (Jackson Laboratories, Bar Harbor, ME).…”