Targeted Replacement of Mouse Apolipoprotein A-I with Human ApoA-I or the Mutant ApoA-I Milano

Despite a pro-atherogenic profile, individuals carrying the molecular variant (R173C) of apolipoprotein (apo)A-I, named apoA-IMilano (apoA-IM), appear to be at reduced risk for cardiovascular disease. To develop an in vivo system to explore, in a controlled manner, the effects of apoA-IM on lipid metabolism, we have used the gene targeting technology, or “gene knock-in” (gene k-in), to replace the murine apoA-I gene with either human apoA-I or apoA-IM genes in embryonic stem cells. As in human carriers, mice expressing apoA-IM (A-IM k-in) are characterized by low concentrations of the human apolipoprotein and reduced high density lipoprotein cholesterol levels, compared with A-I k-in animals. The aim of the present study was to investigate the basic mechanisms of hypoalphalipoproteinemia associated with the apoA-IM mutation. ApoA-I and apoA-IM mRNA expression, as assessed by Northern blot analysis and quantitative real time reverse transcription-PCR, did not exhibit significant differences in either liver or intestine. Moreover, human apolipoprotein synthesis rates were similar in the k-in lines. When the secretion rate of the human apolipoproteins was assessed in cultured hepatocytes from the mouse lines, secretion from apoA-IM-expressing cells was markedly reduced (42% for A-IM k-in and 36% for A-I/A-IM k-in mice) as compared with that of A-I k-in hepatocytes. These results provide the first evidence that the hypoalphalipoproteinemia in apoA-IM human carriers may be partially explained by impaired apoA-IM secretion.