Abstract
Five lines of transgenic mice, which had integrated the human apolipoprotein (apo) A-I gene and various amounts of flanking sequences, were established. Normally, apoA-I is expressed mainly in liver and intestine, but all of the transgenic lines only expressed apoA-I mRNA in liver, strongly suggesting that 256 base pairs of 5'-flanking sequence was sufficient for liver apoA-I gene expression but that 5.5 kilobase pairs was not sufficient for intestinal expression. Mean plasma levels of human apoA-I varied in different lines from approximately 0.1 to 200% of normal mouse levels. This was not dependent on the amount of flanking sequence. Lipoprotein levels were studied in detail in one of the lines with a significantly increased apoA-I pool size. In one study, the total plasma apoA-I level (mouse plus human) was 381 +/- 43 mg/dl in six animals from this line, compared to 153 +/- 17 mg/dl in matched controls. Total and high density lipoprotein cholesterol (HDL-C) levels were increased 60% in transgenic animals, compared to controls (total cholesterol: 125 +/- 12 versus 78 +/- 13 mg/dl, p = 0.0001; HDL-C 90 +/- 7 versus 55 +/- 11 mg/dl, p = 0.0001). The molar ratio of HDL-C/apoA-I was significantly lower in transgenic animals, 17 +/- 1 versus 25 +/- 2 (p = 0.0001), suggesting the increase was in smaller HDL particles. This was confirmed by native gradient gel electrophoresis. This was not due to aberrant metabolism of human apoA-I in the mouse, since human apoA-I was distributed throughout the HDL particle size range and was catabolized at the same rate as mouse apoA-I. In another study of 23 transgenic mice, HDL-C and human apoA-I levels were highly correlated (r = 0.87, p less than 0.001). The slope of the correlation line also indicated the additional HDL particles were in the smaller size range. We conclude that human apoA-I can be incorporated into mouse HDL, and excessive amounts increase HDL-C levels primarily by increasing smaller HDL particles, comparable to human HDL3 (HDL-C/apoA-I molar ratio = 18).
Highlights
Southern blot analysis revealed that thetwo animals that did not express the human apoA-I protein appeared not tohave integrated the entirepiece of DNA
Mouse 139 wasoutbred and offspring positive for the human apoA-I gene were detected by Southern analysis of DNA from tail tissue
To ensure this was not the result of differential human apoA-I metabolism in the mouse, immunoblotting (Fig. 7A, lunes 9-11) indicates that human apoA-I is spread over the entire range of HDL sizes and not just in the lowersize species present in the transgenic animals
Summary
Igene but with differing amounts of 5'- and 3"flanking sequence, were microinjected into fertilized eggs from (C57BL/GJxCBA/J) F1 mice (Fig. 1).There were a total of 61 animals born. One transgenic line was established, Tg(OHSA-A1)427.The mean level of human apoA-I in this line was 339 mg/dl. Five had integrated the human apoA-I gene, but only three had human apoA-I protein in their plasma. These animals were used to establish transgenic lines Tg(YHSA-A1)139, Tg(OHSA-A1)145,and Tg(OHSA-A1)149.The mean levels of human apoA-I in these lines were2.5,0.07, and 94mg/dl, respectively. 30 mice were born from eggs microinjected with the 11.5-kb fragment, but only 2 had integrated the human apoA-I gene Both of these had human apoA-I in plasma. One transgenic line was established, Tg(0HSAAI)179, with a mean human apoA-I level of 245 mg/dl
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