Transgenic mice sing both human apolipoprotein 6 and human CETP have a lipoprotein ch erd dist n similar to that of normolipidemic humans

Transgenic mice expressing both human apolipoprotein (apo) B and human cholesteryl ester transfer protein (CETP) have been developed. When fed a normal mouse chow diet, the apoB/CETP double transgenic animals had threefold higher serum CETP activity than humans and had human apoB levels that were similar to those of normolipidemic humans. When compared with nontransgenic mice, the total serum cholesterol levels in the female apoB/CETP transgenic animals were increased significantly. Serum HDL cholesterol levels were decreased significantly in both male and female apoB/CETP transgenic animals. The percentages of the total cholesterol within the HDL, LDL, and VLDL fractions of the apoB/CETP animals were approximately 30%, 65%, and 5%, respectively, similar to the distribution of cholesterol in the plasma of normolipidemic humans . l Thus, by expressing both human apoB and human CETP, the lipoprotein cholesterol distribution in the serum of a chow-fed mouse was transformed into one that resembles a lated to the risk of developing coronary artery disease (CAD) (4). A specific mutation in the apoB gene that interferes with the ability of LDL to bind to the LDL receptor leads to elevated apoB and LDL cholesterol levels and premature atherosclerosis (5-7). CETP is a 74-kD glycoprotein that mediates the distribution of neutral lipids, including triglycerides and cholesteryl esters, among different classes of lipoproteins (1, 8, 9). Genetic deficiency of CETP in humans results in profound changes in lipoprotein composition and metabolism (10, 11). Humans with complete CETP deficiency have increased levels of HDL cholesterol, increased size of HDL particles, and decreased cholesteryl esters in apoBcontaining lipoproteins (12). It has been suggested that human profile.-Grass, D. S., U. Saini, R. H. Felkner, R. E. CETP deficiency could be anti-atherogenic and may be Wallace, W. JP. Lago, S. G. Young, and Ma E. fkanson. associated with longevity (11). Resistance to atheroscleroTransgenic mice expressing both human apolipoprotein B and sis with CETP deficiency would not be particularly surhuman CETP have a lipoprotein cholesterol distribution similar prising, in view of the well-established inverse relationship to that of normolipidemic humans. J Lipid RES. 1995. 36: 1082-1091. between high density lipoprotein (HDL) cholesterol levels and the risk of CAD (13). Supplementary key words cholesteryl ester transfer protein * apoThe plasma lipoproteins of the normal laboratory mouse are significantly different from those of humans. Whereas normolipidemic humans have 20-30% of their lipoprotein B transgenic mice lipoprotein cholesterol distribution total serum cholesterol in the HDL fraction, mice have approximately 80% and are resistant to the development of atherosclerotic lesions. O n a diet rich in fats and cholesterol, C57BL/6 mice and certain other strains deAbundant genetic, epidemiologic, and experimental studies have established that apolipoprotein (apo) B and cholesteryl ester transfer protein (CETP) play key roles in human lipoprotein metabolism (1-3). ApoB-100, a 512-kD glycoprotein, is an important structural component of the triglyceride-rich very low density lipoproteins (VLDL) (2, 3). ApoB-100 is also the sole protein component of the cholesterol-Ach low density ~ipoproteins (LDL) and is the ligand responsible for the removal of LDL by the liver. Plasma apoB and LDL cholesterol levels are directly re___ Abbreviations: apo, apolipoprotein; CETP, cholesteryl ester transfer protein; HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; CAD, coronary artery disease; PCR, polymerase chain reaction, ITo whom correspondence should be addressed. 1082 Journal of Lipid Research Volume 36, 1995 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 0, 2013 w w w .j.org D ow nladed fom velop a marked reduction in HDL cholesterol levels and are susceptible to atherosclerosis (14, 15). Most other mouse strains are resistant to atherosclerotic lesions under the same conditions (15). The resistance of mice to the development of atherosclerosis may, at least in part, relate to the fact that most strains of mice have low levels of apoB-containing lipoproteins (16) and the fact that mouse serum does not contain CETP activity (1). Over the past several years, transgenic technology has been used to develop mice with altered lipoprotein distribution and metabolism (17, 18). Transgenic mice expressing CETP have been developed by several laboratories (19, 20). In one study, the presence of high levels of monkey CETP activity in these transgenic mice resulted in the redistribution of serum cholesterol from HDL to VLDL and LDL, thus lowering HDL cholesterol levels and raising VLDL and LDL cholesterol levels. These mice were shown to be more susceptible to the development of atherosclerotic lesions (21). More recently, transgenic mice expressing human apoB were developed (22, 23). These mice had plasma apoB-100 levels similar to those of normolipidemic humans. The human apoB-100 was found in the LDL fraction, and the LDL cholesterol levels were increased compared with nontransgenic littermates. However, when fed a chow diet, only approximately 40% of the Serum cholesterol in the plasma of these apoB transgenic mice was contained in the LDL lipoprotein fractions. In addition to the CETP transgenic mice, other genetically modified mice, including mice homozygous for apoE null mutations (24, 25) and mice homozygous for an LDL receptor null mutation (26, 27), have been generated and shown to develop atherosclerotic lesions. The LDL receptor-negative mice had an increase in plasma LDL cholesterol, although the amount of HDL cholesterol still appeared to exceed the amount of LDL cholesterol (26). The apoE-negative mice had marked hypercholesterolemia, with most of the cholesterol located in the VLDL and IDL fractions (24, 25). At the current time, no mice have been produced that have a lipoprotein cholesterol distribution similar to that of normolipidemic humans (Le., a ratio of LDL cholesterol to HDL cholesterol of approximately 2 to 1) when fed a normal chow diet. In order to identify and evaluate new pharmaceuticals for the treatment of human lipid disorders, it would be desirable to have a convenient small laboratory animal model that has lipoprotein composition and metabolism similar to those of humans. Here we report the development of double transgenic mice expressing both human CETP and human apoB. These chow-fed double transgenic mice have a lipoprotein cholesterol distribution similar to that of normolipidemic humans, with an LDL/HDL cholesterol ratio of approximately 2 to 1. MATERIALS AND METHODS Generation of human CETP and apoB transgenic mice A gene construct designed to express the human CETP gene was produced as follows. The human CETP cDNA was amplified by polymerase chain reaction (PCR) from a preparation of human liver cDNA (Clontech Laboratories, Inc., Palo Alto, CA) and inserted into pCR 1000 (a TA cloning vector; Invitrogen Corporation, San Diego, CA). This cDNA contained the complete coding region and 18 bases of 5’ nontranslated sequence, but not the polyadenylation signal sequence. An approximately 1.75-kb PvuII-KpnI fragment (28) containing the human apoA-I promoter was placed into the KpnI and EcoRI sites of the vector, upstream from the 1.5-kb CETP cDNA sequence. A 0.56-kb SnaB1-BamHI fragment from pSVsport (Gib-BRL Life Technologies, Inc., Grand Island, NY) encompassing the SV40 small t splice and polyadenylation signal sequence was placed downstream from the CETP coding sequence. Sal1 and XbaI were utilized to separate the resulting construct from plasmid sequences. The 3.8-kb insert fragment was isolated on an agarose gel and further purified on an Elutip column (Schleicher and Schuell, Keene, NH). This DNA was microinjected into (C57BL/6J x SJL) F2 hybrid zygotes. Transgenic founders were identified by Southern or slot blot analysis using a 32P-labeled KpnI-EcoRI fragment encompassing the CETP cDNA. For the Southern analysis, genomic DNA from potential founders w a s isolated, digested with KpnI, and electrophoresed on a 0.7% agarose gel. The genomic DNA from transgenic founder mice contained a 3.8-kb KpnI fragment that hybridized to the 32P-labeled cDNA probe. The transgenic founders were bred to (C57BL/6J x SJL) F1 mice to produce G1 animals. ApoB transgenic mice (line 1102) were generated using a 79.5-kb genomic fragment encompassing the human apoB gene as previously reported (22). TO produce the apoB/CETP double transgenic mice and additional CETP hemizygous mice, apoB hemizygous mice that had been backcrossed once to C57BL/6J were mated with CETP homozygous mice. Thus, the mice used in these experiments were on a mixed C57BL/6J, SJL background. Serum isolation; cholesterol and triglyceride determinations Mice were bled through the retroorbital plexus during the light cycle. The blood was centrifuged in an Eppendorf microfuge at 14,000 rpm for 10 min to isolate the serum. Standard enzymatic methodologies were used to determine total cholesterol (Sigma Chemical Co., St. Louis, MO), triglycerides (Boehringer Mannheim BioG r m et al. Transgenic mice expressing human apoB and CETP 1083 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 0, 2013 w w w .j.org D ow nladed fom chemicals, Indianapolis, IN), and HDL cholesterol (Sigma Chemical Co., St. Louis, MO). Human serum was isolated from a normolipidemic 35-year-old male. Total cholesterol, HDL cholesterol, and triglyceride levels were 134 mg/dl, 44 mg/dl, and 115 mg/dl, respectively. Fast performance liquid chromatography size exclusion analysis Fifty pl of serum from individual mice or pooled serum from five mice was chromatographed on a Superose 6 column (Pharmacia Fine Chemicals, Piscataway, NJ) equilibrated with 10 mM Tris-C1, pH 7.4, 0.15 M NaCl, 0.01% (w/v) EDTA, 0.02% (w/v) NaN3. The column was run at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected. Aliquots (0.1 ml) from each fraction were assayed for cholesterol and triglyceride as described earlier. Human CETP Construct pvu 1 1 Human Apo A1 Human CETP ECOR I 1 1 promoter I Kpn I cDNA I Sal I A Serum CETP activity determinations and western blot analysis Serum CETP activity assays were performed using a commercial kit (Diagnescent Technologies, Inc., Yonkers, NY) or a modification of the procedure of Agellon et al. (19). Briefly, this modification was performed by incubating 2.5 p1 of serum with 0.867 pg HDL (5000 cpm tritiated cholesterol) and 21.7 pg LDL in 50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM diethyl-pnitrophenylphosphate. The incubation was adjusted to 50 pl and incubated at 37OC for 4 h. After the incubation, the volume was adjusted to 550 pl with TSE (50 mM Tris, pH 7.4, 150 mM NaCI, 2 mM EDTA). The LDL was then precipitated with 200 p1 of a standard precipitation reagent [heparin, 167 units, (Sigma Chemical Co., St. Louis, MO), 333 mM MnCI2, 13.3% BSA] on ice for