Biological insights through genomics: mouse to man.
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Animals↗
Biomedical subjects
E M Rubin
Publications and source records attributed to E M Rubin.
Gene transfer of cytidine deaminase apoBEC-1 lowers lipoprotein(a) in transgenic mice and induces apolipoprotein B editing in rabbits.
Apolipoprotein (apo) B100 is an essential component of low-density lipoproteins (LDL) and lipoprotein(a) [Lp(a)]. In mammals, apoB can be edited post-transcriptionally to encode a truncated form of apoB (apoB48) that is unable to form either of these atherogenic lipoproteins. To study the effect of increasing hepatic apoB editing activity on formation of Lp(a), a recombinant adenovirus encoding rat apoBEC-1, the cytidine deaminase component of the apoB mRNA editing complex, was administered to human apoB/apo(a) transgenic mice. This resulted in expression of apoBEC-1 in hepatocytes of these mice, increased hepatic editing of human apoB mRNA, and decreased plasma levels of human apoB100 and Lp(a). The apoBEC-1 recombinant adenovirus was also administered to rabbits, an animal which, like humans, naturally lacks hepatic apoB editing. Expression of the exogenous apoBEC-1 in rabbit liver resulted in editing of up to 10% of apoB mRNA. Hepatic apoB editing was associated with lower LDL levels in these rabbits relative to those treated with a control adenovirus. However, LDL levels were elevated significantly in both animals as a result of adenovirus injection. These studies demonstrate that introduction of the cytidine deaminase apoBEC-1 is sufficient to induce hepatic apoB editing in an animal lacking this activity, and that induction of editing could serve as a novel approach for lowering plasma concentrations of the atherogenic lipoproteins Lp(a) and LDL.
APOBEC-1 Deaminase↗
Human huntingtin derived from YAC transgenes compensates for loss of murine huntingtin by rescue of the embryonic lethal phenotype.
Huntington disease (HD) is caused by expansion of a CAG trinucleotide repeat in exon 1 of a novel gene. The HD protein (huntingtin) plays a critical role in early embryonic development since homozygous targeted disruption of the murine HD gene results in embryonic lethality by day 7.5. To rescue this phenotype by transgene based huntingtin expression it is therefore essential to express the protein early enough in development in the appropriate cells. Since YAC based transgenes are known to be regulated in an appropriate temporal and tissue-specific manner, we sought to rescue the embryonic lethality by breeding YAC transgenic mice expressing human huntingtin with mice heterozygous for the targeted disruption. We generated viable offspring homozygous for the disrupted murine HD gene but expressing human huntingtin derived from the YAC. This result clearly shows that YAC transgene based expression of huntingtin occurs prior to 7.5 days gestation. Additionally, we show that human huntingtin expression in YAC transgenic mice follows an identical tissue distribution and subcellular localisation pattern as that of the murine endogenous protein and that expression levels of 2-3 times endogenous can be achieved. This shows that human huntingtin under the influence of its native promoter, despite differences to the murine protein, is functional in a murine background and can compensate for loss of the murine protein. These results show that YAC transgenic approaches are a particularly promising route to producing an animal model for disorders associated with CAG expansion.
Animals↗
A simple and efficient method for making site-directed mutants, deletions, and fusions of large DNA such as P1 and BAC clones.
This study addresses two important technical problems: how to perform targeted alterations such as site-directed mutagenesis and deletions in large fragments of DNA and how to construct full-length genes from two partly overlapping bacterial artificial chromosome (BAC) plasmids. Given the size and the lack of convenient unique restriction sites in these large-insert bacterial clones, these are nontrivial tasks. Here we describe a simple and efficient protocol based on RecA-assisted restriction endonuclease (RARE) cleavage, a method that enables sequence-specific cleavage of genomic DNA. The same protocol has been used with minor modifications to introduce site-specific mutations into an apolipoprotein-B 90-kb P1 clone, to generate deletions in a 160-kb BAC, and to generate a 160-kb BAC containing the complete 92-kb gene for low-density lipoprotein-related protein-1 (LRP-1) from two smaller overlapping BACs ("BAC marriage").
Apolipoproteins↗
Site-specific mutagenesis demonstrates that cysteine 4326 of apolipoprotein B is required for covalent linkage with apolipoprotein (a) in vivo.
In the formation of the lipoprotein(a) (Lp(a)) particle, apolipoprotein(a) (apo(a)) and apolipoprotein B (apoB) are covalently linked via a disulfide bond in both humans and human-apo(a)/apoB transgenic mice. Studies based upon fluorescent labeling of free cysteine residues have suggested that cysteine 3734 of the 4 carboxyl-terminal cysteines of apoB (Cys-3734, Cys-3890, Cys-4190, and Cys-4326) is the most likely candidate to form a disulfide bond with apo(a). However, other recent studies using truncated apoB molecules suggest that Cys-4326, the terminal cysteine of apoB, may be implicated in the binding to apo(a). In order to definitively show which of apoB's carboxyl-terminal cysteines is essential in interacting with apo(a) we have used RecA-assisted restriction enzyme digestion coupled with site-specific mutagenesis to convert Cys-3734 and Cys-4326 to serine within separate 90-kilobase pair apoB P1 phagemid clones. Transgenic mice containing the normal or mutated apoB transgenes were created, and the covalent association of mutated apoB with apo(a) was assessed in mice transgenic for both apoB and apo(a). Analysis by ultracentrifugation and immunoblotting revealed that Cys-4326, but not Cys-3734, was essential in the formation of the covalent bond between apo(a) and apoB in vivo.
Amino Acid Sequence↗
A second generation transgenic mouse model expressing both hemoglobin S (HbS) and HbS-Antilles results in increased phenotypic severity.
We report on a second generation of transgenic mice produced by crossing a transgenic mouse line expressing high levels of human alpha and beta S chains (alpha H beta S [beta MDD]) with a line expressing human alpha and beta S-Antilles (beta SAnt). We hypothesized that mice expressing both hemoglobins (Hbs) would have a more severe phenotype because the reduced oxygen affinity and solubility of the beta S-Antilles might enhance the rate and extent of polymer formation. We obtained mice that expressed both beta S and beta S-Antilles. The doubly transgenic mice that are heterozygous for deletion of mouse beta Major (beta MD) occurred with reduced frequency and those that are homozygous for deletion of mouse beta Major (beta MDD) occurred at a much reduced frequency and suffered early mortality. Human alpha was 58% of all alpha globin for all animals, whereas beta S and beta S-Antilles were 34% and 28% of all beta globins for beta MD mice and 42% and 36% for beta MDD mice. Hematocrit, Hb, and mean corpuscular Hb were normal for all transgenic mice, but reticulocyte levels were higher for the doubly transgenic mice versus alpha H beta S [beta MDD] mice older than 30 days (10.0% +/- 1.0% v 4.3% +/- 0.4%; P < .001, mean +/- SE, n = 20 and n = 10, respectively) and control mice (3.9% +/- 0.4%). Reticulocytosis was more severe in mice less than 30 days old ( > 20% for alpha H beta S beta S-Ant[beta MDD] mice). The median mean corpuscular hemoglobin concentration of doubly transgenic mice was higher than that of alpha H beta S[beta MDD] mice with a variable number of very dense cells. Delay times for polymerization of Hb in red blood cells from alpha H beta S beta S-Ant[beta MDD] mice were shorter than those of alpha H beta S[beta MDD] mice, and there were fewer cells with delay times greater than 100 seconds. Urine-concentrating ability in control mice under ambient conditions is 2,846 +/- 294 mOsm and was reduced 30% to 1,958 +/- 240 mOsm, P < 4 x 10(-8) in all mice expressing both transgenes. We conclude that doubly transgenic mice have a more severe phenotype than either of the two parental lines. These mice may be suitable for validating therapeutic intervention in sickle cell disease.
Anemia, Sickle Cell↗
Construction of a panel of transgenic mice containing a contiguous 2-Mb set of YAC/P1 clones from human chromosome 21q22.2.
Libraries of the entire human genome, or regions of the genome, have been made in bacteria, yeast, and somatic cells. We have expanded this strategy using overlapping YACs and P1s from human 21q22.2 (the Down syndrome region) to create a panel of transgenic mice containing DNA that encompasses this region of the human genome. Together the members of the in vivo library, each with a unique transgene (four YACs and four P1s), contain approximately 2 Mb of contiguous DNA. The integrity, stable inheritance, and expression of a coding sequence for each member of the YAC panel are demonstrated, and the uses of the panel are described.
Animals↗
Human apo A-I in transgenic mice is more efficient in activating lecithin:cholesterol acyltransferase than mouse apo A-I.
This study shows that, in control and transgenic mice, there is a parallel increase in LCAT activity and plasma apo A-I concentrations during postnatal development. We also demonstrate that human apo A-I is a much more efficient activator (1.6-fold) of mouse LCAT activity than mouse apo A-I. We propose that the differences in amino acid sequence between human and mouse apo A-I may account for the higher LCAT activity with human apo A-I.
Animals↗
The apolipoprotein(a) gene is regulated by sex hormones and acute-phase inducers in YAC transgenic mice.
High plasma concentrations of apolipoprotein (a) (apo(a)) have been implicated as a major independent risk factor for atherosclerosis in humans. Apo(a) is a large, evolutionarily new gene (present primarily in primates) for which considerable controversy exists concerning the factors that regulate its expression. To investigate the in vivo regulation of apo(a), we have created several lines of YAC transgenic mice containing a 110-kb human apo(a) gene surrounded by greater than 60 kb of 5' and 3' flanking DNA. Studies in humans have suggested that acute-phase inducers increase and sex steroids decrease apo(a) concentrations, but these results are controversial. Analysis of the YAC transgenic mice conclusively supports the hypothesized role of sex steroids and refutes the suggested role of acute-phase inducers in regulating the apo(a) gene.
Acute-Phase Reaction↗
Lethal alpha-thalassaemia created by gene targeting in mice and its genetic rescue.
Mutations at the alpha-globin locus are the most common class of mutations in humans, with deletion of all four adult alpha-globin genes resulting in the perinatal lethal condition haemoglobin Barts hydrops fetalis. Using gene targeting in mice, we have deleted a 16 kilobase region encompassing both adult alpha-globin genes. Animals homozygous for this deletion become hydropic and die late in gestation mimicking humans with hydrops fetalis. Introduction of a human alpha-globin transgene rescued these animals from perinatal death thus demonstrating the utility of this murine model in the development of cellular and gene based approaches for treating this human genetic disease.
Animals↗
Expression of human lecithin-cholesterol acyltransferase in transgenic mice. Effect of human apolipoprotein AI and human apolipoprotein all on plasma lipoprotein cholesterol metabolism.
Human (Hu) lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in the plasma metabolism of cholesterol. To assess the effects of increased plasma levels of LCAT, four lines of transgenic mice were created expressing a Hu LCAT gene driven by either its natural or the mouse albumin enhancer promoter. Plasma LCAT activity increased from 1.2- to 1.6-fold higher than that found in control mouse plasma. Lipid profiles, upon comparing Hu LCAT transgenics to control animals, revealed a 20 t0 60% increase in total and cholesteryl esters that were mainly present in HDL. The in vivo substrate specificity of Hu LCAT was assessed by creating animals expressing Hu apo AI + Hu LCAT (HuAI/ LCAT), Hu apo AI + Hu apo AII + Hu LCAT (HuAI/ AII/LCAT), and Hu apo AII + Hu LCAT (HuAII/LCAT). Plasma cholesterol was increased up to 4.2-fold in HuAI/ LCAT transgenic mice and twofold in the HuAI/AII/LCAT transgenic mice, compared with HuAI and HuAI/AII transgenic mice. HDL cholesteryl ester levels were increased more than twofold in both the HuAI/LCAT and HuAI/AII/LCAT mice compared with the HuAI, HuAI/AII, and HuLCAT animals. The HDL particles were predominantly larger in the HuAI/LCAT and the HuAI/AII/LCAT mice compared with those in HuAI, HuAII/LCAT, and HuLCAT animals. The increase in LCAT activity in the HuAI/LCAT and HuAI/AII/LCAT mice was associated with 62 and 27% reductions respectively, in the proportion of Hu apo AI in the pre beta-HDL fraction, when compared with HuAI and HuAI/AII transgenic mice. These data demonstrate that moderate increases in LCAT activity are associated with significant changes in lipoprotein cholesterol levels and that Hu LCAT has a significant preference for HDL containing Hu apo AI.
Animals↗
Atherogenesis in transgenic mice with human apolipoprotein B and lipoprotein (a).
The engineering of mice that express a human apoB transgene has resulted in animals with high levels of human-like LDL particles and through crosses with human apo(a) transgenics, high levels of human-like lipoprotein (a) (Lp[a]) particles. In this study, these animals have been used to compare the atherogenic properties of apo(a), LDL, and Lp(a). The presence of the high expressing apoB (apoBH) transgene was associated with a 2.5-fold increase in VLDL-LDL cholesterol (primarily in the LDL fraction) and a 15-fold increase in proximal lesions compared with non-transgenic mice (P < or = 0.0001), while the presence of the low expressing human apoB (apoBL) transgene was not associated with major changes in lipoprotein profiles or increases in aortic lesion size. Examination of aortas of apoBH mice demonstrated lesions along the entire length of the aorta and immunochemical analysis of the lesions revealed features characteristically seen in human lesions including the presence of oxidized lipoproteins, macrophages, and immunoglobulins. Unlike animals with the apoBL transgene, animals with the apo(a) transgene had significant increases in proximal aortic fatty streak lesions compared to nontransgenic control animals (threefold; P < 0.02), while animals with both transgenes, the apo(a)/apo BL double transgenics, had lesions 2.5 times greater than animals expressing the apo(a) transgene alone and eightfold (P < 0.0006) greater than nontransgenic animals. These murine studies demonstrate that marked increases in apoB and LDL resulted in atherosclerotic lesions extending down the aorta which resemble human lesions immunochemically and suggest that apo(a) associated with apoB and lipid may result in a more pro-atherogenic state than when apo(a) is free in plasma.
Animals↗
In vivo demonstration of red cell-endothelial interaction, sickling and altered microvascular response to oxygen in the sickle transgenic mouse.
Intravascular sickling, red cell-endothelium interaction, and altered microvascular responses have been suggested to contribute to the pathophysiology of human sickle cell disease, but have never been demonstrated under in vivo flow. To address this issue, we have examined a transgenic mouse line, alphaHbetaSbetaS-Antilles [betaMDD] which has a combined high (78%) expression of beta S and beta S-Antilles globins. In vivo microcirculatory studies using the cremaster muscle preparation showed adhesion of red cells, restricted to postcapillary venules, in transgenic mice but not in control mice. Electron microscopy revealed distinct contacts between the red cell membrane and the endothelium surface. Some red cells exhibiting sickling were regularly observed in the venular flow. Infusion of transgenic mouse red cells into the ex vivo mesocecum vasculature also showed adhesion of mouse red cells exclusively in venules. Under resting conditions (pO2, 15-20 mmHg), there were no differences in the cremaster microvascular diameters of control and transgenic mice; however, transgenic mice showed a drastic reduction in microvascular red cell velocities (Vrbc) with maximal Vrbc decrease (> 60%) occurring in venules, the sites of red cell adhesion and sickling. Local, transient hyperoxia (pO2, 150 mmHg) resulted in striking differences between control and transgenic mice. In controls, oxygen caused a 69% arteriolar constriction, accompanied by 75% reduction in Vrbc. In contrast, in transgenic mice, hyperoxia resulted in only 8% decrease in the arteriolar diameter and in 68% increase in VrBC; the latter is probably due to an improved flow behavior of red cells as a consequence of unsickling. In summary, the high expression of human sickle hemoglobin in the mouse results not only in intravascular sickling but also red cell-endothelium interaction. The altered microvascular response to oxygen could be secondary to blood rheological changes, although possible intrinsic differences in the endothelial cell/vascular smooth muscle function in the transgenic mouse may also contribute. These sickle transgenic mice could serve as a useful model to investigate vasoocclusive mechanisms, as well as to test potential therapies.
Anemia, Sickle Cell↗
Morphometric and immunohistochemical characterization of human liver regeneration.
Regeneration in human liver is characterized in part by the formation of ductular structures, so-called ductular hepatocytes in massive hepatic necrosis and bile ductules in mechanical biliary obstruction. In an attempt to characterize the liver regenerative process, we performed image analysis and immunohistochemical staining of the ductular structures in these well defined human liver disorders, 13 cases of massive hepatic necrosis and 9 cases of mechanical biliary obstruction. The proliferation index was determined and the expression of several antigens was localized by immunohistochemical staining using antibodies to alpha-fetoprotein, alpha-1-antitrypsin, albumin, and cytokeratin 19. The ductular structures in adult human liver were compared with the developing ductal plates in 11 fetal livers, ranging in age from 9 to 36 weeks of gestation. Image analysis demonstrated that the mean total area, mean nuclear area, and mean cell size of ductular hepatocytes were significantly larger than those of bile ductules (p < 0.05). The proliferation index of ductular hepatocytes and bile ductules was significantly higher than that of hepatocytes of normal livers (p < 0.02). Bile ducts, bile ductules in mechanical biliary obstruction, ductular hepatocytes in massive hepatic necrosis, and the ductal plate cells in fetal liver showed strong staining for cytokeratin 19, which characterizes intermediate filaments associated with bile duct epithelial cells. Albumin, a liver-specific protein, and alpha-1-antitrypsin, a protease inhibitor, were strongly expressed in ductal plate cells of fetal liver, hepatocytes, and ductular hepatocytes, whereas bile duct cells and bile ductules were negative for albumin. In summary, ductular hepatocytes demonstrate morphometric and immunophenotypic features of both hepatocytes and biliary epithelial cells, whereas bile ductules share characteristics primarily with fetal ductal plates and mature bile ducts. These findings suggest that ductular hepatocytes in massive hepatic necrosis may serve as bipotential progenitor cells, and bile ductules in mechanical biliary obstruction are related to ductal plates of fetal liver.
Aging↗
Selective uptake of HDL cholesteryl esters is active in transgenic mice expressing human apolipoprotein A-I.
The direct non-endocytotic uptake of cholesteryl esters (CE) from high density lipoprotein (HDL) plays a major role in HDL CE metabolism in rats and rabbits. In vitro evidence indicates it may also play such a role in humans. However, a study in mice (tracing the CE and apoA-I moieties of HDL) concluded that, while selective uptake played a role in normal animals, it did not in transgenic mice which express predominantly human apoA-I (Chajek-Shaul et al., 1991. Proc. Natl. Acad. Sci. USA. 88: 6731-6735);thus human apoA-I was apparently unable to support selective uptake. These conclusions rested on plasma decay data that represent a composite of all tissue and which may obscure tissue-specific factors. Thus we reexamined the matter by measuring the rates of uptake of HDL components by individual tissues using intracellularly trapped tracers. Plasma decay data were much as reported in the referenced study. Nonetheless the fractional rate of uptake of HDL CE was greater than that of apoA-I in adrenal gland and liver, indicating selective uptake. Kidney took up apoA-I tracer at a greater fractional rate than CE tracer, apparently by filtration and reabsorption of free apoA-I, and this uptake was at a greater fractional rate in the transgenic mice than in normal mice. Thus, the lack of evidence for selective uptake in the plasma decay data of the transgenic mice was explained by a higher rate of renal uptake of apoA-I and not by a diminished rate of selective uptake in other tissues.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenal Glands↗
Quantitation of atherosclerosis in murine models: correlation between lesions in the aortic origin and in the entire aorta, and differences in the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice.
Murine strains susceptible to atherosclerosis provide valuable models to study factors involved in atherogenesis. In some murine models, limited hypercholesterolemia can be achieved and lesions develop primarily in the aortic origin, in the vicinity of the aortic valve. In other models such as LDL receptor-deficient and apoE-deficient mice, diet-induced or spontaneous hypercholesterolemia and atherogenesis are much greater. To determine whether lesion formation in the aortic origin, where particular pathogenic conditions may exist, correlates with lesion formation throughout the entire aorta, we measured the extent of atherosclerosis in both areas in 8 apoE- and 11 LDL receptor-deficient mice fed cholesterol-rich diets for 3-6 months, as well as in 9 C57BL/6 mice fed an atherogenic diet for a year, using two different morphometric methods. Both apoE-deficient and LDL receptor-deficient mice developed extensive lesions throughout the aorta, and in these models a significant correlation was observed between the extent of lesions in the entire aorta (measured as percent of surface area) and that at the aortic origin (measured as averaged lesion area per cross-section) (r = 0.77, P < 0.0001). In contrast, the plasma cholesterol levels achieved in C57BL/6 mice were much lower, and atherosclerotic lesions were found almost exclusively in the aortic origin. These results demonstrate that in murine models developing extensive aortic lesions, both morphometric methods provide valid and complementary information on the degree and distribution of atherosclerosis, and suggest that under severe atherogenic conditions lesion formation throughout the aorta is determined by the same pathological factors, in each model. Comparison of the extent of atherosclerosis in the entire aorta between genders also showed that male LDL receptor-deficient mice had significantly more lesions than females (29.2 vs. 14.8%, P < 0.005, n = 16). A similar trend was also seen in apoE-deficient mice.
Animals↗
Single base, site-directed mutagenesis of a 90 kilobase-pair P1 clone.
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Apolipoproteins B↗
Structural and functional properties of human and mouse apolipoprotein A-I.
Mouse and human plasma apolipoprotein A-I (apo A-Im and apo A-Ih, respectively) were investigated to compare their molecular properties in solution, their incorporation into palmitoyloleoylphosphatidylcholine-apo A-I (POPC-apo A-I) discoidal complexes; their structural stability in discoidal complexes and high-density lipoproteins (HDL), and their effect on structural rearrangement of discoidal complexes upon interaction with low-density lipoproteins (LDL). Unlike apo A-Ih, only minimal concentration-dependent self-association was observed for apo A-Im. While both apo A-Im and apo A-Ih formed discoidal complexes of distinct composition and size that reflected reassembly molar ratios of POPC/apo A-I, apo A-Im demonstrated specific deficiencies in formation of larger-sized complexes. Denaturation of both apo A-Im- or apo A-Ih-containing complexes and HDL with guanidine hydrochloride (GuHCl) indicated significantly reduced stabilization of apo A-Im by lipid in these particles. Interaction of apo A-Im- or apo A-Ih-containing discoidal complexes with human plasma LDL revealed a more extensive conversion of apo A-Im-complexes to smaller species. Mean hydrophobicities and mean hydrophobic moments of amphipathic helical segments in apo A-Im and apo A-Ih were compared; differences potentially contributing to differential lipid-binding properties between apo A-Im and apo A-Ih were identified. Our results demonstrate differences between apo A-Im and apo A-Ih that may contribute to the major changes in plasma HDL distribution and function observed in apo A-Ih transgenic mice.
Animals↗