Characterization of transgenic mice expressing apolipoprotein E4(C112R) and apolipoprotein E4(L28P; C112R)

Apolipoprotein E (ApoE), which is genetically polymorphic, is a constituent of different lipoproteins. Two variants, ApoE4(C112R) and ApoE4(L28P; C112R) have been linked to the risk of developing Alzheimer's disease. Transgenic mice carrying ApoE4(C112R) (AD71) and ApoE4(L28P; C112R) (AD61) were generated and compared to wild-type mice. The use of glial fibrillary acidic protein as promoter led to transgene expression mainly in glial cells but also in neurons. Transgene protein levels were approximately three-and-a-half-fold that of endogenous ApoE in the glial fibrillary acidic protein-ApoE4(C112R) (AD71) and nearly twofold in the glial fibrillary acidic protein-ApoE4(L28P; C112R) (AD61) mouse lines. Neither transgenic mouse differed from wild-type in cognitive tests at the age of approximately one-and-a-half years. The locomotor activity of AD61 mice was similar to controls, whereas AD71 mice exhibited a clearly reduced level of motor activity. Immunohistological and biochemical brain protein analyses revealed no difference between strains.Thus, in the absence of morphological changes over-expression of ApoE4(C112R) on a background of endogenous mouse ApoE, may result in behavioral deficits while for the ApoE4(L28P; C112R) transgene higher expression might be required or some compensatory mechanisms might protect these animals from the behavioral abnormalities.     

Spatial learning in transgenic mice expressing human presenilin 1 (PS1) transgenes

Dominant mutations in the Presenilin 1 gene are linked to an aggressive, early-onset form of familial Alzheimer's Disease (FAD). Spatial memory of transgenic (Tg) mice expressing either mutant (lines Tg(M146L)1, Tg(M146L)76, Tg(L286V)198) or wild type (line Tg(PS1wt)195) human PS1 transgenes was investigated in the Morris water maze (WM) test at 6 and 9 months of age. The results showed that the mutated Tg mice had increased swim speed when compared to non-Tg littermates or Tg PS1 wild type mice. The swim speed difference did not, however, significantly affect the spatial learning in the WM test and all groups showed comparable search paths during training and similar spatial bias during probe trials. When re-tested at 9 months, all mice showed significantly improved learning acquisition of spatial information. The lack of progressive spatial learning impairment in mice expressing the mutated human PS1 transgene in the WM does not preclude impairments in other cognitive tasks but suggests that full phenotypic expression of mutant PS1 alleles may require co-expression of human versions of other AD-associated genes.     

人Man2c1转基因小鼠模型的建立

目的 为在体内研究MAN2C1的生物学意义而建立转hMan2c1基因的小鼠。方法 构建pIRKS2-EGFP-hMan2c1重组表达载体，经体外转染实验鉴定转染的基因能在COS-7细胞表达后，注射人ICR小鼠受精卵，以制备转基因小鼠。用基因组PCR鉴定目的基因在宿主基因组DNA的整合。用RT-PCR和Westernblot分析hMan2c1在转基因小鼠的表达。结果 在116只原代小鼠中，有7只hMan2c1基因组PCR阳性。在所检测的20只F1代小鼠中，有9只hMan2c1基因组PCR阳性。在所检测的21只F2代小鼠中，有16只基因组PCR阳性。用鼠尾组织RT-PCR和Western blot检测hMan2c1基因表达，确定基因组PCR阳性的7个系中有4个系阳性。结论 建立了4个稳定表达hMan2c1的转基因小鼠系，为深入研究MAN2C1的生物学意义打下了基础。     

Reversal of presynaptic deficits of apolipoprotein E-deficient mice in human apolipoprotein E transgenic mice

Apolipoprotein E genotype is an important risk factor of Alzheimer's disease, which is associated with the degeneration of distinct brain neuronal systems. In the present study we employed apolipoprotein E-deficient mice and human apolipoprotein E3 and apolipoprotein E4 transgenic mice on a null mouse apolipoprotein E background, to examine the extent to which distinct brain neuronal systems are affected by apolipoprotein E and the isoform specificity of this effect. This was pursued by histological and autoradiographic measurements utilizing neuron specific presynaptic markers. The results thus obtained revealed significant reductions in the levels of brain cholinergic and noradrenergic nerve terminals in young apolipoprotein E-deficient mice and no changes in brain dopaminergic nerve terminals. These cholinergic and noradrenergic presynaptic derangements were ameliorated similarly in human apolipoprotein E3 and apolipoprotein E4 transgenic mice. In the case of the cholinergic system, this resulted in complete reversal of the presynaptic deficits, whereas in the case of the noradrenergic neurons the amelioration was partial.These findings suggest that brain cholinergic and noradrenergic neurons are markedly more dependent on brain apolipoprotein E than brain dopaminergic neurons and that the isoform specificity of these effects is not apparent at a young age under non-challenged conditions.     

Ultrastructural neuronal pathology in transgenic mice expressing mutant (P301L) human tau

Transgenic mice expressing mutant (P301L) human tau develop neurofibrillary tangles, amyotrophy and progressive motor disturbance. We present ultrastructural features of neuronal degeneration in this model that suggests involvement of both neurofibrillary and autophagic processes in neurodegeneration. Neurons undergoing neurofibrillary degeneration contain tau-immunoreactive, 15-20 nm-wide straight or wavy filaments with no periodic twists. Tau filaments were found in two types of affected neurons. One type resembled neurons with neurofibrillary tangles (NFT) that were filled with numerous filaments that displaced sparse cytoplasmic organelles to the periphery. Microtubules were almost completely absent. The nucleus remained centrally located, but showed lobulations due to deep infoldings. The other type resembled ballooned neurons seen in some human tauopathies. The nucleus was peripherally placed, but normal appearing. The cytoplasmic organelles were dispersed throughout the swollen perikarya, the Golgi complex was fragmented and duplicated, while mitochondria and other organelles appeared normal. Tau filaments similar to those in NFT were sparse and not tightly packed. Microtubules were also sparse. Many autophagic vacuoles were present in these cells. Heterogeneous appearing axonal swellings resembling spheroids in human tauopathies were present in gray and white matter. Unlike normal appearing axons, axonal spheroids were filled with tau-immunoreactive filaments and autophagic vacuoles, in addition to normal appearing neurofilaments and microtubules. These P301L transgenic mice exhibit many features common to human tauopathies, making them a valuable model to study the pathogenesis of these uncommon disorders.     

HIV-associated nephropathy in transgenic mice expressing HIV-1 genes

Transgenic mice were produced that bore copies of a defective HIV provirus. The transgenic offspring from three independently derived mouse lines manifested renal disease associated with proteinuria, a high mortality rate, and HIV-specific gene expression in the kidney. An early histopathological lesion in the kidney was focal glomerulosclerosis. Moribund animals had diffuse glomerulosclerosis with prominent microcystic tubular dilatation, tubular epithelial degeneration, and interstitial nephritis. Electron microscopy revealed ultrastructural features consistent with the glomerulosclerosis: effacement of the foot processes of visceral epithelium and an increase in mesangial cell matrix. Transgenic mice variably expressed 6-, 4.3-, and 2-kb HIV-specific RNAs and HIV-related polypeptides in several tissues including kidney. Immunocytostaining revealed the presence of HIV-related protein in the glomeruli of affected animals. Glomerulopathy in these transgenic mice and HIV-associated nephropathy in man have similar features.     

Strategies for expressing human antibody repertoires in transgenic mice

Repertoires of human antibodies can be created in transgenic mice carrying human immunoglobulin-gene loci in germline configuration. These ‘transloci’, introduced either as miniloci or as almost locus-sized regions, undergo rearrangement and hypermutation in mouse lymphoid tissue. Here, Marianne Brüggemann and Michael Neuberger review the use of such mice for raising antigen-specific human monoclonal antibodies, as well as their exploitation for studying regulatory aspects of antibody repertoire formation.     

Comparison of HLA-DR1-restricted T cell response induced in HLA-DR1 transgenic mice deficient for murine MHC class II and HLA-DR1 transgenic mice expressing endogenous murine MHC class II molecules

Transgenic mice expressing human HLA class II molecules provide a useful model for identifying HLA-restricted CD4+ epitopes. However, the influence of endogenous murine H-2-restricted T cell responses on HLA-restricted responses is not known. In the present study, we show that HLA-DR1 transgenic mice deficient for H-2 class II expression (HLA-DR1+/+/IAbeta0/0) exhibit an equivalent expression level of the transgene HLA-DR1 and a similar diversity in the TCR repertoire, but a slightly different number of CD4+ peripheral T cells, when compared to HLA-DR1 transgenic mice in which H-2 class II molecules were retained (HLA-DR1+/+/IAbeta+/+). More importantly, a strong antigen-specific HLA-DR1-restricted response was observed in nearly all HLA-DR1+/+/IAbeta0/0 mice immunized with HBV envelope protein (HBs) or capsid protein (HBc), whereas weak HBs- or HBc-specific HLA-DR1-restricted responses were detected in half of the immunized HLA-DR1+/+/IAbeta+/+ mice. Conversely, strong HBs- or HBc-specific H-2-restricted T cell responses were detected in HLA-DR1+/+/IAbeta+/+ mice but not in HLA-DR1+/+/IAbeta0/0 mice. Our results indicate that the coexpression of endogenous H-2 class II molecules reduces the intensity of HLA-DR1-restricted antigen-specific responses in transgenic mice, by favoring murine over human MHC recognition and education. Thus, HLA-DR1+/+/IAbeta0/0 mice represent a better model for identifying and characterizing HLA-DR1-restricted epitopes relevant for human disease.     

转人溶菌酶基因小鼠乳腺生物反应器的建立

目的 研究人溶菌酶（human lysozyme，hLYZ）动物乳腺生物反应器制备的可行性。方法 将hLYZ基因与动物乳腺特异表达载体p205C3连接，所得重组载体p205C3-hLYZ用显微注射法建立转基因小鼠。结果 共出生了136只F0代小鼠，PER和Southern杂交检测基因整合阳性率分别为5．15％（2♀5♂）和2．94％（1♀3♂）。Western印迹检测结果表明，分泌在小鼠乳汁中的表达产物与正常hLYZ具有相同的分子量。目前转基因小鼠已经繁殖到B代，每一代基因整合阳性母鼠的乳腺均表达hLYZ，乳汁中的表达量最高达750mg/L。斑点杂交试验证明，表达有较强的组织特异性，除在乳腺表达外，仅在脾脏和小肠有一定的异位表达。结论 成功建立了hLYZ小鼠乳腺生物反应器。     

No evidence for cholinergic problems in apolipoprotein E knockout and apolipoprotein E4 transgenic mice

The varepsilon4 allele of the apolipoprotein E gene constitutes the major genetic risk factor to develop Alzheimer's disease. If and how this protein contributes to the pathological cascade of Alzheimer's disease is not known. The varepsilon4 allele particularly affects the cholinergic defect, which is one of the most consistent neurotransmitter problems in an Alzheimer's disease brain.We have analysed several parameters of the cholinergic system in brain of apolipoprotein E knockout mice as well as in transgenic mice overexpressing human apolipoprotein E4. We analysed the distribution of cholinergic fibers, the number and morphology of cholinergic neurons and the enzymatic activity of acetylcholinesterase and choline acetyltransferase in different brain regions. Finally, we analysed the distribution and the binding parameters of [3H]hemicholinium-3, a specific marker for the high affinity choline transporter in different brain sections and regions.This extensive effort failed to show any consistent difference in the cholinergic parameters studied, in either the apolipoprotein E4 transgenic mice or in the apolipoprotein E knockout mice, compared to age-matched non-transgenic mice. We conclude that the apolipoprotein E4 is not deleterious per se for the cholinergic system in mouse brain.     

Prominent axonopathy and disruption of axonal transport in transgenic mice expressing human apolipoprotein E4 in neurons of brain and spinal cord

The epsilon 4 allele of the human apolipoprotein E gene (ApoE4) constitutes an important genetic risk factor for Alzheimer's disease. Recent experimental evidence suggests that human ApoE is expressed in neurons, in addition to being synthesized in glial cells. Moreover, brain regions in which neurons express ApoE seem to be most vulnerable to neurofibrillary pathology. The hypothesis that the expression pattern of human ApoE might be important for the pathogenesis of Alzheimer's disease was tested by generating transgenic mice that express human ApoE4 in neurons or in astrocytes of the central nervous system. Transgenic mice expressing human ApoE4 in neurons developed axonal degeneration and gliosis in brain and in spinal cord, resulting in reduced sensorimotor capacities. In these mice, axonal dilatations with accumulation of synaptophysin, neurofilaments, mitochondria, and vesicles were documented, suggesting impairment of axonal transport. In contrast, transgenic mice expressing human ApoE4 in astrocytes remained normal throughout life. These results suggest that expression of human ApoE in neurons of the central nervous system could contribute to impaired axonal transport and axonal degeneration. The possible contribution of hyperphosphorylation of protein Tau to the resulting phenotype is discussed.     

Differential neurotrophic effects of apolipoprotein E in aged transgenic mice.

The present study seeked to determine whether the neurodegenerative and cognitive alterations in aged apolipoprotein E-deficient mice are differentially reversed by transgenic overexpression of human apolipoprotein-E3 vs. apolipoprotein-E4 in the background of deficient endogenous apolipoprotein E. These studies showed dendritic alterations in pyramidal neurons of apolipoprotein-E4 transgenic mice, similar to the ones observed in apolipoprotein E-deficient mice. However, these mice had a preserved density of synaptophysin-immunoreactive presynaptic terminals. In contrast, mice overexpressing apolipoprotein-E3 showed no synapto-dendritic alterations. Analysis of behavioral performance in the Morris water maze showed that while apolipoprotein E-deficient mice performed poorly, overexpression of apolipoprotein-E3 and, to a lower extent apolipoprotein-E4, resulted in an improved performance. This study supports the contention that, compared with apolipoprotein-E4, apolipoprotein-E3 might have a greater neurotrophic in vivo effect in aged mice.     

Neutrophil response of transgenic mice expressing human group IIA phospholipase A2 in bacterial infections

Group IIA phospholipase A2 (PLA2) is a newly recognized acute phase protein with marked antibacterial properties. We have shown previously that transgenic C57BL/6 J mice expressing human group IIA PLA2 (PLA2+ mice) are more resistant to bacterial infections than nontransgenic C57BL/6 J mice that, among mice, are unusual in that they lack the mouse analogue of group IIA PLA2 (PLA2- mice). To elucidate the possible mechanisms involved in the host response of these mice in bacterial infection, peripheral inflammatory cell responses of PLA2+ and PLA2- mice were studied after i.p. administration of Escherichia coli, E. coli lipopolysaccharide or Staphylococcus aureus. Uninfected PLA2+ mice had higher numbers of lymphocytes and polymorphonuclear neutrophil leukocytes (PMNs) in their blood than PLA2- mice. In PLA2+ mice, the number of PMNs increased in peripheral blood in parallel with the concentration of group IIA PLA2 after the administration of bacteria, whereas these responses were not seen in PLA2- mice. High concentrations of group IIA PLA2 in PLA2+ mice may increase the synthesis of bioactive molecules, such as prostaglandins, which in turn may mobilize PMNs into circulation. Our results support the hypothesis that group IIA PLA2 is an important inflammatory mediator in bacterial infections.     

Premature death and neurologic abnormalities in transgenic mice expressing a mutant huntingtin exon-2 fragment

Huntington's disease (HD) is a fatal neurodegenerative disease characterized pathologically by aggregates composed of N-terminal fragments of the mutant form of the protein huntingtin (htt). The role of these N-terminal fragments in disease pathogenesis has been questioned based in part on studies in transgenic mice. In one important example, mice that express an N-terminal fragment of mutant htt terminating at the C-terminus of exon 2 (termed the Shortstop mouse) were reported to develop robust inclusion pathology without developing phenotypic abnormalities seen in the R6/2 or N171-82Q models of HD, which are also based on expression of mutant N-terminal htt fragments. To further explore the capacity of mutant exon-2 htt fragments to produce neurologic abnormalities (N-terminal 118 amino acids; N118), we generated transgenic mice expressing cDNA that encodes htt N118-82Q with the mouse prion promoter vector. In mice generated in this manner, we demonstrate robust inclusion pathology accompanied by early death and failure to gain weight. These phenotypes are the most robust abnormalities identified in the R6/2 and N171-82Q models. We conclude that the lack of an overt phenotype in the initial Shortstop mice cannot be completely explained by the properties of mutant htt N118 fragments.     

Development of atherosclerosis in Balb/c apolipoprotein E-deficient mice

BACKGROUND: Since its creation in 1992 by gene inactivation via gene targeting, the apolipoprotein E "knockout" mouse has become the most widely used rodent model for the study of atherosclerosis. Commercially available apolipoprotein E(-/-) mice are bred on a C57BL/6J background. The goal of the present study was to investigate the development of atherosclerosis in apolipoprotein E-deficient mice generated on a Balb/c background. METHODS: We compared serum cholesterol concentrations and the development of atherosclerotic lesions in heterozygous Balb/c [apolipoprotein E(+/-)] mice fed regular rodent chow, Balb/c apolipoprotein E-deficient mice fed regular chow, and Balb/c apolipoprotein E-deficient mice fed a high-fat diet for up to 30 weeks. Expression of the chemokine JE (murine homologue of MCP-1), as well as the adhesion molecules E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, in the aortas of knockout mice fed a high-fat diet was measured by enzyme-linked immunosorbent assay. RESULTS: Balb/c apolipoprotein E-deficient mice develop atherosclerotic lesions in a reproducible temporal and morphological pattern. Total serum cholesterol concentrations in Balb/c apolipoprotein E-deficient mice fed regular chow or a high-fat diet, respectively, closely parallel those reported for C57BL/6J apolipoprotein E-deficient mice. The expression of all three adhesion molecules in the aorta follows a similar temporal pattern, peaking in the first 15 weeks, whereas JE concentrations peak around 23 weeks. CONCLUSION: The availability of Balb/c apolipoprotein E-deficient mice will facilitate the study of atherosclerosis in a mouse strain that can concomitantly develop other pathological states that are not readily inducible in mice with the C57BL/6J background.     

Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene

Transgenic mice expressing an inducible suicide gene, which allows systemic and reversible elimination of macrophages, were developed. A macrophage-specific c-fms promoter was used to express enhanced green fluorescent protein and a drug-inducible suicide gene that leads to Fas-mediated apoptosis in resting and cycling cells of the macrophage lineage. Transgenic mice were fertile, of normal weight, and showed no abnormal phenotype before drug exposure. The transgene was expressed constitutively in macrophages and dendritic cells (DC) but not significantly in T cells or B cells. Induction of the suicide gene led to depletion of 70-95% of macrophages and DC in nearly all tissues examined. Depletion reduced the ability to clear bacteria from the blood and led to increased bacterial growth in the liver. Depleted mice displayed several abnormalities, including splenomegaly, lymphadenopathy, thymic atrophy, extramedullary hematopoiesis, and development of peritoneal adhesions. This new, transgenic line will be useful in investigating the role of macrophages and DC.     

Cholesterol homeostatic mechanisms in transgenic mice with altered expression of apoproteins A-I, A-II and A-IV

Our understanding of the in vivo metabolic functions of apoA-I and A-II has greatly advanced with the use of transgenic mice, but the physiological role of apoA-IV remains elusive. Both apoA-I and A-II are necessary for the structural stability of high-density lipoprotein (HDL). Structural differences exist between human and mouse A apoproteins because: i) human cholesterol ester transfer protein, lecithin cholesterol acyl transferase and phospholipid transfer protein interact better with human apoA-I; ii) human apoA-I and A-II, alone or in combination, form polydisperse instead of monodisperse HDL particles. Human apoA-II overexpression has highlighted its inhibitory effect on lipoprotein lipase and hepatic lipase, resulting in hypertriglyceridemia and concomitantly decreased HDL and apoA-I. After long-term challenge with an atherogenic diet, mice are less protected against lesion formation by human apoA-II, mouse apoA-II being overtly proatherogenic. On the other hand, human apoA-I confers great protection against lesion formation and causes reduction of preexisting lesions. Human apoA-IV is also protective, although the mechanisms by which this protection is achieved remain to be determined.     

Generation and characterization of transgenic mice expressing cobra venom factor

Cobra venom factor (CVF), the anticomplementary protein in cobra venom, activates the alternative complement pathway, eventually leading to complement consumption. Here, we describe the development of a transgenic mouse model for CVF. We generated a DNA construct containing the full-length cDNA for single-chain pre-pro-CVF. Expression of CVF was controlled by the alpha(1)-antitrypsin promoter to achieve liver-specific expression. Linearized DNA was microinjected into murine ovary cells (strain CD(2)F(1) (BALB/cxDBA/2J)) and the newborn mice were analyzed for stable integration of CVF DNA. After establishing the transgene, mice were propagated in a BALB/c background. The CVF mRNA was detected in the liver and, in some animals, in the kidney. CVF protein was detected in small amounts in the serum. Serum complement hemolytic activity in CVF-transgenic mice was virtually absent. The concentration of plasma C3 was significantly reduced. The CVF-transgenic animals show no unusual phenotype. They provide an animal model to study the effect of long-term complement depletion by continued activation, as well as the role of complement in host immune response and pathogenesis of disease.     

Schwann cell hyperplasia and tumors in transgenic mice expressing a naturally occurring mutant NF2 protein

Specific mutations in some tumor suppressor genes such as p53 can act in a dominant fashion. We tested whether this mechanism may also apply for the neurofibromatosis type-2 gene (NF2) which, when mutated, leads to schwannoma development. Transgenic mice were generated that express, in Schwann cells, mutant NF2 proteins prototypic of natural mutants observed in humans. Mice expressing a NF2 protein with an interstitial deletion in the amino-terminal domain showed high prevalence of Schwann cell-derived tumors and Schwann cell hyperplasia, whereas those expressing a carboxy-terminally truncated protein were normal. Our results indicate that a subset of mutant NF2 alleles observed in patients may encode products with dominant properties when overexpressed in specific cell lineages.