小鼠乳腺表达抗PD-1抗体对其脾脏T细胞的影响

为从细胞水平研究小鼠乳腺表达抗PD-1抗体对转基因鼠脾脏T细胞表面抗原蛋白、细胞因子表达、脾脏CD4⁺T细胞增殖以及增殖相关通路的影响,将8周龄未经历过怀孕的和18周龄经历过哺乳的表达抗人PD-1抗体的转基因小鼠分成两组,每组以转基因阴性鼠为对照,提取脾脏淋巴细胞,检测脾脏淋巴细胞的变化。与转基因阴性小鼠相比,乳腺表达抗PD-1抗体的转基因小鼠的免疫系统中的脾脏T细胞的效应T细胞比例上升,Treg细胞比例下降,CD4⁺T细胞表达的IFN-γ、IL-17以及IL-2有不同程度的增加。IL-4、IL-10以及TGF-β都没有发生变化。与T细胞刺激相关的一些细胞表面的蛋白分子也没有引起变化。转基因阳性鼠和转基因阴性鼠中T细胞增殖没有显著性差异,转基因阳性鼠中PI3K/Akt/mTOR和Ras/MEK/ERK这两条通路上的磷酸化蛋白只有部分表达上调,整个通路没有完全上调。结果表明,转基因小鼠作为表达抗PD-1抗体这类免疫系统相关单克隆抗体的宿主是可行的。     

Establishment of a transgenic mouse model specifically expressing human serum amyloid A in adipose tissue

Obesity and obesity co-morbidities are associated with a low grade inflammation and elevated serum levels of acute phase proteins, including serum amyloid A (SAA). In the non-acute phase in humans, adipocytes are major producers of SAA but the function of adipocyte-derived SAA is unknown. To clarify the role of adipocyte-derived SAA, a transgenic mouse model expressing human SAA1 (hSAA) in adipocytes was established. hSAA expression was analysed using real-time PCR analysis. Male animals were challenged with a high fat (HF) diet. Plasma samples were subjected to fast protein liquid chromatography (FPLC) separation. hSAA, cholesterol and triglyceride content were measured in plasma and in FPLC fractions. Real-time PCR analysis confirmed an adipose tissue-specific hSAA gene expression. Moreover, the hSAA gene expression was not influenced by HF diet. However, hSAA plasma levels in HF fed animals (37.7±4.0 µg/mL, n = 7) were increased compared to those in normal chow fed animals (4.8±0.5 µg/mL, n = 10; p<0.001), and plasma levels in the two groups were in the same ranges as in obese and lean human subjects, respectively. In FPLC separated plasma samples, the concentration of hSAA peaked in high-density lipoprotein (HDL) containing fractions. In addition, cholesterol distribution over the different lipoprotein subfractions as assessed by FPLC analysis was similar within the two experimental groups. The established transgenic mouse model demonstrates that adipose tissue produced hSAA enters the circulation, resulting in elevated plasma levels of hSAA. This new model will enable further studies of metabolic effects of adipose tissue-derived SAA.     

Tau filament formation in transgenic mice expressing P301L tau

Mutations in the microtubule-associated protein tau, including P301L, are genetically coupled to hereditary frontotemporal dementia with parkinsonism linked to chromosome 17. To determine whether P301L is associated with fibril formation in mice, we expressed the longest human tau isoform, human tau40, with this mutation in transgenic mice by using the neuron-specific mouse Thy1.2 promoter. We obtained mice with high expression of human P301L tau in cortical and hippocampal neurons. Accumulated tau was hyperphosphorylated and translocated from axonal to somatodendritic compartments and was accompanied by astrocytosis and neuronal apoptosis indicated by terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end-labeling staining. Moreover, P301L tau formed abnormal filaments. Electron microscopy of sarcosyl-insoluble protein extracts established that the filaments had a straight or twisted structure of variable length and were approximately 15 nm wide. Immunoelcecton microscopy showed that the tau filaments were phosphorylated at the TG3, AT100, AT8, and AD199 epitopes in vivo. In cortex, brain stem, and spinal cord, neurofibrillary tangles were also identified by thioflavin-S fluorescent microscopy and Gallyas silver stains. Together, our results show that expression of the P301L mutation in mice causes neuronal lesions that are similar to those seen in human tauopathies.     

Posttranslational modifications of tubulin in cultured mouse brain neurons and astroglia

Posttranslational modifications of tubulin were analyzed in mouse brain neurons and glia developing in culture. Purified tubulin was resolved by isoelectric focusing. After 3 weeks of culture, neurons were shown to express a high degree of tubulin heterogeneity (8 alpha and 10 beta isoforms), similar to that found in the brain at the same developmental stage. Astroglial tubulin exhibits a less complex pattern consisting of 4 alpha and 4 beta isoforms. After incubation of neuronal and glial cells with 3H-acetate in the presence of cycloheximide, a major posttranslational label was found associated with alpha-tubulin and a minor one with beta-tubulin. The acetate-labeled isotubulins of neurons were resolved by isoelectric focusing into as many as 6 alpha and 7 beta isoforms, while those of astroglia were resolved into only 2 alpha and 2 beta isoforms. The same alpha isoforms were also shown to react with a monoclonal antibody recognizing selectively the acetylated form(s) of alpha-tubulin. Whether acetate-labeling of alpha-tubulin in these cells corresponds to the acetylation of Lys40, as reported for Chlamydomonas reinhardtii, is discussed according to very recent data obtained by protein sequence analysis. Tubulin phosphorylation was analyzed by incubation of cell cultures with 32PO4. No phosphorylation of alpha-tubulin isoforms was detected. A single beta-tubulin isoform (beta'2), expressed only in neurons, was found to be phosphorylated. This isoform is similar to that previously identified in differentiated mouse neuroblastoma cells.     

含P301L突变的Tau转基因小鼠纯合子品系的建立及鉴定

目的:建立含P301L突变的tau转基因小鼠的纯合子品系。方法:雄原核显微注射法获得含P301L突变的tau转基因阳性首建鼠,通过SYBR Green实时荧光定量PCR法和传统育种方式结合鉴定纯合子和杂合子。结果:共选育出95只纯合子,鉴定出的纯合子具有优于杂合子模拟老年痴呆生物学特性改变的优势。结论:外源性基因tau能稳定遗传,采用的SYBR Green实时荧光定量PCR和传统育种方式结合筛选鉴定纯合子和杂合子快速、经济、可靠。     

Ca(2+) dysregulation in neurons from transgenic mice expressing mutant presenilin 2

Mutations in amyloid precursor protein (APP), and presenilin‐1 and presenilin‐2 (PS1 and PS2) have causally been implicated in Familial Alzheimer’s Disease (FAD), but the mechanistic link between the mutations and the early onset of neurodegeneration is still debated. Although no consensus has yet been reached, most data suggest that both FAD‐linked PS mutants and endogenous PSs are involved in cellular Ca²⁺ homeostasis. We here investigated subcellular Ca²⁺ handling in primary neuronal cultures and acute brain slices from wild type and transgenic mice carrying the FAD‐linked PS2‐N141I mutation, either alone or in the presence of the APP Swedish mutation. Compared with wild type, both types of transgenic neurons show a similar reduction in endoplasmic reticulum (ER) Ca²⁺ content and decreased response to metabotropic agonists, albeit increased Ca²⁺ release induced by caffeine. In both transgenic neurons, we also observed a higher ER–mitochondria juxtaposition that favors increased mitochondrial Ca²⁺ uptake upon ER Ca²⁺ release. A model is described that integrates into a unifying hypothesis the contradictory effects on Ca²⁺ homeostasis of different PS mutations and points to the relevance of these findings in neurodegeneration and aging.     

Increased milk yield in transgenic mice expressing insulin-like growth factor 1

Insulin-like growth factor 1 (IGF-1) mediates many of the actions of growth hormone. Overexpression of IGF-1 was reported to have endocrine and paracrine/autocrine effects on somatic growth in transgenic mice. To study the paracrine/autocrine effects of IGF-1 in mammary gland, transgenic mice were produced by pronuclear microinjection of a construct containing a bovine alpha-lactalbumin (alpha-LA) promoter linked to an ovine IGF-1 cNDA. This alpha-LA promoter has previously been shown to direct expression of a human factor VIII gene specifically to the mammary gland of transgenic mice. Three transgenic mouse lines were established as a result of microinjection of 398 embryos. Transgene expression was found in mammary gland at day 1 of lactation from these three lines. Progeny test were carried out by mating two transgenic males/one transgenic female to two nontransgenic females/one nontransgenic male. Mice from one line (line 1225) were all nonexpressors and the other (line 1372) failed to produce offspring. Milk yield was analyzed in the line 1137 that produced 10 mice, of which three were transgenic females and three nontransgenic females. All of the three transgenic females showed integration of the transgene and expressed transgene IGF-1 mRNA in the mammary gland. Milk yields from days 5, 10, and 15 of lactation were significant greater in transgenic expressors than in their nontransgenic littermates. Specifically, there is 17.9% increase in total milk yield from these three days for transgenics compared with nontransgenics. These results demonstrate that local overexpression of IGF-1 in transgenic mice is capable to stimulating milk yield during the first lactation.     

Generation and characterization of two transgenic mouse lines expressing human ApoE2 in neurons and glial cells

Apolipoprotein E (apoE) isoforms are key determinants of susceptibility to late-onset Alzheimer's disease (AD). The epsilon 4 and epsilon 2 alleles have been associated with increased and decreased risk for AD, respectively. We have generated and characterized transgenic mice in which the human apoE2 gene is expressed under the control of the platelet-derived growth factor B-chain (PDGF-B) promoter, or the transferrin (TF) promoter. S1 nuclease analysis and immunoblotting showed that the PDGF-B apoE2 mice express apoE2 exclusively in the brain whereas the TF apoE2 mice express apoE2 in the liver and in the brain. In the TF apoE2 mouse line, apoE2 is also detected in the plasma. The PDGF-B apoE2 and the TF apoE2 transgenic mice were bred back to apoE(-)(/)(-) background. Immunohistochemical analysis showed that the PDGF apoE2 x apoE(-)(/)(-) and the TF apoE2 x apoE(-)(/)(-) mice express human apoE2 within the neocortex in hippocampal neurons and glial cells, respectively. ApoE(-)(/)(-) mice have been shown to develop age-dependent loss of synaptophysin. Immunoblotting of mouse brain extracts and immunohistochemical analysis of brain sections showed that apoE expression in both apoE2 x apoE(-)(/)(-) transgenic lines was associated with significant recovery of brain synaptophysin levels as compared to the levels of apoE(-)(/)(-) littermates of the same age. These apoE2-expressing mice, when bred back on amyloid precursor protein (APP) transgenic mice or other mouse lines featuring alterations in lipoprotein metabolism, may provide new mouse models for elucidating the role of apoE2 in lipid homeostasis in the brain and in the pathogenesis of AD.     

Muscle atrophy in transgenic mice expressing a human TSC1 transgene

Muscle mass is regulated by a wide range of hormonal and nutritional signals, such as insulin and IGF. Tuberous sclerosis complex (TSC) is an inherited hamartoma disease with tumor growth in numerous organs. TSC is caused by mutation in either TSC1 or TSC2 tumor suppressor genes that negatively regulate insulin-induced S6K activation and cell growth. Here we report that expression of human TSC1 (hTSC1) in mouse skeletal muscle leads to reduction of muscle mass. Expression of hTSC1 stabilizes endogenous TSC2 and leads to inhibition of the mTOR signaling. The hTSC1-mTSC2 hetero-complex and its downstream components remain sensitive to insulin stimulation and nutrition signals. This study suggests that an increase in the steady state level of resident TSC1-TSC2 complex is sufficient to reduce muscle mass and cause atrophy.     

Characterization of transgenic mice expressing cancer-associated variants of human NOTCH1

The Notch1 receptor plays a critical role in cell fate decisions during development. Activation of Notch signaling has been implicated in several types of cancer, particularly T-cell acute lymphoblastic leukemia (T-ALL). Consequently, several transgenic mouse strains have been made to study the role of Notch1 in T-ALL. However, the existing Notch1 transgenic lines mimic a translocation event found in only ～1% of T-ALL cases. Here we describe three novel NOTCH1 transgenic mouse strains that have Cre-inducible expression of the entire human NOTCH1 locus, each possessing a common mutation found in T-ALL. Unlike existing Notch1 transgenic strains, these NOTCH1 transgenic strains express full-length receptors from an endogenous human promoter that should be susceptible to a number of Notch antagonists that have recently been developed. These strains will allow researchers to modulate Notch signaling to study both normal development and cancer biology.     

A transgenic mouse expressing human CYP4B1 in the liver

The human CYP4B1 protein was expressed in the liver of a transgenic mouse line under the control of the promoter of the human apolipoprotein E (apo E) gene. Hepatic microsomes of transgenic mice catalyzed omega-hydroxylation of lauric acid and also activated 2-aminofluorene (2-AF), which is a typical substrate for CYP4B1, to mutagenic compounds detected by an umu gene expression assay. These activities observed in transgenic mouse were efficiently inhibited by CYP4B1 antibody. However, such inhibition was not observed in control mice. This is the first report to indicate catalytic activities of human CYP4B1. For further characterization of human CYP4B1, a fusion protein of CYP4B1 and NADPH-P450 reductase was expressed in yeast cells. It was able to activate 2-AF and was also able to catalyze omega-hydroxylation of lauric acid. This transgenic mouse line and the recombinant fusion protein provide a useful tool to study human CYP4B1 and its relation to chemical toxicity and carcinogenesis.     

Apoptosis in transgenic mice expressing the P301L mutated form of human tau

The rTg4510 mouse is a tauopathy model, characterized by massive neurodegeneration in Alzheimer's disease (AD)-relevant cortical and limbic structures, deficits in spatial reference memory, and progression of neurofibrillary tangles (NFT). In this study, we examined the role of apoptosis in neuronal loss and associated tau pathology. The results showed that DNA fragmentation and caspase-3 activation are common in the hippocampus and frontal cortex of young rTg4510 mice. These changes were associated with cleavage of tau into smaller intermediate fragments, which persist with age. Interestingly, active caspase-3 was often co-localized with cleaved tau. In vitro, fibrillar Abeta(1-42) resulted in nuclear fragmentation, caspase activation, and caspase-3-induced cleavage of tau. Notably, incubation with the antiapoptotic molecule tauroursodeoxycholic acid abrogated apoptosis-mediated cleavage of tau in rat cortical neurons. In conclusion, caspase-3-cleaved intermediate tau species occurred early in rTg54510 brains and preceded cell loss in Abeta-exposed cultured neurons. These results suggest a potential role of apoptosis in neurodegeneration.     

A mouse minialbumin gene is specifically expressed in differentiated hepatoma cells but not in transgenic mice

A mouse genomic DNA fragment including the albumin gene in which central exons 9-12 had been deleted and flanked by 2.2 kb in 5' and 4.3 kb in 3' (minialbumin gene), was introduced into rat hepatoma cells and also into mouse embryos to produce transgenic mice. The minialbumin gene was specifically transcribed in stably transfected differentiated clones and a 47-k Da minialbumin was synthesized and secreted into the culture medium. In contrast, the transgene was not expressed in any of the seven independent transgenic mouse lines examined. This suggests that expression of the albumin gene in developing animals requires cis-regulating elements additional to those located within the immediate flanking regions of the gene, which are sufficient to elicit specific expression in differentiated hepatoma cells in culture.     

离体培养的小鼠脊髓固有神经元的突触构筑

An electron microscopic analysis of the synaptic architecture in propriospinal neurons of cultured fetal mouse spinal cord has been undertaken. The size of the perikarya in the cultured spinal cord represents a range from small- to medium sized neurons, which form many synapses each other. There are many axo-dendritic and axo-somatic synapses in the culture but direct dendro-dendritic apposition is rarely seen. Four morphological types of synaptic boutons, S, F, M and G are classified according to criteria used by previous investigators. The ultrastructural details available suggest that the propriospinal neurons receive synaptic input from propriospinal fibers through simple synapses. It may indicate that their impulses can be controlled only postsynaptically.     

Dendritic cells expressing transgenic galectin-1 delay onset of autoimmune diabetes in mice

Type 1 diabetes (T1D) is a disease caused by the destruction of the beta cells of the pancreas by activated T cells. Dendritic cells (DC) are the APC that initiate the T cell response that triggers T1D. However, DC also participate in T cell tolerance, and genetic engineering of DC to modulate T cell immunity is an area of active research. Galectin-1 (gal-1) is an endogenous lectin with regulatory effects on activated T cells including induction of apoptosis and down-regulation of the Th1 response, characteristics that make gal-1 an ideal transgene to transduce DC to treat T1D. We engineered bone marrow-derived DC to synthesize transgenic gal-1 (gal-1-DC) and tested their potential to prevent T1D through their regulatory effects on activated T cells. NOD-derived gal-1-DC triggered rapid apoptosis of diabetogenic BDC2.5 TCR-transgenic CD4+ T cells by TCR-dependent and -independent mechanisms. Intravenously administered gal-1-DC trafficked to pancreatic lymph nodes and spleen and delayed onset of diabetes and insulitis in the NODrag1(-/-) lymphocyte adoptive transfer model. The therapeutic effect of gal-1-DC was accompanied by increased percentage of apoptotic T cells and reduced number of IFN-gamma-secreting CD4+ T cells in pancreatic lymph nodes. Treatment with gal-1-DC inhibited proliferation and secretion of IFN-gamma of T cells in response to beta cell Ag. Unlike other DC-based approaches to modulate T cell immunity, the use of the regulatory properties of gal-1-DC on activated T cells might help to delete beta cell-reactive T cells at early stages of the disease when the diabetogenic T cells are already activated.     

吗啡对培养海马神经元钙离子作用的机制研究

目的：研究吗啡对海马神经元[Ca^2 ]i影响的机制,为探索吗啡成瘾的神经生物学机制与可能的治疗途径。方法：荧光探针Fluo-4标记细胞内游离钙后,用激光共聚焦显微镜检测吗啡对大鼠原代培养海马神经元[Ca^2 ]i的影响。结果：吗啡急性刺激引起海马神经元[Ca^2 ]i升高,CTOP不能阻断吗啡引起的细胞内[Ca^2 ]i增加,而naltrindole能阻断吗啡引起的细胞内[Ca^2 ]i反应;Thapsigargin预处理阻断吗啡诱导的细胞内[Ca^2 ]i增加,Verapamil预处理不能完全抑制吗啡引起的细胞内[Ca^2 ]i增加;吗啡长时程作用后,海马神经元[Ca^2 ]i升高,加入纳络酮急性戒断后,不能阻断吗啡引起的细胞内[Ca^2 ]i升高,反而引起[Ca^2 ]i异常升高。结论：吗啡急性刺激引起的海马神经元内游离钙增加主要来源于δ2阿片受体介导的IP3敏感的钙库释放。     

Transgenic mouse lines expressing Cre recombinase specifically in posterior notochord and notochord

During development, the organizer provides instructive signals to surrounding cells as well as contributing cells to axial structures. To dissect organizer function at different developmental stages, conditional approaches such as the Cre/loxP system for conditional mutagenesis are particularly useful. Here we describe two new Cre transgenic mouse lines, Foxa2 NFP-Cre and Nodal PNC-Cre, with activity in two organizer domains, the posterior notochord (PNC) and notochord. These lines were made using defined regulatory elements from the Foxa2 and Nodal genes that direct Cre expression in overlapping domains of the PNC and notochord. Our detailed analysis of the timing and location of Foxa2 NFP-Cre and Nodal PNC-Cre activity indicates that these lines are appropriate for conditional mutagenesis of genes expressed from early somite stages onward.     

Progressive adult-onset emphysema in transgenic mice expressing human MMP-1 in the lung

Mice with lung-specific expression of human matrix metalloproteinase-1 (MMP-1) develop emphysematous changes similar to those seen in smoking-induced emphysema in humans. Morphometric analyses of three transgenic lines [homozygous colony (Col) 34, Col 50, and Col 64] with varying temporal expression of MMP-1 were undertaken to determine the validity of this animal as a model of adult-onset emphysema. Line 50 mice, which have early expression of MMP-1 (14 days postconception), exhibited morphometric changes by 5 days of age. In contrast, homozygous line 34 and 64 with delayed expression (birth and 2 wk of age) were normal up until 4 wk of age when progressive changes in their mean linear intercept were first noted. In contrast, heterozygous mice from line 34 with lower transgene expression did not develop emphysema until 1 yr of age. The changes in mean linear intercept coincided with an increase in lung compliance. Emphysema in these mice was associated with decreased immunostaining for type III collagen within the alveolar septa. This study provides evidence that MMP-1 induces progressive adult-onset emphysema by the selective degradation of type III collagen within the alveolar wall.