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Biochemical and morphological characterization of primary kidney cell cultures from beige mutant mice 总被引:2,自引:0,他引:2
Timothy A. Lyerla Sonja K. Gross Thomas B. Shea Peter F. Daniel Robert H. McCluer 《Cell and tissue research》1987,250(3):627-632
Summary Primary kidney cultures from adult beige-J (bg
J/ bg
J) mice were selected for epithelial cell growth using D-valine medium. After 2 weeks of attachment and proliferation in vitro, the cells form a confluent or nearly confluent monolayer that retains several phenotypic characteristics of the beige-J mutant. These include large, multilamellar inclusion bodies that are apparently dysmorphic lysosomes, and higher concentrations of neutral glycosphingolipids and dolichols than control cells. -Glucuronidase activity, used as a lysosomal enzyme marker, is not elevated in beige-J-cultured kidney cells compared with controls, as it is in the intact kidney. The high levels of -glucuronidase activity in both control and mutant cells may mask expression of this difference in vitro. The action of the beige-J mutation in kidney cells is thought to be due to a block in exocytosis that results in the accumulation of abnormal lysosomes and their components. The maintenance of the beige phenotype in vitro indicates that the mutation is not suppressed in primary kidney cell cultures. The expression of the beige phenotype in vitro should be useful for studies concerning the primary lesion of this mutation. 相似文献
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Conditional activation and inactivation of genes using the Cre/loxP recombination system is a powerful tool for the analysis of gene function and for tracking cell fate. Here we report a novel silent EGFP reporter mouse line generated by enhancer trap technology using embryonic stem (ES) cells. Following transfection with the silent EGFP reporter construct, positive ES cell clones were treated with Cre recombinase. These "activated clones" were then further selected on the basis of ubiquitous EGFP expression during in vitro differentiation. The parental "silent" clones were then used for generating mice. Upon Cre-mediated activation in ovo tissues tested from these mice express EGFP. Long-term, strong and sustainable expression of EGFP is observed in most myeloid and lymphoid cells. As shown by in vivo transplantation assays, the majority of hematopoietic stem cells (HSCs) and spleen colony-forming units (CFU-S) reside within the EGFP positive fraction. Most in vitro colony-forming units (CFU-Cs) isolated from bone marrow also express EGFP. Thus, these reporter mice are useful for the analysis of Cre-mediated recombination in HSCs and hematopoietic progenitor cells. This, in combination with the high accessibility of the loxP sites, makes these mice a valuable tool for testing cell/tissue-specific Cre-expressing mice. . 相似文献
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Qiyu Tian Junxing Zhao Qiyuan Yang Bo Wang Jeanene M. Deavila Mei-Jun Zhu Min Du 《Aging cell》2020,19(1)
Aging usually involves the progressive development of certain illnesses, including diabetes and obesity. Due to incapacity to form new white adipocytes, adipose expansion in aged mice primarily depends on adipocyte hypertrophy, which induces metabolic dysfunction. On the other hand, brown adipose tissue burns fatty acids, preventing ectopic lipid accumulation and metabolic diseases. However, the capacity of brown/beige adipogenesis declines inevitably during the aging process. Previously, we reported that DNA demethylation in the Prdm16 promoter is required for beige adipogenesis. DNA methylation is mediated by ten–eleven family proteins (TET) using alpha‐ketoglutarate (AKG) as a cofactor. Here, we demonstrated that the circulatory AKG concentration was reduced in middle‐aged mice (10‐month‐old) compared with young mice (2‐month‐old). Through AKG administration replenishing the AKG pool, aged mice were associated with the lower body weight gain and fat mass, and improved glucose tolerance after challenged with high‐fat diet (HFD). These metabolic changes are accompanied by increased expression of brown adipose genes and proteins in inguinal adipose tissue. Cold‐induced brown/beige adipogenesis was impeded in HFD mice, whereas AKG rescued the impairment of beige adipocyte functionality in middle‐aged mice. Besides, AKG administration up‐regulated Prdm16 expression, which was correlated with an increase of DNA demethylation in the Prdm16 promoter. In summary, AKG supplementation promotes beige adipogenesis and alleviates HFD‐induced obesity in middle‐aged mice, which is associated with enhanced DNA demethylation of the Prdm16 gene. 相似文献
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Young R Le Guillou S Tilly G Passet B Vilotte M Castille J Beringue V Le Provost F Laude H Vilotte JL 《Biochemical and biophysical research communications》2011,(4):184-756
The protein Shadoo (Sho) is a paralogue of prion protein, and encoded by the gene Sprn. Like prion protein it is primarily expressed in central nervous system, and has been shown to have a similar expression pattern in certain regions of the brain. We have generated reporter mice carrying a transgene encompassing the Sprn promoter, exon 1, intron 1 and the 5′-end of exon 2 driving expression of either the LacZ or GFP reporter gene to study the expression profile of Shadoo in mice. Expression of the reporter genes was analysed in brains of these transgenic mice and was shown to mimic that of the endogenous gene expression, previously described by Watts et al. [1]. Consequently, the Sprn-LacZ mice were used to study the spatial expression of Sho in other tissues of the adult mouse. Several tissues were collected and stained for β-gal activity, including the thymus, heart, lung, liver, kidney, spleen, intestine, muscle, and gonads. From this array of tissues, the transgene was consistently expressed only in specific cell types of the testicle and ovary, suggesting a role for Shadoo in fertility and reproduction. These mice may serve as a useful tool in deciphering the regulation of the prion-like gene Sprn and thus, indirectly, of the Shadoo protein. 相似文献
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Girolamo Di Maio Nicola Alessio Alessia Ambrosino Sura H. A. Al Sammarraie Marcellino Monda Giovanni Di Bernardo 《Journal of cellular biochemistry》2024,125(5):e30565
Mammals exhibit two distinct types of adipose depots: white adipose tissue (WAT) and brown adipose tissue (BAT). While WAT primarily functions as a site for energy storage, BAT serves as a thermogenic tissue that utilizes energy and glucose consumption to regulate core body temperature. Under specific stimuli such as exercise, cold exposure, and drug treatment, white adipocytes possess a remarkable ability to undergo transdifferentiation into brown-like cells known as beige adipocytes. This transformation process, known as the “browning of WAT,” leads to the acquisition of new morphological and physiological characteristics by white adipocytes. We investigated the potential role of Irisin, a 12 kDa myokine that is secreted in mice and humans by skeletal muscle after physical activity, in inducing the browning process in mesenchymal stromal cells (MSCs). A subset of the MSCs possesses the remarkable capability to differentiate into different cell types such as adipocytes, osteocytes, and chondrocytes. Consequently, comprehending the effects of Irisin on MSC biology becomes a crucial factor in investigating antiobesity medications. In our study, the primary objective is to evaluate the impact of Irisin on various cell types engaged in distinct stages of the differentiation process, including stem cells, committed precursors, and preadipocytes. By analyzing the effects of Irisin on these specific cell populations, our aim is to gain a comprehensive understanding of its influence throughout the entire differentiation process, rather than solely concentrating on the final differentiated cells. This approach enables us to obtain insights into the broader effects of Irisin on the cellular dynamics and mechanisms involved in adipogenesis. 相似文献
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Donghua Hu Lu Zhang Fan Yang Fuxue Chen Deqiang Li Chen‐Leng Cai 《Genesis (New York, N.Y. : 2000)》2020,58(9)
Endothelial cells are specialized epithelium lining the interior surface of vessels and play fundamental roles in angiogenesis, vascular permeability, and immune response. To identify endothelial cells in vivo, we constructed a Pecam1nlacZ‐H2B‐GFP/+ knock‐in mouse model in which the endothelial cells are labeled by nuclear LacZ (nlacZ) expression. When Pecam1nlacZ‐H2B‐GFP/+ mice are bred with germline Cre deleter mice, Pecam1H2B‐GFP/+ line is created with native nuclear GFP (H2B‐GFP) expression in the endothelium of various organs. This dual reporter mouse provides us with a powerful genetic tool for definitive identification of endothelial cells and monitoring this important cell population throughout development, homeostasis, and disease conditions in mammals. 相似文献
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Andrei M. Vacaru Joan Isern Stuart T. Fraser Margaret H. Baron 《Genesis (New York, N.Y. : 2000)》2013,51(11):751-762
Primitive erythropoiesis is a vital process for mammalian embryonic development. Here we report the generation and characterization of a new transgenic mouse line that expresses a histone H2B‐CFP fusion protein in the nuclei of primitive erythroid cells. We demonstrate the potential of this ε‐globin‐histone H2B‐CFP line for multicolor imaging and flow cytometry analysis. The ε‐globin‐H2B‐CFP line was used to analyze the cell cycle distribution and proliferation of CFP‐expressing primitive erythroblasts from E8.5‐E13.5. We also evaluated phagocytosis of extruded CFP‐positive nuclei by macrophages in fetal liver and placenta. The ε‐globin‐H2B‐CFP transgenic mouse line adds to the available tools for studying the development of the primitive erythroid lineage. genesis 51:751–762. © 2013 Wiley Periodicals, Inc. 相似文献
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Sonja Blumenstock Elena Katharina SchulzTrieglaff Kerstin Voelkl AnnaLena Bolender Paul Lapios Jana Lindner Mark S Hipp F Ulrich Hartl Rüdiger Klein Irina Dudanova 《The EMBO journal》2021,40(19)
The cellular protein quality control machinery is important for preventing protein misfolding and aggregation. Declining protein homeostasis (proteostasis) is believed to play a crucial role in age‐related neurodegenerative disorders. However, how neuronal proteostasis capacity changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP‐fused firefly luciferase (Fluc‐EGFP), a conformationally unstable protein that requires chaperones for proper folding, and that reacts to proteotoxic stress by formation of intracellular Fluc‐EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked reaction of the Fluc‐EGFP sensor in a mouse model of tauopathy, but not in mouse models of Huntington’s disease. Mechanistic investigations in primary neuronal cultures demonstrate that different types of protein aggregates have distinct effects on the cellular protein quality control. Thus, Fluc‐EGFP reporter mice enable new insights into proteostasis alterations in different diseases. 相似文献
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Activation of the antioxidant response element in primary cortical neuronal cultures derived from transgenic reporter mice 总被引:7,自引:0,他引:7
Many phase II protective genes contain a cis -acting enhancer region known as the antioxidant response element (ARE). Increased expression of these genes contributes to the protection of cells from oxidative stress. Transgenic reporter mice were created that carry in their genome the core ARE coupled to the human placental alkaline phosphatase (hPAP) reporter gene. Primary cortical cultures derived from these mice were treated with tBHQ resulting in a dose-dependent increase in hPAP activity. Histochemical staining for hPAP activity was observed in both glia and neurons from tBHQ-treated cultures. The tBHQ-mediated increase in hPAP was not affected by the antioxidant glutathione monoethyl ester (GSHEE), whereas the increase in hPAP following DEM treatment was completely blocked by GSHEE. Pre-treatment of cultures with the PI3-kinase inhibitor LY 294002 demonstrated a dose-dependent decrease in tBHQ-induced hPAP activity. In addition, the tBHQ-mediated expression of ARE-driven genes in primary cortical cultures was blocked by LY 294002. Interestingly, basal expression of Nrf2 was also inhibited by LY 294002. We theorize that increased levels of genes controlled by the ARE are important for cellular protection against oxidative stress. These ARE-hPAP transgenic mice will be an important in vivo model for testing our hypothesis. 相似文献
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Maelandsmo GM Ross PJ Pavliv M Meulenbroek RA Evelegh C Muruve DA Graham FL Parks RJ 《The journal of gene medicine》2005,7(3):307-315
BACKGROUND: The development of any vector system as a gene delivery system requires its optimization in vitro and in vivo. Preliminary studies frequently involve the use of a reporter gene, which allows for the rapid and simple assay of vector function through monitoring expression levels of the reporter gene. However, evaluation of vector efficacy can be compromised by immune responses directed against immunogenic reporter proteins. METHODS: We have cloned a murine secreted alkaline phosphatase (mSEAP), and explored its use as a reporter gene in the context of an early region 1 (E1)-deleted adenovirus (Ad) vector. Studies involved characterization of gene expression in vitro and in vivo, and immunological responses after gene delivery to mice. RESULTS: In tissue culture, we show that mSEAP is easily measured quantitatively using a sensitive, commercially available chemiluminescent assay, or visualized directly using histological staining. The level of transgene expression from AdmSEAP was similar to that observed for an Ad vector encoding the human placental secreted alkaline phosphatase (hSEAP). After intravenous administration in mice, AdmSEAP continued to express at high levels for the duration of the experiment (1 month), whereas expression from AdhSEAP declined to background levels over the course of the experiment. Although cytotoxic T-lymphocytes were not detected against either the murine or human SEAP proteins in mice, antibodies were readily detected against the human protein. No antibodies were detected to mSEAP. CONCLUSIONS: Taken together, these data illustrate that mSEAP is a sensitive, non-immunogenic reporter gene for preclinical mouse studies. 相似文献
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Lin J Arnold HB Della-Fera MA Azain MJ Hartzell DL Baile CA 《Biochemical and biophysical research communications》2002,291(3):701-706
Growth differentiation factor-8 (GDF-8), or Myostatin, plays an important inhibitory role during muscle development. Since muscle and adipose tissue develop from the same mesenchymal stem cells, we hypothesized that Myostatin gene knockout may cause a switch between myogenesis and adipogenesis. Male and female wild type (WT) and Myostatin knockout (KO) mice were sacrificed at 4, 8, and 12 weeks of age. The gluteus muscle (GM) was larger in KO mice compared to WT mice at 8 (P < 0.01) and 12 (P < 0.001) weeks. At 12 weeks, KO mice had decreased fat depots (P < 0.01). Compared to 12-week-old WT mice, serum leptin concentration in KO mice was lower (P < 0.001) and leptin mRNA expression was decreased (P < 0.01) in inguinal adipose tissue. CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma) levels in adipose tissue were significantly lower in KO mice compared to WT mice. Thus, increased muscle development in Myostatin knockout mice is associated with reduced adipogenesis and consequently, decreased leptin secretion. 相似文献
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Ammar Shaker Hamed Hasan Tra Thi Huong Dinh Hoai Thu Le Saori Mizuno-Iijima Yoko Daitoku Miyuki Ishida Yoko Tanimoto Kanako Kato Atsushi Yoshiki Kazuya Murata Seiya Mizuno Fumihiro Sugiyama 《Experimental Animals》2021,70(1):22
Two members of the CDK5 and ABL enzyme substrate (CABLES) family, CABLES1 and CABLES2, share a highly homologous C-terminus. They interact and associate with cyclin-dependent kinase 3 (CDK3), CDK5, and c-ABL. CABLES1 mediates tumor suppression, regulates cell proliferation, and prevents protein degradation. Although Cables2 is ubiquitously expressed in adult mouse tissues at RNA level, the role of CABLES2 in vivo remains unknown. Here, we generated bicistronic Cables2 knock-in reporter mice that expressed CABLES2 tagged with 3×FLAG and 2A-mediated fluorescent reporter tdTomato. Cables2-3×FLAG-2A-tdTomato (Cables2Tom) mice confirmed the expression of Cables2 in various mouse tissues. Interestingly, high intensity of tdTomato fluorescence was observed in the brain, testis and ovary, especially in the corpus luteum. Furthermore, immunoprecipitation analysis using the brain and testis in Cables2Tom/Tom revealed interaction of CABLES2 with CDK5. Collectively, our new Cables2 knock-in reporter model will enable the comprehensive analysis of in vivo CABLES2 function. 相似文献
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Atko Viru Livian Laaneots Kalle Karelson Tamara Smirnova Mehis Viru 《European journal of applied physiology and occupational physiology》1998,77(5):401-408
The dependence of exercise-induced hormone responses on sexual maturation was tested in a 3-year longitudinal experiment
on 34 girls (aged 11–12 years at the beginning). Sexual maturation was evaluated by Tanners five-stage scale. Children cycled
for 20-min at 60% maximal oxygen uptake once a year. Cortisol, insulin, growth hormone, β-oestradiol, progesterone and testosterone
concentrations in venous blood were determined by radioimmunoassay procedures. Basal concentrations of growth hormone increased
and of cortisol decreased when breast stage III was reached. Reaching breast stage IV was associated with an increase in basal
concentrations of β-oestradiol, progesterone and testosterone. The exercise induced significant increases in concentrations
of cortisol, growth hormone and β-oestradiol and a decrease in insulin concentration. At breast stage III the increase in
cortisol concentration was to a lower level [467 (SEM 42) vs 567 (SEM 46)nmol · l−1] and growth hormone concentration to a higher level [29.4 (SEM 0.5) vs 12.8 (SEM 0.4)ng · ml−1], while the fall in insulin concentration was less pronounced [postexercise level 10.6 (SEM 0.9) vs 7.8 (SEM 0.8)mU · l−1] than in stage II. The magnitude of the cortisol response was reduced in the last stage of breast development (+42.1% vs
+55.5% at stage II, +66.2% at stage III, and +50.0% at stage IV). The magnitude of β-oestradiol response was the lowest in
breast stage IV (+15.8%) and the highest at stage V (+41.1%). The progesterone response became significant at stage IV and
testosterone response at stage V. In conclusion, we found that reaching breast stage III was associated with altered responses
of cortisol, insulin and growth hormone concentrations while the responses of the sex hormone concentrations became pronounced
in the last stages of sexual maturation.
Accepted: 17 September 1997 相似文献
16.
Green fluorescent protein as a marker in transgenic mice 总被引:8,自引:0,他引:8
Masahito Ikawa Katsuya Kominami Yasuhide Yoshimura Keiichi Tanaka Yoshitake Nishimune Masaru Okabe 《Development, growth & differentiation》1995,37(4):455-459
Green fluorescent protein (GFP) found in Aequorea victoria absorbs blue light and emits green fluorescence without exogenous substrates or co-factors. We studied the possibility of using the GFP as a marker in mammals. Transgenic mice were produced using the GFP coding sequence, ligated with the chicken beta-actin promoter. Green fluorescence was observed in muscle, pancreas, kidney, heart and other organs in all the three transgenic mouse lines. Detection of the transgenic mouse was possible by observing a tail or fingers of new born pups under a fluorescent microscope. The marker also enabled us to detect localized expression of the transgene in intact tissues without preliminary steps. It was also demonstrated that the GFP expression could be quantified by measuring the fluorescence in tissue extracts. 相似文献
17.
双荧光素酶报告基因系统能够提供灵敏的读数,但该系统需要依赖组成型表达的内参对读数进行归一化。然而,大多数内参并不是在所有条件下都组成型表达。为此,文中建立了一个有效的方法制备适于家蚕细胞双荧光素酶报告基因系统的内参质粒。首先,突变BmV gP78启动子上的激素应答相关元件,获得了在家蚕细胞中稳定表达的组成型启动子BmV gP78M;然后,用BmV gP78M替换pRL-SV40质粒上的SV40启动子和嵌合内含子序列,成功构建了pRL-V gP78M内参质粒;最后,通过细胞转染实验证实pRL-V gP78M内参在家蚕细胞系中稳定表达,并且pRL-V gP78M内参的表达活性不受蜕皮激素、保幼激素及激素相关转录因子的影响。最终,获得了在家蚕细胞中稳定表达且表达量适中的内参质粒pRL-V gP78M。该内参可以有效地作为双荧光素酶报告基因系统的内参质粒用于家蚕细胞系中激素的研究。同时,该内参质粒的构建方法也为构建适于其他物种细胞系的双荧光素酶报告基因系统的内参质粒提供了参考。 相似文献
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Lyst(beige) mice crossed with hyperlipidemic low density lipoprotein receptor-deficient mice (BgLDLr(-/-)) display increased lesion area and a more stable lesion morphology. To verify that the beige phenotype is not unique to LDLr(-/-) mice, we examined atherosclerosis in beige, apolipoprotein E-deficient mutant mice (BgApoE(-/-)). Severe diet-induced hyperlipidemia in BgApoE(-/-) mice resulted in increased aortic sinus lesion areas compared with controls. Minimal aortic lesions were observed in both genotypes on a chow diet. Nevertheless, BgApoE(-/-) mice displayed drastically reduced aortic sinus lesion growth. Reconstitution with bone marrow (BM) from green fluorescent protein mice created chimeric animals that allowed for the identification of donor-derived cells within lesions. Expressing the beige mutation exclusively in BM-derived cells had no impact on plaque development, yet the beige mutation in all cells except the BM-derived cells led to significantly larger aortic sinus lesion areas. Both mRNA and secreted protein levels of monocyte chemoattractant protein 1 were altered in quiescent and phorbol ester-stimulated cultured macrophages, vascular smooth muscle cells, and aortic endothelial cells isolated from BgApoE(-/-) mice. Thus, expression of the beige mutation in all cell types involved in lesion development contributed to atheroprotection in chow-fed ApoE(-/-) mice. 相似文献
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Anna Alvarez-Guaita Satish Patel Koini Lim Afreen Haider Liang Dong Olivia J. Conway Marcella K.L. Ma Davide Chiarugi Vladimir Saudek Stephen O’Rahilly David B. Savage 《Cell reports》2021,34(10):108810
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20.
Objective: Epidermal growth factor (EGF) stimulates proliferation in 3T3‐L1 preadipocytes, but EGF action in differentiation is less clear. EGF promotes differentiation at concentrations <1 nM but inhibits differentiation at higher concentrations, suggesting a dual role in adipogenesis. We hypothesized that differences in EGF receptor activation and downstream signaling mediate distinct biological effects of EGF at low vs. high abundance. Research Methods and Procedures: We compared the effects of low (0.1 nM) vs. high (10 nM) EGF on the activation of EGF receptors, proximal signaling molecules Src and Shc, and the downstream mitogen‐activated protein kinase (MAPK) pathways extracellular regulated kinase (ERK) and p38 in proliferating and differentiated 3T3‐L1 cells. Results: Both low and high EGF activated ERK and p38 in preadipocytes. Src inhibitors PP1 and PP2 blocked ERK and p38 activation by low but not high EGF, and only high EGF increased Shc phosphorylation. Selective inhibition of the EGF receptor (EGFR) with AG1478 blocked ERK and p38 activation at both concentrations; however, selective inhibition of the ErbB2 receptor (EB2R) with AG825 or small interfering RNA (siRNA) blocked low but not high EGF activation of ERK and p38. Coimmunoprecipitation of EGFR with EB2R and Src was observed with low EGF in preadipocytes but at both concentrations in adipocytes. EB2R inhibition during differentiation decreased p38 activity and peroxisome proliferator‐activated receptor γ (PPARγ) abundance. Discussion: Our results show that EGFR homodimers mediate action of EGF at high abundance, but at low abundance, EGF promotes differentiation through EGFR/EB2R heterodimer activation of Src and p38. These results may partially explain the observations that high EGF concentrations inhibit, whereas low concentrations support, preadipocyte differentiation. 相似文献