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Gimenez GA, Romijn M, van den Herik J, Meijer W, Eggers R, Hobo B, De Zeeuw CI, Canto CB, Verhaagen J, Carulli D. A Study on Potential Sources of Perineuronal Net-Associated Sema3A in Cerebellar Nuclei Reveals Toxicity of Non-Invasive AAV-Mediated Cre Expression in the Central Nervous System. Int J Mol Sci 2025; 26:819. [PMID: 39859534 PMCID: PMC11765860 DOI: 10.3390/ijms26020819] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2024] [Revised: 01/13/2025] [Accepted: 01/16/2025] [Indexed: 01/27/2025] Open
Abstract
Semaphorin 3A (Sema3A) is an axon guidance molecule, which is also abundant in the adult central nervous system (CNS), particularly in perineuronal nets (PNNs). PNNs are extracellular matrix structures that restrict plasticity. The cellular sources of Sema3A in PNNs are unknown. Most Sema3A-bearing neurons do not express Sema3A mRNA, suggesting that Sema3A may be released from other neurons. Another potential source of Sema3A is the choroid plexus. To identify sources of PNN-associated Sema3A, we focused on the cerebellar nuclei, which contain Sema3A+ PNNs. Cerebellar nuclei neurons receive prominent input from Purkinje cells (PCs), which express high levels of Sema3A mRNA. By using a non-invasive viral vector approach, we overexpressed Cre in PCs, the choroid plexus, or throughout the CNS of Sema3Afl/fl mice. Knocking out Sema3A in PCs or the choroid plexus was not sufficient to decrease the amount of PNN-associated Sema3A. Alternatively, knocking out Sema3A throughout the CNS induced a decrease in PNN-associated Sema3A. However, motor deficits, microgliosis, and neurodegeneration were observed, which were due to Cre toxicity. Our study represents the first attempt to unravel cellular sources of PNN-associated Sema3A and shows that non-invasive viral-mediated Cre expression throughout the CNS could lead to toxicity, complicating the interpretation of Cre-mediated Sema3A knock-out.
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Affiliation(s)
- Geoffrey-Alexander Gimenez
- Department of Neuroregeneration, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands; (G.-A.G.); (M.R.); (J.v.d.H.); (W.M.); (R.E.); (B.H.); (J.V.)
- Department of Cerebellar Coordination & Cognition, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands; (C.I.D.Z.); (C.B.C.)
| | - Maurits Romijn
- Department of Neuroregeneration, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands; (G.-A.G.); (M.R.); (J.v.d.H.); (W.M.); (R.E.); (B.H.); (J.V.)
| | - Joëlle van den Herik
- Department of Neuroregeneration, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands; (G.-A.G.); (M.R.); (J.v.d.H.); (W.M.); (R.E.); (B.H.); (J.V.)
| | - Wouter Meijer
- Department of Neuroregeneration, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands; (G.-A.G.); (M.R.); (J.v.d.H.); (W.M.); (R.E.); (B.H.); (J.V.)
| | - Ruben Eggers
- Department of Neuroregeneration, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands; (G.-A.G.); (M.R.); (J.v.d.H.); (W.M.); (R.E.); (B.H.); (J.V.)
| | - Barbara Hobo
- Department of Neuroregeneration, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands; (G.-A.G.); (M.R.); (J.v.d.H.); (W.M.); (R.E.); (B.H.); (J.V.)
| | - Chris I. De Zeeuw
- Department of Cerebellar Coordination & Cognition, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands; (C.I.D.Z.); (C.B.C.)
- Department of Neuroscience, Erasmus MC, 3015 GD Rotterdam, The Netherlands
| | - Cathrin B. Canto
- Department of Cerebellar Coordination & Cognition, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands; (C.I.D.Z.); (C.B.C.)
| | - Joost Verhaagen
- Department of Neuroregeneration, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands; (G.-A.G.); (M.R.); (J.v.d.H.); (W.M.); (R.E.); (B.H.); (J.V.)
- Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, Vrije Universiteit Amsterdam, 1081 HZ Amsterdam, The Netherlands
| | - Daniela Carulli
- Department of Neuroregeneration, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands; (G.-A.G.); (M.R.); (J.v.d.H.); (W.M.); (R.E.); (B.H.); (J.V.)
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Jiang Z, Wang Q, Zhao J, Wang J, Li Y, Dai W, Zhang X, Fang Z, Hou W, Xiong L. Sex-specific cannabinoid 1 receptors on GABAergic neurons in the ventrolateral periaqueductal gray mediate analgesia in mice. J Comp Neurol 2022; 530:2315-2334. [PMID: 35716006 DOI: 10.1002/cne.25334] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2021] [Revised: 04/04/2022] [Accepted: 04/13/2022] [Indexed: 11/07/2022]
Abstract
Sex differences in analgesic effects have gradually attracted public attention in preclinical and clinical studies. Both human and animal females are more sensitive to cannabinoid antinociception than males. Expression of the cannabinoid 1 receptor (CB1 R) and the function of the endocannabinoid system have been explored in both male and female mice and CB1 Rs in the ventrolateral periaqueductal gray (vlPAG) participate in antinociception. However, whether there are cell-type- and sex-specific patterns of vlPAG CB1 R expression that affect analgesia is unknown. In the current study, we either activated or inhibited CB1 Rs in the vlPAG and found that female mice produced stronger analgesia or developed more robust mechanical allodynia than males did. Specific deletion of GABAergic CB1 Rs in the vlPAG promoted stronger mechanical allodynia in female mice than that in male mice. However, no sex differences in cannabinoid antinociception were found following chemogenetic inhibition of GABAergic neurons. Using fluorescence in situ hybridization, we found that the sex difference in cannabinoid antinociception was due to females having higher expression of GABAergic CB1 Rs in the vlPAG than males. Furthermore, activation of CB1 Rs in the vlPAG significantly reduced the frequency of GABA-mediated spontaneous inhibitory postsynaptic currents recorded in vGlut2-tdTomato positive neurons in both sexes. This effect was greater in females than males and this reduction was closely related to CB1 R expression difference between sexes. Our work indicates that vlPAG GABAergic CB1 Rs modulate cannabinoid-mediated analgesia in a sex-specific manner, which may provide a potential explanation of sex difference found in the analgesic effect of cannabinoids.
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Affiliation(s)
- Zhenhua Jiang
- Department of Anesthesiology and Perioperative Medicine, Xijing Hospital, Fourth Military Medical University, Xian, Shaanxi Province, China
| | - Qun Wang
- Department of Anesthesiology and Perioperative Medicine, Xijing Hospital, Fourth Military Medical University, Xian, Shaanxi Province, China
| | - Jianshuai Zhao
- Department of Anesthesiology and Perioperative Medicine, Xijing Hospital, Fourth Military Medical University, Xian, Shaanxi Province, China
| | - Jiajia Wang
- Department of Anesthesiology and Perioperative Medicine, Xijing Hospital, Fourth Military Medical University, Xian, Shaanxi Province, China
| | - You Li
- Department of Anesthesiology and Perioperative Medicine, Xijing Hospital, Fourth Military Medical University, Xian, Shaanxi Province, China
| | - Wei Dai
- Hangzhou Sanatorium Health Management Center, Hangzhou, People's Republic of China
| | - Xiao Zhang
- Department of Anesthesiology and Perioperative Medicine, Xijing Hospital, Fourth Military Medical University, Xian, Shaanxi Province, China
| | - Zongping Fang
- Department of Anesthesiology and Perioperative Medicine, Xijing Hospital, Fourth Military Medical University, Xian, Shaanxi Province, China
| | - Wugang Hou
- Department of Anesthesiology and Perioperative Medicine, Xijing Hospital, Fourth Military Medical University, Xian, Shaanxi Province, China
| | - Lize Xiong
- Department of Anesthesiology and Perioperative Medicine, Xijing Hospital, Fourth Military Medical University, Xian, Shaanxi Province, China
- Department of Anesthesiology and Perioperative Medicine, Translational Research Institute of Brain and Brain-Like Intelligence, Shanghai Fourth People's Hospital Affiliated to Tongji University School of Medicine, Shanghai, China
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Cho K, Ro SW, Seo SH, Jeon Y, Moon H, Kim DY, Kim SU. Genetically Engineered Mouse Models for Liver Cancer. Cancers (Basel) 2019; 12:14. [PMID: 31861541 PMCID: PMC7016809 DOI: 10.3390/cancers12010014] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2019] [Revised: 12/16/2019] [Accepted: 12/16/2019] [Indexed: 02/07/2023] Open
Abstract
Liver cancer is the fourth leading cause of cancer-related death globally, accounting for approximately 800,000 deaths annually. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, comprising approximately 80% of cases. Murine models of HCC, such as chemically-induced models, xenograft models, and genetically engineered mouse (GEM) models, are valuable tools to reproduce human HCC biopathology and biochemistry. These models can be used to identify potential biomarkers, evaluate potential novel therapeutic drugs in pre-clinical trials, and develop molecular target therapies. Considering molecular target therapies, a novel approach has been developed to create genetically engineered murine models for HCC, employing hydrodynamics-based transfection (HT). The HT method, coupled with the Sleeping Beauty transposon system or the CRISPR/Cas9 genome editing tool, has been used to rapidly and cost-effectively produce a variety of HCC models containing diverse oncogenes or inactivated tumor suppressor genes. The versatility of these models is expected to broaden our knowledge of the genetic mechanisms underlying human hepatocarcinogenesis, allowing the study of premalignant and malignant liver lesions and the evaluation of new therapeutic strategies. Here, we review recent advances in GEM models of HCC with an emphasis on new technologies.
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Affiliation(s)
- Kyungjoo Cho
- Yonsei Liver Center, Yonsei University College of Medicine, Seoul 03722, Korea; (K.C.); (S.W.R.); (S.H.S.); (H.M.)
- Brain Korea 21 PLUS Project for Medical Science College of Medicine, Yonsei University, Seoul 03722, Korea
| | - Simon Weonsang Ro
- Yonsei Liver Center, Yonsei University College of Medicine, Seoul 03722, Korea; (K.C.); (S.W.R.); (S.H.S.); (H.M.)
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul 03722, Korea
| | - Sang Hyun Seo
- Yonsei Liver Center, Yonsei University College of Medicine, Seoul 03722, Korea; (K.C.); (S.W.R.); (S.H.S.); (H.M.)
| | - Youjin Jeon
- Department of Life Science, Sahmyook University, Seoul 03722, Korea;
| | - Hyuk Moon
- Yonsei Liver Center, Yonsei University College of Medicine, Seoul 03722, Korea; (K.C.); (S.W.R.); (S.H.S.); (H.M.)
- Brain Korea 21 PLUS Project for Medical Science College of Medicine, Yonsei University, Seoul 03722, Korea
| | - Do Young Kim
- Yonsei Liver Center, Yonsei University College of Medicine, Seoul 03722, Korea; (K.C.); (S.W.R.); (S.H.S.); (H.M.)
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul 03722, Korea
- Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Korea
| | - Seung Up Kim
- Yonsei Liver Center, Yonsei University College of Medicine, Seoul 03722, Korea; (K.C.); (S.W.R.); (S.H.S.); (H.M.)
- Institute of Gastroenterology, Yonsei University College of Medicine, Seoul 03722, Korea
- Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Korea
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He L, Tian DA, Li PY, He XX. Mouse models of liver cancer: Progress and recommendations. Oncotarget 2016; 6:23306-22. [PMID: 26259234 PMCID: PMC4695120 DOI: 10.18632/oncotarget.4202] [Citation(s) in RCA: 88] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2015] [Accepted: 05/23/2015] [Indexed: 02/06/2023] Open
Abstract
To clarify the pathogenesis of hepatocellular carcinoma (HCC) and investigate the effects of potential therapies, a number of mouse models have been developed. Subcutaneous xenograft models are widely used in the past decades. Yet, with the advent of in vivo imaging technology, investigators are more and more concerned with the orthotopic models nowadays. Genetically engineered mouse models (GEM) have greatly facilitated studies of gene function in HCC development. Recently, GEM of miR-122 and miR-221 provided new approaches for better understanding of the in vivo functions of microRNA in hepatocarcinogenesis. Chemically induced liver tumors in animals share many of the morphological, histogenic, and biochemical features of human HCC. Yet, the complicated and obscure genomic alternation restricts their applications. In this review, we highlight both the frequently used mouse models and some emerging ones with emphasis on their merits or defects, and give advises for investigators to chose a “best-fit” animal model in HCC research.
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Affiliation(s)
- Li He
- Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - De-An Tian
- Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Pei-Yuan Li
- Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Xing-Xing He
- Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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Mangiavini L, Merceron C, Schipani E. Analysis of Mouse Growth Plate Development. CURRENT PROTOCOLS IN MOUSE BIOLOGY 2016; 6:67-130. [PMID: 26928664 PMCID: PMC5493209 DOI: 10.1002/9780470942390.mo150094] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
To investigate skeletal development, pathophysiological mechanisms of cartilage and bone disease, and eventually assess innovative treatments, the mouse is a very important resource. During embryonic development, mesenchymal condensations are formed, and cells within these mesenchymal condensations either directly differentiate into osteoblasts and give origin to intramembranous bone, or differentiate into chondrocytes and form a cartilaginous anlage. The cartilaginous anlage or fetal growth plate is then replaced with bone. This process is also called endochondral bone development, and it is responsible for the generation of most of our skeleton. Here we discuss in detail the most common in vivo and in vitro techniques our laboratory is currently using for the analysis of the mouse fetal growth plate during development.
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Affiliation(s)
- Laura Mangiavini
- Department of Orthopaedic Surgery, Department of Medicine, Division of Endocrinology and Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan
| | - Christophe Merceron
- Department of Orthopaedic Surgery, Department of Medicine, Division of Endocrinology and Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan
- Inserm, UMRS 791-LIOAD, Centre for Osteoarticular and Dental Tissue Engineering, Group STEP ‘Skeletal Tissue Engineering and Physiopathology’, Nantes, France
- Faculty of Dental Surgery, LUNAM, Nantes University, Nantes, France
| | - Ernestina Schipani
- Department of Orthopaedic Surgery, Department of Medicine, Division of Endocrinology and Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan
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Targeted axonal import (TAxI) peptide delivers functional proteins into spinal cord motor neurons after peripheral administration. Proc Natl Acad Sci U S A 2016; 113:2514-9. [PMID: 26888285 DOI: 10.1073/pnas.1515526113] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
A significant unmet need in treating neurodegenerative disease is effective methods for delivery of biologic drugs, such as peptides, proteins, or nucleic acids into the central nervous system (CNS). To date, there are no operative technologies for the delivery of macromolecular drugs to the CNS via peripheral administration routes. Using an in vivo phage-display screen, we identify a peptide, targeted axonal import (TAxI), that enriched recombinant bacteriophage accumulation and delivered protein cargo into spinal cord motor neurons after intramuscular injection. In animals with transected peripheral nerve roots, TAxI delivery into motor neurons after peripheral administration was inhibited, suggesting a retrograde axonal transport mechanism for delivery into the CNS. Notably, TAxI-Cre recombinase fusion proteins induced selective recombination and tdTomato-reporter expression in motor neurons after intramuscular injections. Furthermore, TAxI peptide was shown to label motor neurons in the human tissue. The demonstration of a nonviral-mediated delivery of functional proteins into the spinal cord establishes the clinical potential of this technology for minimally invasive administration of CNS-targeted therapeutics.
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Zhang M, Kirsch DG. The generation and characterization of novel Col1a1FRT-Cre-ER-T2-FRT and Col1a1FRT-STOP-FRT-Cre-ER-T2 mice for sequential mutagenesis. Dis Model Mech 2015; 8:1155-66. [PMID: 26183214 PMCID: PMC4582108 DOI: 10.1242/dmm.021204] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2015] [Accepted: 07/07/2015] [Indexed: 01/10/2023] Open
Abstract
Novel genetically engineered mouse models using the Cre-loxP or the Flp-FRT systems have generated useful reagents to manipulate the mouse genome in a temporally-regulated and tissue-specific manner. By incorporating a constitutive Cre driver line into a mouse model in which FRT-regulated genes in other cell types are regulated by Flp-FRT recombinase, gene expression can be manipulated simultaneously in separate tissue compartments. This application of dual recombinase technology can be used to dissect the role of stromal cells in tumor development and cancer therapy. Generating mice in which Cre-ERT2 is expressed under Flp-FRT-mediated regulation would enable step-wise manipulation of the mouse genome using dual recombinase technology. Such next-generation mouse models would enable sequential mutagenesis to better model cancer and define genes required for tumor maintenance. Here, we generated novel genetically engineered mice that activate or delete Cre-ERT2 in response to Flp recombinase. To potentially utilize the large number of Cre-loxP-regulated transgenic alleles that have already been targeted into the Rosa26 locus, such as different reporters and mutant genes, we targeted the two novel Cre-ERT2 alleles into the endogenous Col1a1 locus for ubiquitous expression. In the Col1a1FRT-Cre-ER-T2-FRT mice, Flp deletes Cre-ERT2, so that Cre-ERT2 is only expressed in cells that have never expressed Flp. In contrast, in the Col1a1FRT-STOP-FRT-Cre-ER-T2 mice, Flp removes the STOP cassette to allow Cre-ERT2 expression so that Cre-ERT2 is only expressed in cells that previously expressed Flp. These two new novel mouse strains will be complementary to each other and will enable the exploration of complex biological questions in development, normal tissue homeostasis and cancer. Summary: We generated two mouse strains expressing Cre-ERT2 under Flp-FRT regulation. These tools enable sequential mutagenesis in the same or different cells to study development, tissue homeostasis and diseases such as cancer.
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Affiliation(s)
- Minsi Zhang
- Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27708, USA
| | - David G Kirsch
- Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27708, USA Department of Radiation Oncology, Duke University Medical Center, Box 91006, LSRC Bldg, Rm B230, Durham, NC 27708, USA
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Li L, Hu J, He T, Zhang Q, Yang X, Lan X, Zhang D, Mei H, Chen B, Huang Y. P38/MAPK contributes to endothelial barrier dysfunction via MAP4 phosphorylation-dependent microtubule disassembly in inflammation-induced acute lung injury. Sci Rep 2015; 5:8895. [PMID: 25746230 PMCID: PMC4352893 DOI: 10.1038/srep08895] [Citation(s) in RCA: 62] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2014] [Accepted: 02/10/2015] [Indexed: 02/07/2023] Open
Abstract
Excessive activation of inflammation and the accompanying lung vascular endothelial barrier disruption are primary pathogenic features of acute lung injury (ALI). Microtubule-associated protein 4 (MAP4), a tubulin assembly-promoting protein, is important for maintaining the microtubule (MT) cytoskeleton and cell-cell junctional structures. However, both the involvement and exact mechanism of MAP4 in the development of endothelial barrier disruption in ALI remains unknown. In this study, lipopolysaccharide (LPS) and tumour necrosis factor-α (TNF-α) were applied to human pulmonary microvascular endothelial cells (HPMECs) to mimic the endothelial damage during inflammation in vitro. We demonstrated that the MAP4 (Ser696 and Ser787) phosphorylation increased concomitantly with the p38/MAPK pathway activation by the LPS and TNF-α stimulation of HPMECs, which induced MT disassembly followed by hyperpermeability. Moreover, the application of taxol, the overexpression of a MAP4 (Ala) mutant, or the application of the p38/MAPK inhibitor SB203580 inhibited the MT disruption and the intracellular junction dysfunction. In contrast, MKK6 (Glu), which constitutively activated p38/MAPK, resulted in microtubule depolymerisation and, subsequently, hyperpermeability. Our findings reveal a novel role of MAP4 in endothelial barrier dysfunction.
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Affiliation(s)
- Lingfei Li
- Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing, China
| | - Jiongyu Hu
- Endocrinology Department, Southwest Hospital, Third Military Medical University, Chongqing, China
| | - Ting He
- Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing, China
| | - Qiong Zhang
- Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing, China
| | - Xu Yang
- Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University, Chongqing, China
| | - Xiaodong Lan
- Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing, China
| | - Dongxia Zhang
- Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing, China
| | - Hao Mei
- Department of Biostatistics in the School of Public Health, Yale University
| | - Bing Chen
- Endocrinology Department, Southwest Hospital, Third Military Medical University, Chongqing, China
| | - Yuesheng Huang
- Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing, China
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Werfel S, Jungmann A, Lehmann L, Ksienzyk J, Bekeredjian R, Kaya Z, Leuchs B, Nordheim A, Backs J, Engelhardt S, Katus HA, Müller OJ. Rapid and highly efficient inducible cardiac gene knockout in adult mice using AAV-mediated expression of Cre recombinase. Cardiovasc Res 2014; 104:15-23. [PMID: 25082846 DOI: 10.1093/cvr/cvu174] [Citation(s) in RCA: 71] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
AIMS Inducible gene targeting in mice using the Cre/LoxP system has become a valuable tool to analyse the roles of specific genes in the adult heart. However, the commonly used Myh6-MerCreMer system requires time-consuming breeding schedules and is potentially associated with cardiac side effects, which may result in transient cardiac dysfunction. The aim of our study was to establish a rapid and simple system for cardiac gene inactivation in conditional knockout mice by gene transfer of a Cre recombinase gene using adeno-associated viral vectors of serotype 9 (AAV9). METHODS AND RESULTS AAV9 vectors expressing Cre under the control of a human cardiac troponin T promoter (AAV-TnT-Cre) enabled a highly efficient Cre/LoxP switching in cardiomyocytes 2 weeks after injection into 5- to 6-week-old ROSA26-LacZ reporter mice. Recombination efficiency was at least as high as observed with the Myh6-MerCreMer system. No adverse side effects were detected upon application of AAV-TnT-Cre. As proof of principle, we studied AAV-TnT-Cre in a conditional knockout model (Srf-flex1 mice) to deplete the myocardium of the transcription factor serum response factor (SRF). Four weeks after AAV-TnT-Cre injection, a strong decrease in the cardiac expression of SRF mRNA and protein was observed. Furthermore, mice developed a severe cardiac dysfunction with increased interstitial fibrosis in accordance with the central role of SRF for the expression of contractile and calcium trafficking proteins in the heart. CONCLUSIONS AAV9-mediated expression of Cre is a promising approach for rapid and efficient conditional cardiac gene knockout in adult mice.
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Affiliation(s)
- Stanislas Werfel
- Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany Institute for Pharmacology and Toxicology, Technische Universität München, Biedersteiner Str. 29, Munich 80802, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany
| | - Andreas Jungmann
- Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany
| | - Lorenz Lehmann
- Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Germany
| | - Jan Ksienzyk
- Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany
| | - Raffi Bekeredjian
- Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Germany
| | - Ziya Kaya
- Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Germany
| | - Barbara Leuchs
- Applied Tumorvirology, German Cancer Research Center, Heidelberg, Germany
| | - Alfred Nordheim
- Interfaculty Institute for Cell Biology, Department of Molecular Biology, University of Tübingen, Tübingen, Germany
| | - Johannes Backs
- Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Germany
| | - Stefan Engelhardt
- Institute for Pharmacology and Toxicology, Technische Universität München, Biedersteiner Str. 29, Munich 80802, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany
| | - Hugo A Katus
- Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Germany
| | - Oliver J Müller
- Department of Internal Medicine III, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg 69120, Germany DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Germany
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Sharma S, Zhu J. Immunologic applications of conditional gene modification technology in the mouse. ACTA ACUST UNITED AC 2014; 105:10.34.1-10.34.13. [PMID: 24700321 DOI: 10.1002/0471142735.im1034s105] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Since the success of homologous recombination in altering mouse genome and the discovery of Cre-loxP system, the combination of these two breakthroughs has created important applications for studying the immune system in the mouse. Here, we briefly summarize the general principles of this technology and its applications in studying immune cell development and responses; such implications include conditional gene knockout and inducible and/or tissue-specific gene over-expression, as well as lineage fate mapping. We then discuss the pros and cons of a few commonly used Cre-expressing mouse lines for studying lymphocyte development and functions. We also raise several general issues, such as efficiency of gene deletion, leaky activity of Cre, and Cre toxicity, all of which may have profound impacts on data interpretation. Finally, we selectively list some useful links to the Web sites as valuable mouse resources.
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Affiliation(s)
- Suveena Sharma
- Molecular and Cellular Immunoregulation Unit, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
| | - Jinfang Zhu
- Molecular and Cellular Immunoregulation Unit, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
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11
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Gierut JJ, Jacks TE, Haigis KM. Strategies to achieve conditional gene mutation in mice. Cold Spring Harb Protoc 2014; 2014:339-49. [PMID: 24692485 DOI: 10.1101/pdb.top069807] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
The laboratory mouse is an ideal model organism for studying disease because it is physiologically similar to human and also because its genome is readily manipulated. Genetic engineering allows researchers to introduce specific loss-of-function or gain-of-function mutations into genes and then to study the resulting phenotypes in an in vivo context. One drawback of using traditional transgenic and knockout mice to study human diseases is that many mutations passed through the germline can profoundly affect development, thus impeding the study of disease phenotypes in adults. New technology has made it possible to generate conditional mutations that can be introduced in a spatially and/or temporally restricted manner. Mouse strains carrying conditional mutations represent valuable experimental models for the study of human diseases and they can be used to develop strategies for prevention and treatment of these diseases. In this article, we will describe the most widely used DNA recombinase systems used to achieve conditional gene mutation in mouse models and discuss how these systems can be employed in vivo.
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Affiliation(s)
- Jessica J Gierut
- Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Department of Pathology, Harvard Medical School, Charlestown, Massachusetts 02129
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12
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Li Q, He TC. Recombinant Adenovirus in Neurobiology. NEUROMETHODS 2014:11-25. [DOI: 10.1007/978-1-62703-610-8_2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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13
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Baba Y, Nakano M, Yamada Y, Saito I, Kanegae Y. Practical Range of Effective Dose for Cre Recombinase-Expressing Recombinant Adenovirus without Cell Toxicity in Mammalian Cells. Microbiol Immunol 2013; 49:559-70. [PMID: 15965304 DOI: 10.1111/j.1348-0421.2005.tb03753.x] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
The site-specific recombinase Cre is valuable for regulation of gene expression not only in vitro but also in vivo. We previously reported that replication-deficient recombinant adenovirus (rAd) expressing Cre can mediate efficient and strict regulation in 100% of cultured cells. Recently, the constitutive-expression of Cre using retrovirus or lentivirus vector reportedly inhibited cell-growth, but the effect of transient Cre expression have not yet been examined. Here we showed that an excess amount of Cre produced from Cre-expressing rAd caused a deleterious effect in cells even when Cre was transiently expressed. We used three rAds carrying promoters with different activities: the SV40 early promoter (AxSVENCre), the SR alpha promoter (AxSRCre) and the CAG promoter (AxCANCre). Cell toxicity was clearly caused by Cre itself and was distinguishable from that caused by rAd virions when the cytopathic effects of these rAds were compared with that of a control virus lacking the Cre expression unit. Cre toxicity was strongly correlated with the expression level of Cre. Importantly, AxSRCre and AxCANCre gave a 60-fold range of effective MOIs ("effective range") sufficient for gene activation without causing cell toxicity from either the rAd particles or Cre itself, while AxSVENCre failed to give such a range because the expression level of Cre was too low. When Cre was tagged with a nuclear localization signal (NLS), not only its activity but also Cre toxicity was increased fourfold, and the effective range was unchanged. Therefore, AxSRNCre might be more useful to control cell toxicity from the rAd virions than AxSRCre. Cre-induced cell toxicity can be avoided by pre-examining the "effective range" using the purpose cell lines before starting experiments utilizing the experiment of Cre-expressing rAd.
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Affiliation(s)
- Yasuko Baba
- Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, Japan
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14
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Gaj T, Sirk SJ, Barbas CF. Expanding the scope of site-specific recombinases for genetic and metabolic engineering. Biotechnol Bioeng 2013; 111:1-15. [PMID: 23982993 DOI: 10.1002/bit.25096] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2013] [Revised: 08/12/2013] [Accepted: 08/13/2013] [Indexed: 12/20/2022]
Abstract
Site-specific recombinases are tremendously valuable tools for basic research and genetic engineering. By promoting high-fidelity DNA modifications, site-specific recombination systems have empowered researchers with unprecedented control over diverse biological functions, enabling countless insights into cellular structure and function. The rigid target specificities of many sites-specific recombinases, however, have limited their adoption in fields that require highly flexible recognition abilities. As a result, intense effort has been directed toward altering the properties of site-specific recombination systems by protein engineering. Here, we review key developments in the rational design and directed molecular evolution of site-specific recombinases, highlighting the numerous applications of these enzymes across diverse fields of study.
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Affiliation(s)
- Thomas Gaj
- The Skaggs Institute for Chemical Biology and the Departments of Chemistry and Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, 92037
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15
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Breen EC, Malloy JL, Tang K, Xia F, Fu Z, Hancock REW, Overhage J, Wagner PD, Spragg RG. Impaired pulmonary defense against Pseudomonas aeruginosa in VEGF gene inactivated mouse lung. J Cell Physiol 2013; 228:371-9. [PMID: 22718316 DOI: 10.1002/jcp.24140] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Repeated bacterial and viral infections are known to contribute to worsening lung function in several respiratory diseases, including asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). Previous studies have reported alveolar wall cell apoptosis and parenchymal damage in adult pulmonary VEGF gene ablated mice. We hypothesized that VEGF expressed by type II cells is also necessary to provide an effective host defense against bacteria in part by maintaining surfactant homeostasis. Therefore, Pseudomonas aeruginosa (PAO1) levels were evaluated in mice following lung-targeted VEGF gene inactivation, and alterations in VEGF-dependent type II cell function were evaluated by measuring surfactant homeostasis in mouse lungs and isolated type II cells. In VEGF-deficient lungs increased PAO1 levels and pro-inflammatory cytokines, TNFα and IL-6, were detected 24 h after bacterial instillation compared to control lungs. In vivo lung-targeted VEGF gene deletion (57% decrease in total pulmonary VEGF) did not alter alveolar surfactant or tissue disaturated phosphatidylcholine (DSPC) levels. However, sphingomyelin content, choline phosphate cytidylyltransferase (CCT) mRNA, and SP-D expression were decreased. In isolated type II cells an 80% reduction of VEGF protein resulted in decreases in total phospholipids (PL), DSPC, DSPC synthesis, surfactant associated proteins (SP)-B and -D, and the lipid transporters, ABCA1 and Rab3D. TPA-induced DSPC secretion and apoptosis were elevated in VEGF-deficient type II cells. These results suggest a potential protective role for type II cell-expressed VEGF against bacterial initiated infection.
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Affiliation(s)
- Ellen C Breen
- Division of Physiology, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0623, USA.
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16
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Nafissi N, Slavcev R. Construction and characterization of an in-vivo linear covalently closed DNA vector production system. Microb Cell Fact 2012; 11:154. [PMID: 23216697 PMCID: PMC3540006 DOI: 10.1186/1475-2859-11-154] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2012] [Accepted: 11/25/2012] [Indexed: 12/30/2022] Open
Abstract
BACKGROUND While safer than their viral counterparts, conventional non-viral gene delivery DNA vectors offer a limited safety profile. They often result in the delivery of unwanted prokaryotic sequences, antibiotic resistance genes, and the bacterial origins of replication to the target, which may lead to the stimulation of unwanted immunological responses due to their chimeric DNA composition. Such vectors may also impart the potential for chromosomal integration, thus potentiating oncogenesis. We sought to engineer an in vivo system for the quick and simple production of safer DNA vector alternatives that were devoid of non-transgene bacterial sequences and would lethally disrupt the host chromosome in the event of an unwanted vector integration event. RESULTS We constructed a parent eukaryotic expression vector possessing a specialized manufactured multi-target site called "Super Sequence", and engineered E. coli cells (R-cell) that conditionally produce phage-derived recombinase Tel (PY54), TelN (N15), or Cre (P1). Passage of the parent plasmid vector through R-cells under optimized conditions, resulted in rapid, efficient, and one step in vivo generation of mini lcc--linear covalently closed (Tel/TelN-cell), or mini ccc--circular covalently closed (Cre-cell), DNA constructs, separated from the backbone plasmid DNA. Site-specific integration of lcc plasmids into the host chromosome resulted in chromosomal disruption and 10(5) fold lower viability than that seen with the ccc counterpart. CONCLUSION We offer a high efficiency mini DNA vector production system that confers simple, rapid and scalable in vivo production of mini lcc DNA vectors that possess all the benefits of "minicircle" DNA vectors and virtually eliminate the potential for undesirable vector integration events.
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Affiliation(s)
- Nafiseh Nafissi
- School of Pharmacy, Faculty of Science, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada
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17
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Sabbir MG, Prieditis H, Ravinsky E, Mowat MRA. The role of Dlc1 isoform 2 in K-Ras2(G12D) induced thymic cancer. PLoS One 2012; 7:e40302. [PMID: 22792269 PMCID: PMC3390377 DOI: 10.1371/journal.pone.0040302] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2011] [Accepted: 06/07/2012] [Indexed: 01/08/2023] Open
Abstract
The Deleted in liver cancer one (Dlc1) tumor suppressor gene encodes a RhoGTPase activating protein (RhoGAP). The Dlc1 gene has multiple transcriptional isoforms and we have previously established a mouse strain containing a gene trap (gt) insertion, which specifically reduces the expression of the 6.1 kb isoform (isoform 2). This gene trapped allele when homozygous results in embryonic lethality and the heterozygous gene trapped mice do not show an increased incidence of cancers, suggesting that cooperating oncogenic changes may be required for transformation. In the present work, we have studied the in vivo cooperation between oncogenic K-Ras2 and Dlc1 genes in tumourigenesis. We have observed an increase in invasive thymic cancers, including both thymomas and lymphomas, resulting in significantly shortened life spans in mice heterozygous for the gt Dlc1 allele and an inducible LSL-K-Ras2G12D allele compared with the LSL-K-Ras2G12D only mice. The heterozygous mice showed a high degree of metastasis in the lung. We have found tumour specific selective hypermethylation of the Dlc1 isoform 2 promoter and reduction of the corresponding protein expression in thymic lymphoma (TL) and thymic epithelial carcinoma (TEC) derived from the thymic tumours. The Dlc1 deficient thymic lymphoma cell lines exhibited increased trans-endothelial cell migration. TEC cell lines also exhibited increased stress fiber formation and Rho activity. Introduction of the three Dlc1 isoforms tagged with GFP into these cells resulted in different morphological changes. These results suggest that loss of expression of only isoform 2 may be sufficient for the development of thymic tumors and metastasis.
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MESH Headings
- Animals
- Base Sequence
- Cell Shape
- Cell Surface Extensions
- Chromosome Mapping
- CpG Islands
- Cyclin-Dependent Kinase Inhibitor p21/metabolism
- DNA Methylation
- GTPase-Activating Proteins/genetics
- GTPase-Activating Proteins/metabolism
- GTPase-Activating Proteins/physiology
- Gene Expression Regulation, Neoplastic
- Lung Neoplasms/genetics
- Lung Neoplasms/metabolism
- Lung Neoplasms/secondary
- Lymphoma, T-Cell/genetics
- Lymphoma, T-Cell/metabolism
- Lymphoma, T-Cell/pathology
- Mice
- Mice, Transgenic
- Molecular Sequence Data
- Mutation, Missense
- Neoplasms, Experimental/genetics
- Neoplasms, Experimental/metabolism
- Neoplasms, Experimental/pathology
- Promoter Regions, Genetic
- Protein Isoforms/genetics
- Protein Isoforms/metabolism
- Protein Isoforms/physiology
- Proto-Oncogene Proteins p21(ras)/genetics
- Stress Fibers/metabolism
- Thymoma/genetics
- Thymoma/metabolism
- Thymoma/secondary
- Thymus Neoplasms/genetics
- Thymus Neoplasms/metabolism
- Thymus Neoplasms/pathology
- Transendothelial and Transepithelial Migration
- Tumor Cells, Cultured
- Tumor Suppressor Proteins/genetics
- Tumor Suppressor Proteins/metabolism
- Tumor Suppressor Proteins/physiology
- rho GTP-Binding Proteins/metabolism
- rhoA GTP-Binding Protein
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Affiliation(s)
- Mohammad Golam Sabbir
- Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Manitoba, Canada
- Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada
| | - Heather Prieditis
- Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Manitoba, Canada
| | - Esther Ravinsky
- Department of Pathology, Health Sciences Centre, Winnipeg, Manitoba, Canada
| | - Michael R. A. Mowat
- Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Manitoba, Canada
- Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada
- * E-mail:
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18
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Abstract
Genetically engineered mouse models have significantly contributed to our understanding of cancer biology. They have proven to be useful in validating gene functions, identifying novel cancer genes and tumor biomarkers, gaining insight into the molecular and cellular mechanisms underlying tumor initiation and multistage processes of tumorigenesis, and providing better clinical models in which to test novel therapeutic strategies. However, mice still have significant limitations in modeling human cancer, including species-specific differences and inaccurate recapitulation of de novo human tumor development. Future challenges in mouse modeling include the generation of clinically relevant mouse models that recapitulate the molecular, cellular, and genomic events of human cancers and clinical response as well as the development of technologies that allow for efficient in vivo imaging and high-throughput screening in mice.
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Affiliation(s)
- Dong-Joo Cheon
- Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA
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19
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Cazzin C, Zanderigo F, Costantini VJA, Zambello E, Ring CJA, Morrison AD, Caberlotto L, Kew JNC. Adenoviral-mediated Cre expression effectively suppresses GlyT1 binding in the thalamic area of GlyT1 conditional knock-out mice. J Neurosci Methods 2010; 193:254-63. [PMID: 20832426 DOI: 10.1016/j.jneumeth.2010.09.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2010] [Revised: 08/31/2010] [Accepted: 09/01/2010] [Indexed: 12/20/2022]
Abstract
To properly understand the function of genes of neurological interest, in vivo manipulation in the adult is essential, particularly when the target gene is involved in brain development. Moreover, since the physiological effects of target protein may be region-specific, targeting a distinct brain region could be required to dissect these effects in specific brain locations. Infection of somatic tissues of transgenic mice bearing loxP-flanked gene sequences with a viral vector expressing Cre recombinase provides a means of allowing flexible spatio-temporal control of target gene expression. Viral vector-mediated Cre expression could be used to mediate localized gene modulation in a specific brain region. In the present study this technology was applied to the glycine transporter type-1 (GlyT1) protein which is responsible for the uptake of synaptic glycine in the forebrain and has been implicated as a therapeutic target for the treatment of schizophrenia. Since GlyT1 is widely expressed in glial cells, we employed an adenoviral-based vector (Ad5) to deliver Cre protein, due to the preferentially transduction of glial cells by adenoviral vectors in rodent brain. We show significant reduced GlyT1 binding specifically in the thalamic area of conditional GlyT1 (GlyT1c) transgenic mice injected with Ad5-Cre virus, as measured by GlyT1 autoradiography. In conclusion, we demonstrated the validity of viral vector-mediated delivery of Cre to loxP targeted transgenic mice as a novel strategy to investigate target gene function in selected subregions of the adult brain, which provides a valuable technique to investigate gene function both in normal physiology and in disease models.
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Affiliation(s)
- Chiara Cazzin
- Biology Department A&S DPU, Neurosciences CEDD, GlaxoSmithKline, Medicines Research Center, Verona, Italy.
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20
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McPherson CS, Lawrence AJ. The nuclear transcription factor CREB: involvement in addiction, deletion models and looking forward. Curr Neuropharmacol 2010; 5:202-12. [PMID: 19305803 PMCID: PMC2656817 DOI: 10.2174/157015907781695937] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2006] [Revised: 03/16/2007] [Accepted: 03/29/2007] [Indexed: 01/26/2023] Open
Abstract
Addiction involves complex physiological processes, and is characterised not only by broad phenotypic and behavioural traits, but also by ongoing molecular and cellular adaptations. In recent years, increasingly effective and novel techniques have been developed to unravel the molecular implications of addiction. Increasing evidence has supported a contribution of the nuclear transcription factor CREB in the development of addiction, both in contribution to phenotype and expression in brain regions critical to various aspects of drug-seeking behaviour and drug reward. Abstracting from this, models have exploited these data by removing the CREB gene from the developing or developed mouse, to crucially determine its impact upon addiction-related processes. More recent models, however, hold greater promise in unveiling the contribution of CREB to disorders such as addiction.
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Affiliation(s)
- Cameron S McPherson
- Brain Injury and Repair Group, Howard Florey Institute, University of Melbourne, Parkville, Victoria 3010, Australia
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21
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Hu JY, Chu ZG, Han J, Dang YM, Yan H, Zhang Q, Liang GP, Huang YS. The p38/MAPK pathway regulates microtubule polymerization through phosphorylation of MAP4 and Op18 in hypoxic cells. Cell Mol Life Sci 2010; 67:321-33. [PMID: 19915797 PMCID: PMC11115776 DOI: 10.1007/s00018-009-0187-z] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2009] [Revised: 10/13/2009] [Accepted: 10/16/2009] [Indexed: 02/07/2023]
Abstract
In both cardiomyocytes and HeLa cells, hypoxia (1% O(2)) quickly leads to microtubule disruption, but little is known about how microtubule dynamics change during the early stages of hypoxia. We demonstrate that microtubule associated protein 4 (MAP4) phosphorylation increases while oncoprotein 18/stathmin (Op18) phosphorylation decreases after hypoxia, but their protein levels do not change. p38/MAPK activity increases quickly after hypoxia concomitant with MAP4 phosphorylation, and the activated p38/MAPK signaling leads to MAP4 phosphorylation and to Op18 dephosphorylation, both of which induce microtubule disruption. We confirmed the interaction between phospho-p38 and MAP4 using immunoprecipitation and found that SB203580, a p38/MAPK inhibitor, increases and MKK6(Glu) overexpression decreases hypoxic cell viability. Our results demonstrate that hypoxia induces microtubule depolymerization and decreased cell viability via the activation of the p38/MAPK signaling pathway and changes the phosphorylation levels of its downstream effectors, MAP4 and Op18.
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Affiliation(s)
- Jiong-Yu Hu
- State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, Southwest Hospital, The Third Military Medical University, 400038 Chongqing, People’s Republic of China
| | - Zhi-Gang Chu
- State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, Southwest Hospital, The Third Military Medical University, 400038 Chongqing, People’s Republic of China
| | - Jian Han
- Department of Gynecology and Obstetrics, Daping Hospital, The Third Military Medical University, 400038 Chongqing, People’s Republic of China
| | - Yong-ming Dang
- State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, Southwest Hospital, The Third Military Medical University, 400038 Chongqing, People’s Republic of China
| | - Hong Yan
- State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, Southwest Hospital, The Third Military Medical University, 400038 Chongqing, People’s Republic of China
| | - Qiong Zhang
- State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, Southwest Hospital, The Third Military Medical University, 400038 Chongqing, People’s Republic of China
| | - Guang-ping Liang
- State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, Southwest Hospital, The Third Military Medical University, 400038 Chongqing, People’s Republic of China
| | - Yue-Sheng Huang
- State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, Southwest Hospital, The Third Military Medical University, 400038 Chongqing, People’s Republic of China
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22
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Lu G, Sun H, Korge P, Koehler CM, Weiss JN, Wang Y. Functional characterization of a mitochondrial Ser/Thr protein phosphatase in cell death regulation. Methods Enzymol 2009; 457:255-73. [PMID: 19426872 DOI: 10.1016/s0076-6879(09)05014-9] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Protein phosphorylation is a major form of posttranslational modification critical to cell signaling that also occurs in mitochondrial proteome. Yet, only very limited studies have been performed to characterize mitochondrial-targeted protein kinases or phosphatases. Recently, we identified a novel member of PP2C family (PP2Cm) that is a resident mitochondrial protein phosphatase which plays an important role in normal development and cell survival. In this chapter, we will describe the methods applied in the identification of PP2Cm as a resident mitochondrial protein phosphatase based on sequence analysis and biochemical characterization. We will also provide experimental protocols used to establish the intracellular localization of PP2Cm, to achieve loss and gain function of PP2Cm in cultured cells and intact tissue, and to assess the impact of PP2Cm deficiency on cell death, mitochondria oxidative phosphorylation and permeability transition pore opening.
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Affiliation(s)
- Gang Lu
- Department of Anesthesiology, Division of Molecular Medicine, UCLA, Los Angeles, California, USA
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23
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Šale S. Genetically Engineered Mouse Models of Ovarian Cancer and Their Utility in Drug Discovery. ACTA ACUST UNITED AC 2009; Chapter 14:Unit14.11. [DOI: 10.1002/0471141755.ph1411s45] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Affiliation(s)
- Sanja Šale
- Department of Cell Biology, Harvard Medical School Boston Massachusetts
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24
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Vargas NB, Brewer BY, Rogers TB, Wilson GM. Protein kinase C activation stabilizes LDL receptor mRNA via the JNK pathway in HepG2 cells. J Lipid Res 2009; 50:386-397. [PMID: 18936517 PMCID: PMC2638102 DOI: 10.1194/jlr.m800316-jlr200] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2008] [Revised: 09/30/2008] [Indexed: 01/20/2023] Open
Abstract
LDL is the most abundant cholesterol transport vehicle in plasma and a major prognostic indicator of atherosclerosis. Hepatic LDL receptors limit circulating LDL levels, since cholesterol internalized by the liver can be excreted. As such, mechanisms regulating LDL receptor expression in liver cells are appealing targets for cholesterol-lowering therapeutic strategies. Activation of HepG2 cells with phorbol esters enhances LDL receptor mRNA levels through transcriptional and posttranscriptional mechanisms. Here, we show that 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced stabilization of receptor mRNA requires the activity of protein kinase C and is accompanied by activation of the major mitogen activated protein kinase pathways. Inhibitor studies demonstrated that receptor mRNA stabilization is independent of the extracellular signal-regulated kinase or p38(MAPK), but requires activation of the c-Jun N-terminal kinase (JNK). An essential role for JNK in stabilizing receptor mRNA was further confirmed through small interfering RNA (siRNA) experiments and by activating JNK through two protein kinase C-independent mechanisms. Finally, prolonged JNK activation increased steady-state levels of receptor mRNA and protein, and significantly enhanced cellular LDL-binding activity. These data suggest that JNK may play an important role in posttranscriptional control of LDL receptor expression, thus constituting a novel mechanism to enhance plasma LDL clearance by liver cells.
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Affiliation(s)
- Noelle B Vargas
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201
| | - Brandy Y Brewer
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201
| | - Terry B Rogers
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201
| | - Gerald M Wilson
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201; Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201.
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25
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Mena KD, Gerba CP. Waterborne adenovirus. REVIEWS OF ENVIRONMENTAL CONTAMINATION AND TOXICOLOGY 2009; 198:133-167. [PMID: 19253037 DOI: 10.1007/978-0-387-09647-6_4] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
Abstract
Adenoviruses are associated with numerous disease outbreaks, particularly those involving d-cares, schools, children's camps, hospitals and other health care centers, and military settings. In addition, adenoviruses have been responsible for many recreational water outbreaks, including a great number of swimming pool outbreaks than any other waterborne virus (Gerba and Enriquez 1997). Two drinking water outbreaks have been documented for adenovirus (Divizia et al. 2004; Kukkula et al. 1997) but none for food. Of the 51 known adenovirus serotypes, one third are associated with human disease, while other infections are asymptomatic. Human disease associated with adenovirus infections include gastroenteritis, respiratory infections, eye infections, acute hemorrhagic cystitis, and meningoencephalitis (Table 2). Children and the immunocompromised are more severely impacted by adenovirus infections. Subsequently, adenovirus is included in the EPA's Drinking Water Contaminant Candidate List (CCL), which is a list of unregulated contaminants found in public water systems that may pose a risk to public health (National Research Council 1999). Adenoviruses have been detected in various waters worldwide including wastewater, river water, oceans, and swimming pools (Hurst et al. 1988; Irving and Smith 1981; Pina et al. 1998). Adenoviruses typically outnumber the enteroviruses, when both are detected in surface waters. Chapron et al. (2000) found that 38% of 29 surface water samples were positive for infectious Ad40 and Ad41. Data are lacking regarding the occurrence of adenovirus in water in the US, particularly for groundwater and drinking water. Studies have shown, however, that adenoviruses survive longer in water than enteroviruses and hepatitis A virus (Enriquez et al. 1995), which may be due to their double-stranded DNA. Risk assessments have been conducted on waterborne adenovirus (Crabtree et al. 1997; van Heerden et al. 2005c). Using dose-response data for inhalation from Couch et al. (1966), human health risks of infection, illness and death have been determined for various adenovirus exposures. Crabtree et al. (1997) conclude that, even at an adenovirus concentration of 1 per 1,000 L of drinking water, annual risks of infection exceed the suggested risk recommendation of 1 x 10(-4) per yr (Regli et al. 1991) (Table 8). Using the same exposure and dose-response assumptions, van Heerden et al. (2005c) determined annual risks of infection to be 1-1.7 x 10(-1) for two drinking water samples from South Africa containing 1.40 and 2.45 adenoviruses per 10,000 L, respectively. This present study estimated annual risks of infection associated with varying levels of adenoviruses per 100 L (Table 9). By assuming a 2 L/d exposure and utilizing the exponential model at r = 0.4172 (Haas et al. 1993), yearly risks exceed the risk recommendation of 1 x 10(-4) at every exposure level. There are limited data regarding the removal of adenoviruses by conventional water treatment or other physical-chemical treatment processes, but studies do suggest that adenoviruses are of equal or greater sensitivity to oxidizing disinfectants, when compared to waterborne viruses (the most resistant to ultraviolet light). Data suggest that the chlorine doses applied to control other waterborne viruses are more effective against adenovirus, resulting in a greater than 4-log10 removal of adenoviruses by conventional treatment and chlorination. If treatment can achieve a 4-log10 removal of adenoviruses, then, based on the risk levels presented in Table 9, surface water concentrations should not exceed 0.5 adenoviruses per 100 L (Fig. 2). More data are needed regarding effectiveness of water treatment against adenovirus and the human-virus dose-response relationship to fully understand the role of adenovirus as a waterborne public health threat.
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Affiliation(s)
- Kristina D Mena
- University of Texas, Houston School of Public Health, Houston, Texas, USA
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Wang J, Wang H, Chen J, Wang X, Sun K, Wang Y, Wang J, Yang X, Song X, Xin Y, Liu Z, Hui R. GADD45B inhibits MKK7-induced cardiac hypertrophy and the polymorphisms of GADD45B is associated with inter-ventricular septum hypertrophy. Biochem Biophys Res Commun 2008; 372:623-8. [DOI: 10.1016/j.bbrc.2008.05.122] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2008] [Accepted: 05/15/2008] [Indexed: 11/15/2022]
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Ho KJ, Bass CE, Kroemer AHK, Ma C, Terwilliger E, Karp SJ. Optimized adeno-associated virus 8 produces hepatocyte-specific Cre-mediated recombination without toxicity or affecting liver regeneration. Am J Physiol Gastrointest Liver Physiol 2008; 295:G412-9. [PMID: 18535290 PMCID: PMC2519860 DOI: 10.1152/ajpgi.00590.2007] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
UNLABELLED Engineering viral vectors to produce liver-specific protein expression may help advance understanding of hepatic regeneration and disease states. In addition to introducing genes of interest to the liver, these vectors can be adapted for gene deletion when designed to express Cre recombinase. The ability to use this system requires high, liver-restricted expression, low toxicity, and no effect on the process of interest. We developed an adeno-associated virus 8 (AAV8) with a codon-optimized Cre recombinase under a hepatocyte-specific major urinary protein (MUP) promoter (MUP-iCre-AAV8) that fulfills these requirements. A single intravenous injection of ROSA26R reporter mice, which express lacZ after Cre-mediated recombination, demonstrated homogeneous beta-galactosidase expression limited to hepatocytes after only 7 days. Cre protein expression remained strong for at least 31 days. Serum liver function tests and histology demonstrated minimal liver toxicity. The presence of MUP-iCre-AAV8 did not affect hepatocyte proliferation after partial hepatectomy as measured by Ki67 staining. CONCLUSION AAV8 with the MUP promoter, by virtue of its lack of hepatic toxicity or effect on liver regeneration, may be an efficient alternative to complex transgenic methodologies for studies of the mouse liver.
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Affiliation(s)
- Karen J. Ho
- Departments of Surgery and Medicine, Beth Israel Deaconess Medical Center, Department of Surgery, Brigham and Women's Hospital, Boston, Massachusetts
| | - Caroline E. Bass
- Departments of Surgery and Medicine, Beth Israel Deaconess Medical Center, Department of Surgery, Brigham and Women's Hospital, Boston, Massachusetts
| | - Alexander H. K. Kroemer
- Departments of Surgery and Medicine, Beth Israel Deaconess Medical Center, Department of Surgery, Brigham and Women's Hospital, Boston, Massachusetts
| | - Chunyan Ma
- Departments of Surgery and Medicine, Beth Israel Deaconess Medical Center, Department of Surgery, Brigham and Women's Hospital, Boston, Massachusetts
| | - Ernest Terwilliger
- Departments of Surgery and Medicine, Beth Israel Deaconess Medical Center, Department of Surgery, Brigham and Women's Hospital, Boston, Massachusetts
| | - Seth J. Karp
- Departments of Surgery and Medicine, Beth Israel Deaconess Medical Center, Department of Surgery, Brigham and Women's Hospital, Boston, Massachusetts
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Sacher T, Jordan S, Mohr CA, Vidy A, Weyn AMG, Ruszics Z, Koszinowski UH. Conditional gene expression systems to study herpesvirus biology in vivo. Med Microbiol Immunol 2008; 197:269-276. [PMID: 18324415 DOI: 10.1007/s00430-008-0086-1] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2008] [Indexed: 12/28/2022]
Abstract
Cytomegalovirus (CMV), a prototypic beta-herpesvirus, is an important human pathogen causing protean clinical manifestations in immature and immunocompromised patients. Mechanisms of infection can be studied in a mouse model. Mouse cytomegalovirus (MCMV) resembles in pathogenesis its human counterpart in many ways. Although MCMV infection is studied extensively on the level of organs, the contribution of specific cell types to viral replication in vivo is still elusive. Here we describe our approach based on the the Cre/loxP-system to investigate MCMV infection at the level of cell types in vivo. Using bacterial artificial chromosome (BAC)-technology, we created an MCMV virus containing an enhanced green fluorescent protein (egfp) reporter-gene which is not expressed due to a 'Stop' cassette flanked by two loxP-sites between promoter and coding sequence. Infection of cre-transgenic mice with this reporter virus results in the deletion of the 'Stop' cassette and expression of EGFP in a cell type-specific manner. Using this conditional gene expression system we are able to quantify viral productivity in specific cell types and to determine their contribution to viral dissemination in vivo. Furthermore, the deletion of viral genes can be used to screen for cell type-specificity of viral gene functions. Hence, conditional MCMV mutants allow the study of herpesvirus biology on the level of cell types in vivo.
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Affiliation(s)
- Torsten Sacher
- Max von Pettenkofer-Institute, Ludwig Maximilians-University, Pettenkoferstrasse 9a, 80336 Munich, Germany
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Abstract
The analysis of mutant organisms and cell lines is important in determining the function of specific proteins. Recent technological advances in gene targeting by homologous recombination in mammalian systems enable the production of mutants in any desired gene, and can be used to produce mutant mouse strains and mutant cell lines. The yeast Flp/FRT recombinase system and bacteriophage recombinases such as Cre and its recognition sequence, loxP, allow spatial and temporal control of knockouts. This unit discusses crucial issues for homologous recombination experiments, including requirements for the source of DNA, criteria for the targeting constructs, methods of enrichment for homologous recombinants, (positive and negative selection, and the use of endogenous promoters), and the types of mutations that can be created.
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Mortensen R. Overview of gene targeting by homologous recombination. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 2008; Chapter 23:Unit 23.1. [PMID: 18265202 DOI: 10.1002/0471142727.mb2301s51] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Formerly UNIT 9.15, this unit has been moved to the opening spot of our new chapter on Embryonic Stem Cell technology. The unit has also been updated, and now includes information about the Cre-lox and FlP/FRT recombinase systems.
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Affiliation(s)
- R Mortensen
- University of Michigan Medical Center, Ann Arbor, Michigan, USA
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Gkretsi V, Mars WM, Bowen WC, Barua L, Yang Y, Guo L, St-Arnaud R, Dedhar S, Wu C, Michalopoulos GK. Loss of integrin linked kinase from mouse hepatocytes in vitro and in vivo results in apoptosis and hepatitis. Hepatology 2007; 45:1025-1034. [PMID: 17385211 DOI: 10.1002/hep.21540] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
UNLABELLED Extracellular matrix (ECM) is fundamental for the survival of cells within a tissue. Loss of contact with the surrounding ECM often causes altered cell differentiation or cell death. Hepatocytes cultured without matrix lose patterns of hepatocyte-specific gene expression and characteristic cellular micro-architecture. However, differentiation is restored after the addition of hydrated matrix preparations to dedifferentiated hepatocytes. Integrin-linked kinase (ILK) is an important component of cell-ECM adhesions transmitting integrin signaling to the interior of the cell. ILK has been implicated in many fundamental cellular processes such as differentiation, proliferation, and survival. In this study, we investigated the role of ILK in mouse hepatocytes in vitro as well as in vivo. Depletion of ILK from primary mouse hepatocytes resulted in enhanced apoptosis. This was accompanied by increased caspase 3 activity and a significant decrease in expression of PINCH and alpha-parvin, which, along with ILK, form a stable well-characterized ternary complex at cell-ECM adhesions. The induction of apoptosis caused by ILK depletion could be substantially reversed by simultaneous overexpression of ILK, indicating that apoptosis is indeed a consequence of ILK removal. These results were further corroborated via in vivo data showing that adenoviral delivery of Cre-recombinase in ILK-floxed animals by tail vein injection resulted in acute hepatitis, with a variety of pathological findings including inflammation, fatty change, and apoptosis, abnormal mitoses, hydropic degeneration, and necrosis. CONCLUSION Our results demonstrate the importance of ILK and integrin signaling for the survival of hepatocytes and the maintenance of normal liver function.
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Affiliation(s)
- Vasiliki Gkretsi
- Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
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Abstract
The ability to generate genetically manipulated mice has revolutionized the study of development, cell biology, immunobiology and transplantation. Conventional gene targeting approaches lead to inactivation of the target gene in all tissues. This approach often has unintended consequences, such as embryonic lethality, which preclude studying the originally intended tissue. Newer approaches allowing conditional gene expression in a tissue-specific or temporally controlled fashion have the advantage of normal development with gene deletion only in the desired tissues. While nuances to these techniques continue to be developed, the underlying concepts remain consistent. This minireview focuses on the use of conditional gene targeting in mice using the Cre-loxP system and drug inducible gene expression using the tetracycline system.
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Affiliation(s)
- J S Maltzman
- Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
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Lu G, Ren S, Korge P, Choi J, Dong Y, Weiss J, Koehler C, Chen JN, Wang Y. A novel mitochondrial matrix serine/threonine protein phosphatase regulates the mitochondria permeability transition pore and is essential for cellular survival and development. Genes Dev 2007; 21:784-96. [PMID: 17374715 PMCID: PMC1838530 DOI: 10.1101/gad.1499107] [Citation(s) in RCA: 108] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Mitochondria play a central role in the regulation of programmed cell death signaling. Here, we report the finding of a mitochondrial matrix-targeted protein phosphatase 2C family member (PP2Cm) that regulates mitochondrial membrane permeability transition pore (MPTP) opening and is essential for cell survival, embryonic development, and cardiac function. PP2Cm is highly conserved among vertebrates, with the highest expression levels detected in the heart and brain. Small hairpin RNA (shRNA)-mediated knockdown of PP2Cm resulted in cell death associated with loss of mitochondrial membrane potential in cultured cardiac mycoytes and an induction of hepatocyte apoptosis in vivo. PP2Cm-deficient mitochondria showed elevated susceptibility to calcium-induced MPTP opening, whereas mitochondrial oxidative phosphorylation activities were not affected. Finally, inactivation of PP2Cm in developing zebrafish embryos caused abnormal cardiac and neural development as well as heart failure associated with induced apoptosis. These data suggest that PP2Cm is a novel mitochondrial protein phosphatase that has a critical function in cell death and survival, and may play a role in regulating the MPTP opening.
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Affiliation(s)
- Gang Lu
- Department of Anesthesiology, University of California at Los Angeles, Los Angeles, California 90095, USA
- Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, USA
| | - Shuxun Ren
- Department of Anesthesiology, University of California at Los Angeles, Los Angeles, California 90095, USA
- Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, USA
| | - Paavo Korge
- Department of Medicine, University of California at Los Angeles, Los Angeles, California 90095, USA
| | - Jayoung Choi
- Department of Molecular, Cellular, and Developmental Biology, University of California at Los Angeles, Los Angeles, California 90095, USA
| | - Yuan Dong
- Department of Molecular, Cellular, and Developmental Biology, University of California at Los Angeles, Los Angeles, California 90095, USA
| | - James Weiss
- Department of Physiology, University of California at Los Angeles, Los Angeles, California 90095, USA
- Department of Medicine, University of California at Los Angeles, Los Angeles, California 90095, USA
| | - Carla Koehler
- Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, USA
- Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90095, USA
| | - Jau-nian Chen
- Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, USA
- Department of Molecular, Cellular, and Developmental Biology, University of California at Los Angeles, Los Angeles, California 90095, USA
| | - Yibin Wang
- Department of Anesthesiology, University of California at Los Angeles, Los Angeles, California 90095, USA
- Department of Physiology, University of California at Los Angeles, Los Angeles, California 90095, USA
- Department of Medicine, University of California at Los Angeles, Los Angeles, California 90095, USA
- Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, USA
- Corresponding author.E-MAIL ; FAX (310) 206-5097
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Pomiès P, Pashmforoush M, Vegezzi C, Chien KR, Auffray C, Beckerle MC. The cytoskeleton-associated PDZ-LIM protein, ALP, acts on serum response factor activity to regulate muscle differentiation. Mol Biol Cell 2007; 18:1723-33. [PMID: 17332502 PMCID: PMC1855033 DOI: 10.1091/mbc.e06-09-0815] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
In this report, an antisense RNA strategy has allowed us to show that disruption of ALP expression affects the expression of the muscle transcription factors myogenin and MyoD, resulting in the inhibition of muscle differentiation. Introduction of a MyoD expression construct into ALP-antisense cells is sufficient to restore the capacity of the cells to differentiate, illustrating that ALP function occurs upstream of MyoD. It is known that MyoD is under the control of serum response factor (SRF), a transcriptional regulator whose activity is modulated by actin dynamics. A dramatic reduction of actin filament bundles is observed in ALP-antisense cells and treatment of these cells with the actin-stabilizing drug jasplakinolide stimulates SRF activity and restores the capacity of the cells to differentiate. Furthermore, we show that modulation of ALP expression influences SRF activity, the level of its coactivator, MAL, and muscle differentiation. Collectively, these results suggest a critical role of ALP on muscle differentiation, likely via cytoskeletal regulation of SRF.
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Affiliation(s)
- Pascal Pomiès
- Centre National de la Recherche Scientifique Unité Mixte de Recherche 5237, Centre de Recherches de Biochimie Macromoléculaire, 34293 Montpellier, France.
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35
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Mortensen R. Overview of Gene Targeting by Homologous Recombination. ACTA ACUST UNITED AC 2006; Chapter 23:Unit 23.1. [DOI: 10.1002/0471142727.mb2301s76] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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Li X, Jung C, Liu YH, Bae KH, Zhang YP, Zhang HJ, Vanderputten D, Jeng MH, Gardner TA, Kao C. Anti-tumor efficacy of a transcriptional replication-competent adenovirus, Ad-OC-E1a, for osteosarcoma pulmonary metastasis. J Gene Med 2006; 8:679-89. [PMID: 16570242 DOI: 10.1002/jgm.904] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
BACKGROUND Osteosarcoma (OSA) is the most frequent type of primary malignant bone tumor and is apt to occur in children and young adults. Pulmonary metastasis (OSPM) is the major reason for its fatal outcome. Osteocalcin (OC) is a major noncollagenous bone protein whose expression is limited almost exclusively to bone marrow and osteotropic tumors. OC is also known to express in cell lines with bone metastasis feathers. Gene therapy strategies with the OC promoter directing the replication of adenovirus in a tumor-specific manner are a potential modality for OSPM therapy. METHODS We detected OC mRNA expression by RNA in situ hybridization in OSA and OSPM samples from patients, and tested OC promoter transcriptional activity in OSA and non-OSA cell lines. Then we used a transcriptional replication-competent adenovirus, Ad-OC-E1a, to treat OSPM, and evaluated its tumor-specific replication and killing activities in vitro as well as anti-OSPM efficacy in vivo via systemic delivery. RESULTS OC mRNA was detected in all types of OSA tissues, including OSPM tissues. The transcriptional activity of the OC promoter was much higher in a OSPM cell line SAOS-2LM7 and primary OSA cell line MG63 than in non-OSA cell lines, including cell lines from breast cancer, colon cancer, and liver cancer. Ad-OC-E1a expressed E1a protein only in MG63 and SAOS-2LM7, which indicated that adenovirus E1a was under strict control by the OC promoter. Ad-OC-E1a demonstrated killing and viral replication activity close to wild-type adenovirus levels in MG63 and SAOS-2LM7, but the killing and viral replication activities were attenuated significantly in cells expressing low OC transcriptional activity. To test whether Ad-OC-E1a could be used to target human OSPM in vivo, SAOS-2LM7 pulmonary metastasis models in nude mice were induced and treated by tail-vein injection with Ad-OC-E1a. Compared to tumor nodules in the lung in groups treated with PBS or control virus, the quantity of metastasized tumor nodules decreased significantly. Adenovirus-infected cells were stained immunohistochemically only inside and around the OSPM nodules but spared normal lung tissue and other organs. CONCLUSIONS These data demonstrated that OC promoter could direct adenovirus replication by controlling the E1a gene to target human OSPM in a tumor-specific manner, providing an efficient tool to develop a feasible therapeutic modality for OSPM.
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Affiliation(s)
- Xiong Li
- Department of Urology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
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Zhu HZ, Wang W, Feng DM, Sai Y, Xue JL. Conditional gene modification in mouse liver using hydrodynamic delivery of plasmid DNA encoding Cre recombinase. FEBS Lett 2006; 580:4346-52. [PMID: 16846600 DOI: 10.1016/j.febslet.2006.06.094] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2006] [Revised: 06/30/2006] [Accepted: 06/30/2006] [Indexed: 10/24/2022]
Abstract
The success of Cre-mediated conditional gene targeting in liver of mice has until now depended on the generation of Cre recombinase transgenic mice or on viral-mediated transduction. Here, we sought to establish the feasibility of using hydrodynamic gene delivery of Cre recombinase into liver, using a ROSA26 EGFP mouse. The expression of EGFP and beta-galactosidase was exclusively detected in the liver of mice treated with hydrodynamic gene delivery of Cre recombinase, as assessed with fluorescence microscopy and X-Gal staining, respectively; Southern blotting also showed that Cre mediated recombination occurred specifically in the liver and not in other organs. The Cre mediated recombination reached about 61% of hepatocytes of mouse after repeated injection, as analyzed by flow cytometry. These results demonstrate that Cre recombinase can be transferred to the liver of mice through a simple hydrodynamic gene-delivery approach and can mediate efficient recombination in hepatocytes. Thus, hydrodynamic gene delivery of the Cre recombinase provides a valuable approach for Cre-loxP-mediated conditional gene modification in the liver of mice.
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Affiliation(s)
- Huan Zhang Zhu
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China.
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38
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Wang H, Xie H, Zhang H, Das SK, Dey SK. Conditional gene recombination by adenovirus-driven Cre in the mouse uterus. Genesis 2006; 44:51-6. [PMID: 16416422 PMCID: PMC4267753 DOI: 10.1002/gene.20185] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Cre-mediated conditional gene targeting has been shown to be successful in many cell and tissue types. However, gene recombination in the uterus with heterogeneous cell types by Cre activation is not yet well established. Using recombinant adenoviruses expressing a functional Cre (ADV-Cre) and ROSA26 reporter mice, we show here that ADV-Cre infused intraluminally in a small volume (10 microl) conditionally excises the loxP site, resulting in lacZ expression in uterine luminal epithelial cells without significantly affecting pregnancy. In contrast, a similar intraluminal infusion of ADV-Cre in a larger volume (50 microl) damages the normal architecture and integrity of the luminal epithelium, inducing gene recombination in the underneath stromal cells, with disruption of pregnancy. Further, decidualizing stromal cells at the implantation sites can be targeted by ADV-Cre after intravenous administration on days 5-6. This route of administration also elicits Cre activity in other tissues, including the liver, spleen, ovary, and, more remarkably, in the adrenal cortex. These findings demonstrate the feasibility of achieving conditional expression or deletion of specific genes in uterine cells at desired times and physiological states.
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Affiliation(s)
- Haibin Wang
- Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee
| | - Huirong Xie
- Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee
| | - Hao Zhang
- Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee
| | - Sanjoy K. Das
- Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee
- Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, Tennessee
| | - Sudhansu K. Dey
- Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee
- Department of Cell & Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee
- Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee
- Correspondence to: Sudhansu K. Dey, Department of Pediatrics, Division of Reproductive and Developmental Biology, Vanderbilt University Medical Center, MCN-D4100, Nashville, TN 37232-2678,
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Glaser ND, Lukyanenko YO, Wang Y, Wilson GM, Rogers TB. JNK activation decreases PP2A regulatory subunit B56alpha expression and mRNA stability and increases AUF1 expression in cardiomyocytes. Am J Physiol Heart Circ Physiol 2006; 291:H1183-92. [PMID: 16603688 PMCID: PMC1564198 DOI: 10.1152/ajpheart.01162.2005] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
A central feature of heart disease is a molecular remodeling of signaling pathways in cardiac myocytes. This study focused on novel molecular elements of MAPK-mediated alterations in the pattern of gene expression of the protein phosphatase 2A (PP2A). In an established model of sustained JNK activation, a 70% decrease in expression of the targeting subunit of PP2A, B56alpha, was observed in either neonatal or adult cardiomyocytes. This loss in protein abundance was accompanied by a decrease of 69% in B56alpha mRNA steady-state levels. Given that the 3'-untranslated region of this transcript contains adenylate-uridylate-rich elements known to regulate mRNA degradation, experiments explored the notion that instability of B56alpha mRNA accounts for the response. mRNA time-course analyses with real-time PCR methods showed that B56alpha transcript was transformed from a stable (no significant decay over 1 h) to a labile form that rapidly degraded within minutes. These results were supported by complementary experiments that revealed that the RNA-binding protein AUF1, known to destabilize target mRNA, was increased fourfold in JNK-activated cells. A variety of other stress-related stimuli, such as p38 MAPK activation and phorbol ester, upregulated AUF1 expression in cultured cardiac cells as well. In addition, gel mobility shift assays demonstrated that p37AUF1 binds with nanomolar affinity to segments of the B56alpha 3'-untranslated region. Thus these studies provide new evidence that signaling-induced mRNA instability is an important mechanism that underlies the changes in the pattern of gene expression evoked by stress-activated pathways in cardiac cells.
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Affiliation(s)
- Nicole D. Glaser
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland and
| | - Yevgeniya O. Lukyanenko
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland and
| | - Yibin Wang
- Departments of Anesthesiology and Medicine, University of California at Los Angeles, Los Angeles, California
| | - Gerald M. Wilson
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland and
| | - Terry B. Rogers
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland and
- Institute of Molecular Cardiology, Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland
- To whom correspondence should be addressed: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene Street, Baltimore, MD 21201. Tel: 410-706-3169; Fax: 410-706-6676;
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Ungrin MD, Harrington L. Strict control of telomerase activation using Cre-mediated inversion. BMC Biotechnol 2006; 6:10. [PMID: 16504006 PMCID: PMC1403769 DOI: 10.1186/1472-6750-6-10] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2005] [Accepted: 02/20/2006] [Indexed: 01/25/2023] Open
Abstract
Background Human cells appear exquisitely sensitive to the levels of hTERT expression, the telomerase reverse transcriptase. In primary cells that do not express hTERT, telomeres erode with each successive cell division, leading to the eventual loss of telomere DNA, an induction of a telomere DNA damage response, and the onset of cellular senescence or crisis. In some instances, an average of less than one appropriately spliced hTERT transcript per cell appears sufficient to restore telomerase activity and telomere maintenance, and overcome finite replicative capacity. Results To underscore this sensitivity, we showed that a widely used system of transcriptional induction involving ecdysone (muristerone) led to sufficient expression of hTERT to immortalize human fibroblasts, even in the absence of induction. To permit tightly regulated expression of hTERT, or any other gene of interest, we developed a method of transcriptional control using an invertible expression cassette flanked by antiparallel loxP recombination sites. When introduced into human fibroblasts with the hTERT cDNA positioned in the opposite orientation relative to a constitutively active promoter, no telomerase activity was detected, and the cell population retained a mortal phenotype. Upon inversion of the hTERT cDNA to a transcriptionally competent orientation via the action of Cre recombinase, cells acquired telomerase activity, telomere DNA was replenished, and the population was immortalized. Further, using expression of a fluorescent protein marker, we demonstrated the ability to repeatedly invert specific transcripts between an active and inactive state in an otherwise isogenic cell background. Conclusion This binary expression system thus provides a useful genetic means to strictly regulate the expression of a given gene, or to control the expression of at least two different genes in a mutually exclusive manner.
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Affiliation(s)
- Mark D Ungrin
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Terrence Donnelly Centre for Cellular and Biomolecular Research, 160 College Street, Toronto, Ontario, M5S 3E1, USA
| | - Lea Harrington
- Department of Medical Biophysics, University of Toronto, Ontario Cancer Institute, and Campbell Family Institute for Breast Cancer Research, 620 University Avenue, Toronto, ON M5G 2C1, USA
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Satoh Y, Kanda Y, Terakawa M, Obara M, Mizuno K, Watanabe Y, Endo S, Ooigawa H, Nawashiro H, Sato S, Takishima K. Targeted DNA transfection into the mouse central nervous system using laser-induced stress waves. JOURNAL OF BIOMEDICAL OPTICS 2005; 10:060501. [PMID: 16409064 DOI: 10.1117/1.2128432] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/06/2023]
Abstract
We investigated the feasibility of gene transfer into the mouse central nervous system (CNS) by applying nanosecond pulsed laser-induced stress waves (LISWs). Intraventricular or hippocampal injection of a reporter gene [enhanced green fluorescent protein (EGFP)] followed by application of LISWs showed this method to be efficient in the CNS of newborn and adult mice. Cells expressing EGFP reside at least 3.5 mm from the surface of the tissue, while no apparent damage was detected. Additionally, expression of EGFP was limited to the area that was exposed to LISWs. Using this method, the formulation of plasmid DNA by cationic transfer reagent polyethylenimine proved to be effective for improving transfer efficiency into the CNS.
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Leow CC, Wang XD, Gao WQ. Novel method of generating prostate-specific Cre-LoxP gene switching via intraductal delivery of adenovirus. Prostate 2005; 65:1-9. [PMID: 15791629 DOI: 10.1002/pros.20244] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
BACKGROUND In order to facilitate elucidation of oncogene or tumor suppressor gene function on initiation and progression of prostate cancer, it would be advantageous to develop an effective method to generate spatially and temporally controlled gene modification in murine prostates. METHODS Adenovirus expressing Cre-recombinase (Adeno-Cre) was intraductally injected into the prostate of ROSA26 reporter mice. Immmunohistochemical and X-gal staining were performed on prostate tissue sections harvested from mice at various time points following viral injection to confirm expression and activity of Cre-recombinase, respectively. RESULTS Adenovirus was intraductally delivered to the anterior lobe of the mouse prostate. Using this method of intraductal injection, we were able to precisely obtain Adeno-Cre infection to a majority of epithelial cells but not in the stromal cells or other organs. We further demonstrated that Adeno-Cre infected epithelial cells not only expressed Cre-recombinase enzyme but more importantly, Cre-recombinase activity was revealed through positive X-gal staining in Rosa26 reporter mice, thus, confirming epithelial-specific Cre-loxP recombination in Adeno-Cre infected prostate tissue sections. CONCLUSIONS This novel method of direct genetic delivery into adult murine prostates could provide an alternative to the more expensive and time-consuming transgenic/knockout approaches. The latter also have other limitations such as the availability of cell-type specific or temporally-regulated promoters, and the complication of genetic background differences, which can potentially be complemented by the technology we describe here.
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Affiliation(s)
- Ching Ching Leow
- Department of Molecular Biology, Genentech, Inc., South San Francisco, California 94080, USA
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Guan Y, Hao C, Cha DR, Rao R, Lu W, Kohan DE, Magnuson MA, Redha R, Zhang Y, Breyer MD. Thiazolidinediones expand body fluid volume through PPARgamma stimulation of ENaC-mediated renal salt absorption. Nat Med 2005; 11:861-6. [PMID: 16007095 DOI: 10.1038/nm1278] [Citation(s) in RCA: 471] [Impact Index Per Article: 23.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2005] [Accepted: 06/24/2005] [Indexed: 12/13/2022]
Abstract
Thiazolidinediones (TZDs) are widely used to treat type 2 diabetes mellitus; however, their use is complicated by systemic fluid retention. Along the nephron, the pharmacological target of TZDs, peroxisome proliferator-activated receptor-gamma (PPARgamma, encoded by Pparg), is most abundant in the collecting duct. Here we show that mice treated with TZDs experience early weight gain from increased total body water. Weight gain was blocked by the collecting duct-specific diuretic amiloride and was also prevented by deletion of Pparg from the collecting duct, using Pparg (flox/flox) mice. Deletion of collecting duct Pparg decreased renal Na(+) avidity and increased plasma aldosterone. Treating cultured collecting ducts with TZDs increased amiloride-sensitive Na(+) absorption and Scnn1g mRNA (encoding the epithelial Na(+) channel ENaCgamma) expression through a PPARgamma-dependent pathway. These studies identify Scnn1g as a PPARgamma target gene in the collecting duct. Activation of this pathway mediates fluid retention associated with TZDs, and suggests amiloride might provide a specific therapy.
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Affiliation(s)
- YouFei Guan
- Division of Nephrology, Department of Medicine, Vanderbilt University School of Medicine, 21st Avenue South at Garland Avenue, Nashville, Tennessee 37232, USA
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Gigena MS, Ito A, Nojima H, Rogers TB. A B56 regulatory subunit of protein phosphatase 2A localizes to nuclear speckles in cardiomyocytes. Am J Physiol Heart Circ Physiol 2005; 289:H285-94. [PMID: 15778281 DOI: 10.1152/ajpheart.01291.2004] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Protein phosphatase 2A (PP2A) is widely distributed in heart tissues, yet its precise cellular functions are poorly understood. This study is based on the notion that PP2A action is governed by interactions of the core enzyme with B targeting/regulatory subunits. The subcellular localizations of two B subunits, B56α and B56γ1, were assessed using adenovirus-driven expression of epitope-tagged (hemagglutinin, HA) in cultured neonatal and adult rat ventricular myocytes. Confocal imaging revealed that HA-B56α was excluded from the nucleus and decorated striated structures, whereas HA-B56γ1 was principally found in the nucleus. Precise immunolabeling studies showed that B56γ1 was concentrated in intranuclear structures known as nuclear speckles, macromolecular structures that accumulate transcription and splicing factors. Western blot analyses revealed that overexpression of either B subunit had no effect on the levels of other PP2A subunits in cultured neonatal cardiac cells. However, overexpression of only B56γ1 increased whole cell PP2A activity by 40% when measured in cell extracts. Finally, B56γ1 did not alter global gene expression or expression of hypertrophic gene markers such as α-skeletal actin. However, morphometric analyses of confocal images revealed that B56γ1 alters the dynamic assembly/disassembly process of nuclear speckles in heart cells. These studies provide new insight into mechanisms of PP2A targeting in the subnuclear architecture in cardiomyocytes and into the role of this phosphatase in nuclear signaling.
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Affiliation(s)
- Marisa S Gigena
- Dept. of Biochemistry and Molecular Biology, Univ. of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201, USA
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Aoyama M, Agari K, Sun-Wada GH, Futai M, Wada Y. Simple and straightforward construction of a mouse gene targeting vector using in vitro transposition reactions. Nucleic Acids Res 2005; 33:e52. [PMID: 15784610 PMCID: PMC1069132 DOI: 10.1093/nar/gni055] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2005] [Revised: 03/07/2005] [Accepted: 03/07/2005] [Indexed: 11/12/2022] Open
Abstract
In a gene targeting experiment, the generation of a targeting construct often requires complex DNA manipulations. We developed a set of cassettes and plasmids useful for creating targeting vectors to modify the mammalian genome. A positive selection marker cassette (PGK/EM7p-npt), which included dual prokaryotic and eukaryotic promoters to permit consecutive selection for recombination in Escherichia coli and then in mouse embryonic stem cells, was flanked by two FRT-loxP sequences. The PGK/EM7p-npt cassette was placed between the minimum regions of a Tn7 transposable element for insertion into another DNA by means of Tn7 transposase in vitro. We also constructed a plasmid having a loxP-Zeo-loxP cassette between the modified Tn5 outer elements. These cassettes can be integrated randomly into a given genomic DNA through the in vitro transposition reaction, thus producing a collection of genomic segments flanked by loxP sites (floxed) at various positions without the use of restriction enzymes and DNA ligase. We confirmed that this system remarkably reduced the time and labor for the construction of complex gene targeting vectors.
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Affiliation(s)
- Minako Aoyama
- Division of Biological Science, Institute for Scientific and Industrial Research, Osaka University and CREST, Japan Science and Technology AgencyMihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan
- Futai Special Laboratory, Microbial Chemistry Research Center, Microbial Chemistry Research Foundation and CREST, Japan Science and Technology Agency3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
| | - Kazuko Agari
- Division of Biological Science, Institute for Scientific and Industrial Research, Osaka University and CREST, Japan Science and Technology AgencyMihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan
- Futai Special Laboratory, Microbial Chemistry Research Center, Microbial Chemistry Research Foundation and CREST, Japan Science and Technology Agency3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
| | - Ge-Hong Sun-Wada
- Division of Biological Science, Institute for Scientific and Industrial Research, Osaka University and CREST, Japan Science and Technology AgencyMihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan
- Futai Special Laboratory, Microbial Chemistry Research Center, Microbial Chemistry Research Foundation and CREST, Japan Science and Technology Agency3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
| | - Masamitsu Futai
- Futai Special Laboratory, Microbial Chemistry Research Center, Microbial Chemistry Research Foundation and CREST, Japan Science and Technology Agency3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
| | - Yoh Wada
- To whom correspondence should be addressed. Tel: +81 6 6879 8481; Fax: +81 6 6875 5724;
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Heine HL, Leong HS, Rossi FMV, McManus BM, Podor TJ. Strategies of Conditional Gene Expression in Myocardium. MOLECULAR CARDIOLOGY 2005; 112:109-54. [PMID: 16010014 DOI: 10.1007/978-1-59259-879-3_8] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The use of specialized reporter genes to monitor real-time, tissue-specific transgene expression in animal models offers an opportunity to circumvent current limitations associated with the establishment of transgenic mouse models. The Cre-loxP and the tetracycline (Tet)-inducible systems are useful methods of conditional gene expression that allow spatial (cell-type-specific) and temporal (inducer-dependent) control. Most often, the alpha-myosin heavy chain (alpha-MHC) promoter is used in these inducible systems to restrict expression of reporter genes and transgenes to the myocardium. An overview of each inducible system is described, along with suggested reporter genes for real-time, noninvasive imaging in the myocardium. Effective gene delivery of the inducible gene expression system is carried out by lentiviral vectors, which offer high transduction efficiency, long-term transgene expression, and low immunogenicity. This chapter outlines the packaging of myocardium-specific inducible expression systems into lentiviral vectors, in which a transgene and a reporter gene are transduced into cardiomyocytes. In doing so, transgene and reporter expression can be monitored/tracked with bioluminescence imaging (BLI) and positron emission tomography (PET).
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Affiliation(s)
- Heather L Heine
- The James Hogg iCAPTURE Center for Cardiovascular and Pulmonary Research/MRL, University of British Columbia, St. Paul's Hospital, Vancouver, Canada
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Colnot S, Decaens T, Niwa-Kawakita M, Godard C, Hamard G, Kahn A, Giovannini M, Perret C. Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas. Proc Natl Acad Sci U S A 2004; 101:17216-21. [PMID: 15563600 PMCID: PMC535370 DOI: 10.1073/pnas.0404761101] [Citation(s) in RCA: 250] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Although inappropriate activation of the Wnt/beta-catenin pathway has been implicated in the development of hepatocellular carcinoma (HCC), the role of this signaling in liver carcinogenesis remains unclear. To investigate this issue, we constructed a mutant mouse strain, Apc(lox/lox), in which exon 14 of the tumor-suppressor gene adenomatous polyposis coli (Apc) is flanked by loxP sequences. i.v. injection of adenovirus encoding Cre recombinase (AdCre) at high multiplicity [10(9) plaque-forming units (pfu) per mouse] inactivated the Apc gene in the liver and resulted in marked hepatomegaly, hepatocyte hyperplasia, and rapid mortality. beta-Catenin signaling activation was demonstrated by nuclear and cytoplasmic accumulation of beta-catenin in the hepatocytes and by the induction of beta-catenin target genes (glutamine synthetase, glutamate transporter 1, ornithine aminotransferase, and leukocyte cell-derived chemotaxin 2) in the liver. To test a long-term oncogenic effect, we inoculated mice with lower doses of AdCre (0.5 x 10(9) pfu per mouse), compatible with both survival and persistence of beta-catenin-activated cells. In these conditions, 67% of mice developed HCC. beta-Catenin signaling was strongly activated in these Apc-inactivated HCCs. The HCCs were well, moderately, or poorly differentiated. Indeed, their histological and molecular features mimicked human HCC. Thus, deletion of Apc in the liver provides a valuable model of human HCC, and, in this model, activation of the Wnt/beta-catenin pathway by invalidation of Apc is required for liver tumorigenesis.
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Affiliation(s)
- S Colnot
- Institut Cochin, Institut National de la Santé et de la Recherche Médicale U567, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Université Paris V, 24 Rue du Faubourg St. Jacques, 75014 Paris, France
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Salbaum JM, Cirelli C, Walcott E, Krushel LA, Edelman GM, Tononi G. Chlorotoxin-mediated disinhibition of noradrenergic locus coeruleus neurons using a conditional transgenic approach. Brain Res 2004; 1016:20-32. [PMID: 15234248 DOI: 10.1016/j.brainres.2004.03.078] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/02/2004] [Indexed: 10/26/2022]
Abstract
The noradrenergic locus coeruleus (LC) has been implicated in the promotion of arousal, in focused attention and learning, and in the regulation of the sleep/waking cycle. The complex biological functions of the central noradrenergic system have been investigated largely through electrophysiological recordings and neurotoxic lesions of LC neurons. Activation of LC neurons through electrical or chemical stimulation has also led to important insights, although these techniques have limited cellular specificity and short-term effects. Here, we describe a novel method aimed at stimulating the central noradrenergic system in a highly selective manner for prolonged periods of time. This was achieved through the conditional expression of a transgene for chlorotoxin (Cltx) in the LC of adult mice. Chlorotoxin is a component of scorpion venom that partially blocks small conductance chloride channels. In this manner, the influence of GABAergic and glycinergic inhibitory inputs on LC cells is greatly reduced, while their ability to respond to excitatory inputs is unaffected. We demonstrate that the unilateral induction of Cltx expression in the LC is associated with a concomitant ipsilateral increase in the expression of markers of noradrenergic activity in LC neurons. Moreover, LC disinhibition is associated with the ipsilateral induction of the immediate early gene NGFI-A in cortical and subcortical target areas. Unlike previous gain of function approaches, transgenic disinhibition of LC cells is highly selective and persists for at least several weeks. This method represents a powerful new tool to assess the long-term effects of LC activation and is potentially applicable to other neuronal systems.
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Affiliation(s)
- J Michael Salbaum
- The Neurosciences Institute, 10640 John J. Hopkins Drive, San Diego, CA 92121, USA.
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Kopertekh L, Jüttner G, Schiemann J. PVX-Cre-mediated marker gene elimination from transgenic plants. PLANT MOLECULAR BIOLOGY 2004; 55:491-500. [PMID: 15604695 DOI: 10.1007/s11103-004-0237-8] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
Cre recombinase gene from bacteriophage P1 was transiently expressed by a Potato Virus X (PVX)-based vector in transgenic lox -target Nicotiana benthamiana plants to remove the selectable marker gene. The target construct consisted of two directly oriented lox sites flanking a bar gene located between a gfp coding region and an upstream CaMV 35S promoter. The Cre-mediated excision of intervening sequence placed the gfp coding region under the transcriptional control of the CaMV 35S promoter. GFP activity was observed in PVX-Cre systemically infected leaves, regenerants from PVX-Cre infected explants and T1 progeny of these regenerants. PVX-Cre was removed efficiently from the regenerants by adding the nucleoside analogue ribavirin to the culture medium. Molecular data proved a correlation between gfp expression and precise site-specific excision of the bar gene in all examined transgenic lines. The frequency of recombination expressed as a percentage of regenerated plants exhibiting marker gene excision varied from 48% to 82%. These results demonstrate that a plant virus vector can be used efficiently to express cre recombinase in vivo providing an alternative method for the production of transgenic plants without marker genes.
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Affiliation(s)
- L Kopertekh
- Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Virology, Microbiology and Biosafety, Messeweg 11-12, Braunschweig, Germany
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Abstract
The lung is a complex organ consisting of numerous cell types that function to ensure sufficient gas exchange to oxygenate the blood. In order to accomplish this function, the lung must be exposed to the external environment and at the same time maintain a homeostatic balance between its function in gas exchange and the maintenance of inflammatory balance. During the past two decades, as molecular methodologies have evolved with the sequencing of entire genomes, the use of in vivo models to elucidate the molecular mechanisms involved in pulmonary physiology and disease have increased. The mouse has emerged as a potent model to investigate pulmonary physiology due to the explosion in molecular methods that now allow for the developmental and tissue-specific regulation of gene transcription. Initial efforts to manipulate gene expression in the mouse genome resulted in the generation of transgenic mice characterized by the constitutive expression of a specific gene and knockout mice characterized by the ablation of a specific gene. The utility of these original mouse models was limited, in many cases, by phenotypes resulting in embryonic or neonatal lethality that prevented analysis of the impact of the genetic manipulation on pulmonary biology. Second-generation transgenic mouse models employ multiple strategies that can either activate or silence gene expression thereby providing extensive temporal and spatial control of the experimental parameters of gene expression. These highly regulated mouse models are intended to serve as a foundation for further investigation of the molecular basis of human disease such as tumorigenesis. This review describes the principles, progress, and application of systems that are currently employed in the conditional regulation of gene expression in the investigation of lung cancer.
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Affiliation(s)
- I Kwak
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
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