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Toshio Kitamura - One of the best experts on this subject based on the ideXlab platform.
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retrovirus mediated gene transfer and Expression Cloning powerful tools in functional genomics
Experimental Hematology, 2003Co-Authors: Toshio Kitamura, Yuko 越野 裕子 Koshino, Fumi Shibata, Hideaki Nakajima, Tetsuya Nosaka, Hidetoshi KumagaiAbstract:Most of the human genome has now been sequenced and about 30,000 potential open reading frames have been identified, indicating that we use these 30,000 genes to functionally organize our biologic activities. However, functions of many genes are still unknown despite intensive efforts using bioinformatics as well as transgenic and knockout mice. Retrovirus-mediated gene transfer is a powerful tool that can be used to understand gene functions. We have developed a variety of retrovirus vectors and efficient packaging cell lines that have facilitated the development of efficient functional Expression Cloning methods. In this review, we describe retrovirus-mediated strategies used for investigation of gene functions and function-based screening strategies.
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A method to identify cDNAs based on localization of green fluorescent protein fusion products
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Kazuhide Misawa, Tetsuya Nosaka, Sumiyo Morita, Azusa Kaneko, Tatsutoshi Nakahata, Shigetaka Asano, Toshio KitamuraAbstract:We previously established a high-efficiency, retrovirus-mediated Expression Cloning method. Using this system, we now have developed an Expression Cloning method (FL-REX; fluorescence localization-based retrovirus-mediated Expression Cloning) in which cDNAs can be isolated based on the subcellular localization of their protein products. Complementary DNAs generated from mRNA using random hexamers were fused to the cDNA of green fluorescent protein (GFP) in the pMX retrovirus vector. The resulting cDNA-GFP fusion library was transfected into retrovirus-packaging cells, and the derived retroviruses were used to infect NIH 3T3 cells. Infected cells then were screened to identify cDNAs of interest through the subcellular localization of the GFP-fusion products. Using FL-REX, we have identified 25 cDNAs, most of which showed reasonable subcellular localization as GFP-fusion proteins, indicating that FL-REX is useful for identification of proteins that show specific intracellular localization.
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New experimental approaches in retrovirus-mediated Expression screening.
International Journal of Hematology, 1998Co-Authors: Toshio KitamuraAbstract:: We have established an Expression screening system that utilizes retrovirus-mediated gene transfer. The system is based on a high-efficiency packaging cell line BOSC23 and retrovirus vectors pBabeX and pMX which are suitable for cDNA library construction. The combination of BOSC23 packaging cells and the pBabeX and pMX retrovirus vectors produces high-titer recombinant retroviruses, which are able to cover large complexities of cDNA libraries. These retroviruses gave 100% infection efficiency in NIH3T3 cells and 10-100% infection efficiency in various hemopoietic cell lines. In contrast to the conventional mammalian Expression Cloning system where it is required to transduce cDNA transiently into particular cell types such as COS cells, the retrovirus-mediated Expression Cloning method allows stable transduction of cDNAs into a wide variety of cell types. This method, therefore, makes it possible to select cells expressing the desired cDNA by various functional assays. In addition, when combined with PCR-driven random mutagenesis, this system is also useful in searching for mutations of various molecules which will result in functional alterations. Other potential applications will also be discussed.
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applications of retrovirus mediated Expression Cloning
Experimental Hematology, 1996Co-Authors: Mayumi Onishi, Garry P. Nolan, S Kinoshita, Yoshihiro Morikawa, Akira Shibuya, Joseph H Phillips, Lewis L Lanier, Daniel M Gorman, Atsushi Miyajima, Toshio KitamuraAbstract:: We have recently established a novel Expression Cloning system using retroviral vectors. The system is based on a high-efficiency packaging cell line, BOSC23, and a simplified retroviral vector, pBabeX, carrying no selection marker. cDNA libraries, constructed in the pBabeX vector, are transiently transfected into BOSC23 cells. The supernatant contains more than 3X10(6)/mL, which would cover large complexities of cDNA libraries. The retrovirus stock gave 100% infection efficiency in NIH3T3 cells and 5-40% infection efficiency in various hematopoietic cell lines. In contrast to the conventional Expression Cloning system, in which it is necessary to transfect cDNA libraries transiently into particular cell types such as COS cells, retrovirus-mediated Expression Cloning allows us to transduce cDNAs into a wide variety of cell types. This method therefore makes it possible to select cells expressing a cDNA of interest by various functional assays. When combined with polymerase chain reaction (PCR)-driven random mutagenesis, this system is also useful in searching for mutations of various molecules that will result in alterations of their functions.
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Expression Cloning of the human il 3 receptor cdna reveals a shared β subunit for the human il 3 and gm csf receptors
Cell, 1991Co-Authors: Toshio Kitamura, Noriko Sato, Kenichi Arai, Atsushi MiyajimaAbstract:A cDNA for a human interleukin-3 (hIL-3) binding protein has been isolated by a novel Expression Cloning strategy: a cDNA library was coexpressed with the cDNA for the beta subunit of human granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (hGMR beta) in COS7 cells and screened by binding of 125I-labeled IL-3. The cloned cDNA (DUK-1) encodes a mature protein of 70 kd, which belongs to the cytokine receptor family and which alone binds hIL-3 with extremely low affinity (Kd = 120 +/- 60 nM). A high affinity IL-3-binding site (Kd = 140 +/- 30 pM) was reconstituted by coexpressing the DUK-1 protein and hGMR beta, indicating that hIL-3R and hGMR share the beta subunit. Therefore, we designated DUK-1 as the alpha subunit of the hIL-3R. As in human hematopoietic cells, hIL-3 and hGM-CSF complete for binding in fibroblasts expressing the cDNAs for hIL-3R alpha, GMR alpha, and the common beta subunit, indicating that different alpha subunits compete for a common beta subunit.
Bernard C. Rossier - One of the best experts on this subject based on the ideXlab platform.
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Epithelial sodium channel regulatory proteins identified by functional Expression Cloning
Kidney International, 1998Co-Authors: Veronique Vallet, Jean-daniel Horisberger, Bernard C. RossierAbstract:Epithelial sodium channel regulatory proteins identified by functional Expression Cloning. We describe here our current strategy for identifying and Cloning proteins involved in the regulation of the epithelial sodium channel (ENaC). We have set up a complementation functional assay in the Xenopus laevis oocyte Expression system. Using this assay, we have been able to identify a channel-activating protease (CAP-1) that can increase ENaC activity threefold. We propose a novel extracellular signal transduction pathway controlling ionic channels of the ENaC gene family that include genes involved in mechanotransduction (degenerins), in peptide-gated channels involved in neurotransmission (FaNaCh), in proton-gated channels involved in pH sensing (ASIC) or pain sensation (DRASIC).
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Expression Cloning of the epithelial sodium channel
Kidney International, 1995Co-Authors: Cecilia M. Canessa, Laurent Schild, Jean-daniel Horisberger, Bernard C. RossierAbstract:Complex organisms take sodium from the external environment to achieve and maintain extracellular volume and blood pressure. Many sodium transporter systems have evolved, and among them the epithelial sodium channel plays a fundamental role in determining the final amount of sodium reabsorbed. Epithelial sodium channels are present in the apical membrane of cells lining the distal segment of the nephron, urinary bladder, distal colon, and the airways. In the skin they are found in the sweat ducts and in certain animals (amphibians) they are also present in the epidermal cells. All these tissues absorb sodium via an amiloride-sensitive, highly selective Na + channel. This paper discusses recent efforts that have lead to the Cloning and the elucidation of the structure of the epithelial sodium channel.
Michael J. Briskin - One of the best experts on this subject based on the ideXlab platform.
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Expression Cloning of the STRL33/BONZO/TYMSTR Ligand Reveals Elements of CC, CXC, and CX3C Chemokines
Journal of immunology (Baltimore Md. : 1950), 2001Co-Authors: Alyson M. Wilbanks, Susan Carr Zondlo, Kristine E. Murphy, Simona Mak, Dulce Soler, Patricia Langdon, David P. Andrew, Michael J. BriskinAbstract:STRL33/BONZO/TYMSTR is an orphan chemokine and HIV/SIV coreceptor receptor that is expressed on activated T lymphocytes. We describe an Expression Cloning strategy whereby we isolated a novel chemokine, which we name CXCL16. CXCL16 is an alpha (CXC) chemokine but also has characteristics of CC chemokines and a structure similar to fractalkine (neurotactin) in having a transmembrane region and a chemokine domain suspended by a mucin-like stalk. A recombinant version of CXCL16 fails to mediate chemotaxis to all known chemokine receptor transfectants tested but does mediate robust chemotaxis, high affinity binding, and calcium mobilization to Bonzo receptor transfectants, indicating that this is a unique receptor ligand interaction. In vitro polarized T cell subsets including Th1, Th2, and Tr1 cells express functional Bonzo, suggesting Expression of this receptor in chronic inflammation, which we further verified by demonstration of CXCL16-mediated migration of tonsil-derived CD4(+) T lymphocytes. CXCL16 is expressed on the surface of APCs including subsets of CD19(+) B cells and CD14(+) monocyte/macrophages, and functional CXCL16 is also shed from macrophages. The combination of unique structural features of both Bonzo and CXCL16 suggest that this interaction may represent a new class of ligands for this receptor family. Additionally, this chemokine might play a unique dual role of attracting activated lymphocyte subsets during inflammation as well as facilitating immune responses via cell-cell contact.
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Expression Cloning of the strl33 bonzo tymstr ligand reveals elements of cc cxc and cx3c chemokines
Journal of Immunology, 2001Co-Authors: Alyson M. Wilbanks, Susan Carr Zondlo, Kristine E. Murphy, Simona Mak, Dulce Soler, Patricia Langdon, David P. Andrew, Michael J. BriskinAbstract:STRL33/BONZO/TYMSTR is an orphan chemokine and HIV/SIV coreceptor receptor that is expressed on activated T lymphocytes. We describe an Expression Cloning strategy whereby we isolated a novel chemokine, which we name CXCL16. CXCL16 is an alpha (CXC) chemokine but also has characteristics of CC chemokines and a structure similar to fractalkine (neurotactin) in having a transmembrane region and a chemokine domain suspended by a mucin-like stalk. A recombinant version of CXCL16 fails to mediate chemotaxis to all known chemokine receptor transfectants tested but does mediate robust chemotaxis, high affinity binding, and calcium mobilization to Bonzo receptor transfectants, indicating that this is a unique receptor ligand interaction. In vitro polarized T cell subsets including Th1, Th2, and Tr1 cells express functional Bonzo, suggesting Expression of this receptor in chronic inflammation, which we further verified by demonstration of CXCL16-mediated migration of tonsil-derived CD4(+) T lymphocytes. CXCL16 is expressed on the surface of APCs including subsets of CD19(+) B cells and CD14(+) monocyte/macrophages, and functional CXCL16 is also shed from macrophages. The combination of unique structural features of both Bonzo and CXCL16 suggest that this interaction may represent a new class of ligands for this receptor family. Additionally, this chemokine might play a unique dual role of attracting activated lymphocyte subsets during inflammation as well as facilitating immune responses via cell-cell contact.
Alyson M. Wilbanks - One of the best experts on this subject based on the ideXlab platform.
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Expression Cloning of the STRL33/BONZO/TYMSTR Ligand Reveals Elements of CC, CXC, and CX3C Chemokines
Journal of immunology (Baltimore Md. : 1950), 2001Co-Authors: Alyson M. Wilbanks, Susan Carr Zondlo, Kristine E. Murphy, Simona Mak, Dulce Soler, Patricia Langdon, David P. Andrew, Michael J. BriskinAbstract:STRL33/BONZO/TYMSTR is an orphan chemokine and HIV/SIV coreceptor receptor that is expressed on activated T lymphocytes. We describe an Expression Cloning strategy whereby we isolated a novel chemokine, which we name CXCL16. CXCL16 is an alpha (CXC) chemokine but also has characteristics of CC chemokines and a structure similar to fractalkine (neurotactin) in having a transmembrane region and a chemokine domain suspended by a mucin-like stalk. A recombinant version of CXCL16 fails to mediate chemotaxis to all known chemokine receptor transfectants tested but does mediate robust chemotaxis, high affinity binding, and calcium mobilization to Bonzo receptor transfectants, indicating that this is a unique receptor ligand interaction. In vitro polarized T cell subsets including Th1, Th2, and Tr1 cells express functional Bonzo, suggesting Expression of this receptor in chronic inflammation, which we further verified by demonstration of CXCL16-mediated migration of tonsil-derived CD4(+) T lymphocytes. CXCL16 is expressed on the surface of APCs including subsets of CD19(+) B cells and CD14(+) monocyte/macrophages, and functional CXCL16 is also shed from macrophages. The combination of unique structural features of both Bonzo and CXCL16 suggest that this interaction may represent a new class of ligands for this receptor family. Additionally, this chemokine might play a unique dual role of attracting activated lymphocyte subsets during inflammation as well as facilitating immune responses via cell-cell contact.
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Expression Cloning of the strl33 bonzo tymstr ligand reveals elements of cc cxc and cx3c chemokines
Journal of Immunology, 2001Co-Authors: Alyson M. Wilbanks, Susan Carr Zondlo, Kristine E. Murphy, Simona Mak, Dulce Soler, Patricia Langdon, David P. Andrew, Michael J. BriskinAbstract:STRL33/BONZO/TYMSTR is an orphan chemokine and HIV/SIV coreceptor receptor that is expressed on activated T lymphocytes. We describe an Expression Cloning strategy whereby we isolated a novel chemokine, which we name CXCL16. CXCL16 is an alpha (CXC) chemokine but also has characteristics of CC chemokines and a structure similar to fractalkine (neurotactin) in having a transmembrane region and a chemokine domain suspended by a mucin-like stalk. A recombinant version of CXCL16 fails to mediate chemotaxis to all known chemokine receptor transfectants tested but does mediate robust chemotaxis, high affinity binding, and calcium mobilization to Bonzo receptor transfectants, indicating that this is a unique receptor ligand interaction. In vitro polarized T cell subsets including Th1, Th2, and Tr1 cells express functional Bonzo, suggesting Expression of this receptor in chronic inflammation, which we further verified by demonstration of CXCL16-mediated migration of tonsil-derived CD4(+) T lymphocytes. CXCL16 is expressed on the surface of APCs including subsets of CD19(+) B cells and CD14(+) monocyte/macrophages, and functional CXCL16 is also shed from macrophages. The combination of unique structural features of both Bonzo and CXCL16 suggest that this interaction may represent a new class of ligands for this receptor family. Additionally, this chemokine might play a unique dual role of attracting activated lymphocyte subsets during inflammation as well as facilitating immune responses via cell-cell contact.
Garry P. Nolan - One of the best experts on this subject based on the ideXlab platform.
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Genetic selection and the lure of SIN
Nature Biotechnology, 2001Co-Authors: Garry P. NolanAbstract:A rapid method for exploring gene function uses alphavirus Expression Cloning.
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applications of retrovirus mediated Expression Cloning
Experimental Hematology, 1996Co-Authors: Mayumi Onishi, Garry P. Nolan, S Kinoshita, Yoshihiro Morikawa, Akira Shibuya, Joseph H Phillips, Lewis L Lanier, Daniel M Gorman, Atsushi Miyajima, Toshio KitamuraAbstract:: We have recently established a novel Expression Cloning system using retroviral vectors. The system is based on a high-efficiency packaging cell line, BOSC23, and a simplified retroviral vector, pBabeX, carrying no selection marker. cDNA libraries, constructed in the pBabeX vector, are transiently transfected into BOSC23 cells. The supernatant contains more than 3X10(6)/mL, which would cover large complexities of cDNA libraries. The retrovirus stock gave 100% infection efficiency in NIH3T3 cells and 5-40% infection efficiency in various hematopoietic cell lines. In contrast to the conventional Expression Cloning system, in which it is necessary to transfect cDNA libraries transiently into particular cell types such as COS cells, retrovirus-mediated Expression Cloning allows us to transduce cDNAs into a wide variety of cell types. This method therefore makes it possible to select cells expressing a cDNA of interest by various functional assays. When combined with polymerase chain reaction (PCR)-driven random mutagenesis, this system is also useful in searching for mutations of various molecules that will result in alterations of their functions.
