The Experts below are selected from a list of 5151 Experts worldwide ranked by ideXlab platform
Nicholas C. Brecha - One of the best experts on this subject based on the ideXlab platform.
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A Thy1-CFP DBA/2J mouse line with Cyan Fluorescent Protein expression in retinal ganglion cells
Visual neuroscience, 2009Co-Authors: Iona D. Raymond, Angela L. Pool, Alejandro Vila, Nicholas C. BrechaAbstract:A DBA/2J (D2) transgenic mouse line with Cyan Fluorescent Protein (CFP) reporter expression in ganglion cells was developed for the analysis of ganglion cells during progressive glaucoma. The Thy1- CFP D2 (CFP-D2) line was created by congenically breeding the D2 line, which develops pigmentary glaucoma, and the Thy1 -CFP line, which expresses CFP in ganglion cells. Microsatellite marker analysis of CFP-D2 progeny verified the genetic inclusion of the D2 isa and ipd loci. Specific mutations within these loci lead to dysfunctional melanosomal Proteins and glaucomatous phenotype in D2 mice. Polymerase chain reaction analysis confirmed the inclusion of the Thy1 -CFP transgene. CFP-Fluorescent ganglion cells, 6–20 μ m in diameter, were distributed in all retinal regions, CFP processes were throughout the inner plexiform layer, and CFP-Fluorescent axons were in the fiber layer and optic nerve head. Immunohistochemistry with antibodies to ganglion cell markers NF-L, NeuN, Brn3a, and SMI32 was used to confirm CFP expression in ganglion cells. Immunohistochemistry with antibodies to amacrine cell markers HPC-1 and ChAT was used to confirm weak CFP expression in cholinergic amacrine cells. CFP-D2 mice developed a glaucomatous phenotype, including iris disease, ganglion cell loss, attrition of the fiber layer, and elevated intraocular pressure. A CFP-D2 transgenic line with CFP-expressing ganglion cells was developed, which has (1) a predominantly D2 genetic background, (2) CFP-expressing ganglion cells, and (3) age-related progressive glaucoma. This line will be of value for experimental studies investigating ganglion cells and their axons in vivo and in vitro during the progressive development of glaucoma.
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a thy1 cfp dba 2j mouse line with Cyan Fluorescent Protein expression in retinal ganglion cells
Visual Neuroscience, 2009Co-Authors: Iona D. Raymond, Angela L. Pool, Alejandro Vila, Nicholas C. BrechaAbstract:A DBA/2J (D2) transgenic mouse line with Cyan Fluorescent Protein (CFP) reporter expression in ganglion cells was developed for the analysis of ganglion cells during progressive glaucoma. The Thy1- CFP D2 (CFP-D2) line was created by congenically breeding the D2 line, which develops pigmentary glaucoma, and the Thy1 -CFP line, which expresses CFP in ganglion cells. Microsatellite marker analysis of CFP-D2 progeny verified the genetic inclusion of the D2 isa and ipd loci. Specific mutations within these loci lead to dysfunctional melanosomal Proteins and glaucomatous phenotype in D2 mice. Polymerase chain reaction analysis confirmed the inclusion of the Thy1 -CFP transgene. CFP-Fluorescent ganglion cells, 6–20 μ m in diameter, were distributed in all retinal regions, CFP processes were throughout the inner plexiform layer, and CFP-Fluorescent axons were in the fiber layer and optic nerve head. Immunohistochemistry with antibodies to ganglion cell markers NF-L, NeuN, Brn3a, and SMI32 was used to confirm CFP expression in ganglion cells. Immunohistochemistry with antibodies to amacrine cell markers HPC-1 and ChAT was used to confirm weak CFP expression in cholinergic amacrine cells. CFP-D2 mice developed a glaucomatous phenotype, including iris disease, ganglion cell loss, attrition of the fiber layer, and elevated intraocular pressure. A CFP-D2 transgenic line with CFP-expressing ganglion cells was developed, which has (1) a predominantly D2 genetic background, (2) CFP-expressing ganglion cells, and (3) age-related progressive glaucoma. This line will be of value for experimental studies investigating ganglion cells and their axons in vivo and in vitro during the progressive development of glaucoma.
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Cyan Fluorescent Protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina
Molecular vision, 2008Co-Authors: Iona D. Raymond, Alejandro Vila, Uyen-chi N Huynh, Nicholas C. BrechaAbstract:Purpose To characterize Cyan Fluorescent Protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. Methods CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain Protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD(67)), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. Results CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP Fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP Fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 microm in diameter, and they had a density of 2636+/-347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD(67), GAT-1, and ChAT indicated that the small, weakly Fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. Conclusions The total number and density of CFP-Fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells.
Iona D. Raymond - One of the best experts on this subject based on the ideXlab platform.
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A Thy1-CFP DBA/2J mouse line with Cyan Fluorescent Protein expression in retinal ganglion cells
Visual neuroscience, 2009Co-Authors: Iona D. Raymond, Angela L. Pool, Alejandro Vila, Nicholas C. BrechaAbstract:A DBA/2J (D2) transgenic mouse line with Cyan Fluorescent Protein (CFP) reporter expression in ganglion cells was developed for the analysis of ganglion cells during progressive glaucoma. The Thy1- CFP D2 (CFP-D2) line was created by congenically breeding the D2 line, which develops pigmentary glaucoma, and the Thy1 -CFP line, which expresses CFP in ganglion cells. Microsatellite marker analysis of CFP-D2 progeny verified the genetic inclusion of the D2 isa and ipd loci. Specific mutations within these loci lead to dysfunctional melanosomal Proteins and glaucomatous phenotype in D2 mice. Polymerase chain reaction analysis confirmed the inclusion of the Thy1 -CFP transgene. CFP-Fluorescent ganglion cells, 6–20 μ m in diameter, were distributed in all retinal regions, CFP processes were throughout the inner plexiform layer, and CFP-Fluorescent axons were in the fiber layer and optic nerve head. Immunohistochemistry with antibodies to ganglion cell markers NF-L, NeuN, Brn3a, and SMI32 was used to confirm CFP expression in ganglion cells. Immunohistochemistry with antibodies to amacrine cell markers HPC-1 and ChAT was used to confirm weak CFP expression in cholinergic amacrine cells. CFP-D2 mice developed a glaucomatous phenotype, including iris disease, ganglion cell loss, attrition of the fiber layer, and elevated intraocular pressure. A CFP-D2 transgenic line with CFP-expressing ganglion cells was developed, which has (1) a predominantly D2 genetic background, (2) CFP-expressing ganglion cells, and (3) age-related progressive glaucoma. This line will be of value for experimental studies investigating ganglion cells and their axons in vivo and in vitro during the progressive development of glaucoma.
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a thy1 cfp dba 2j mouse line with Cyan Fluorescent Protein expression in retinal ganglion cells
Visual Neuroscience, 2009Co-Authors: Iona D. Raymond, Angela L. Pool, Alejandro Vila, Nicholas C. BrechaAbstract:A DBA/2J (D2) transgenic mouse line with Cyan Fluorescent Protein (CFP) reporter expression in ganglion cells was developed for the analysis of ganglion cells during progressive glaucoma. The Thy1- CFP D2 (CFP-D2) line was created by congenically breeding the D2 line, which develops pigmentary glaucoma, and the Thy1 -CFP line, which expresses CFP in ganglion cells. Microsatellite marker analysis of CFP-D2 progeny verified the genetic inclusion of the D2 isa and ipd loci. Specific mutations within these loci lead to dysfunctional melanosomal Proteins and glaucomatous phenotype in D2 mice. Polymerase chain reaction analysis confirmed the inclusion of the Thy1 -CFP transgene. CFP-Fluorescent ganglion cells, 6–20 μ m in diameter, were distributed in all retinal regions, CFP processes were throughout the inner plexiform layer, and CFP-Fluorescent axons were in the fiber layer and optic nerve head. Immunohistochemistry with antibodies to ganglion cell markers NF-L, NeuN, Brn3a, and SMI32 was used to confirm CFP expression in ganglion cells. Immunohistochemistry with antibodies to amacrine cell markers HPC-1 and ChAT was used to confirm weak CFP expression in cholinergic amacrine cells. CFP-D2 mice developed a glaucomatous phenotype, including iris disease, ganglion cell loss, attrition of the fiber layer, and elevated intraocular pressure. A CFP-D2 transgenic line with CFP-expressing ganglion cells was developed, which has (1) a predominantly D2 genetic background, (2) CFP-expressing ganglion cells, and (3) age-related progressive glaucoma. This line will be of value for experimental studies investigating ganglion cells and their axons in vivo and in vitro during the progressive development of glaucoma.
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Cyan Fluorescent Protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina
Molecular vision, 2008Co-Authors: Iona D. Raymond, Alejandro Vila, Uyen-chi N Huynh, Nicholas C. BrechaAbstract:Purpose To characterize Cyan Fluorescent Protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. Methods CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain Protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD(67)), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. Results CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP Fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP Fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 microm in diameter, and they had a density of 2636+/-347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD(67), GAT-1, and ChAT indicated that the small, weakly Fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. Conclusions The total number and density of CFP-Fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells.
Alejandro Vila - One of the best experts on this subject based on the ideXlab platform.
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A Thy1-CFP DBA/2J mouse line with Cyan Fluorescent Protein expression in retinal ganglion cells
Visual neuroscience, 2009Co-Authors: Iona D. Raymond, Angela L. Pool, Alejandro Vila, Nicholas C. BrechaAbstract:A DBA/2J (D2) transgenic mouse line with Cyan Fluorescent Protein (CFP) reporter expression in ganglion cells was developed for the analysis of ganglion cells during progressive glaucoma. The Thy1- CFP D2 (CFP-D2) line was created by congenically breeding the D2 line, which develops pigmentary glaucoma, and the Thy1 -CFP line, which expresses CFP in ganglion cells. Microsatellite marker analysis of CFP-D2 progeny verified the genetic inclusion of the D2 isa and ipd loci. Specific mutations within these loci lead to dysfunctional melanosomal Proteins and glaucomatous phenotype in D2 mice. Polymerase chain reaction analysis confirmed the inclusion of the Thy1 -CFP transgene. CFP-Fluorescent ganglion cells, 6–20 μ m in diameter, were distributed in all retinal regions, CFP processes were throughout the inner plexiform layer, and CFP-Fluorescent axons were in the fiber layer and optic nerve head. Immunohistochemistry with antibodies to ganglion cell markers NF-L, NeuN, Brn3a, and SMI32 was used to confirm CFP expression in ganglion cells. Immunohistochemistry with antibodies to amacrine cell markers HPC-1 and ChAT was used to confirm weak CFP expression in cholinergic amacrine cells. CFP-D2 mice developed a glaucomatous phenotype, including iris disease, ganglion cell loss, attrition of the fiber layer, and elevated intraocular pressure. A CFP-D2 transgenic line with CFP-expressing ganglion cells was developed, which has (1) a predominantly D2 genetic background, (2) CFP-expressing ganglion cells, and (3) age-related progressive glaucoma. This line will be of value for experimental studies investigating ganglion cells and their axons in vivo and in vitro during the progressive development of glaucoma.
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a thy1 cfp dba 2j mouse line with Cyan Fluorescent Protein expression in retinal ganglion cells
Visual Neuroscience, 2009Co-Authors: Iona D. Raymond, Angela L. Pool, Alejandro Vila, Nicholas C. BrechaAbstract:A DBA/2J (D2) transgenic mouse line with Cyan Fluorescent Protein (CFP) reporter expression in ganglion cells was developed for the analysis of ganglion cells during progressive glaucoma. The Thy1- CFP D2 (CFP-D2) line was created by congenically breeding the D2 line, which develops pigmentary glaucoma, and the Thy1 -CFP line, which expresses CFP in ganglion cells. Microsatellite marker analysis of CFP-D2 progeny verified the genetic inclusion of the D2 isa and ipd loci. Specific mutations within these loci lead to dysfunctional melanosomal Proteins and glaucomatous phenotype in D2 mice. Polymerase chain reaction analysis confirmed the inclusion of the Thy1 -CFP transgene. CFP-Fluorescent ganglion cells, 6–20 μ m in diameter, were distributed in all retinal regions, CFP processes were throughout the inner plexiform layer, and CFP-Fluorescent axons were in the fiber layer and optic nerve head. Immunohistochemistry with antibodies to ganglion cell markers NF-L, NeuN, Brn3a, and SMI32 was used to confirm CFP expression in ganglion cells. Immunohistochemistry with antibodies to amacrine cell markers HPC-1 and ChAT was used to confirm weak CFP expression in cholinergic amacrine cells. CFP-D2 mice developed a glaucomatous phenotype, including iris disease, ganglion cell loss, attrition of the fiber layer, and elevated intraocular pressure. A CFP-D2 transgenic line with CFP-expressing ganglion cells was developed, which has (1) a predominantly D2 genetic background, (2) CFP-expressing ganglion cells, and (3) age-related progressive glaucoma. This line will be of value for experimental studies investigating ganglion cells and their axons in vivo and in vitro during the progressive development of glaucoma.
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Cyan Fluorescent Protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina
Molecular vision, 2008Co-Authors: Iona D. Raymond, Alejandro Vila, Uyen-chi N Huynh, Nicholas C. BrechaAbstract:Purpose To characterize Cyan Fluorescent Protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. Methods CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain Protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD(67)), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. Results CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP Fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP Fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 microm in diameter, and they had a density of 2636+/-347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD(67), GAT-1, and ChAT indicated that the small, weakly Fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. Conclusions The total number and density of CFP-Fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells.
Michael Bouvet - One of the best experts on this subject based on the ideXlab platform.
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The Cyan Fluorescent Protein Nude Mouse as a Host for Multicolor-coded Imaging Models of Primary and Metastatic Tumor Microenvironments
Anticancer research, 2012Co-Authors: Atsushi Suetsugu, Jose Reynoso, Michael Bouvet, Mohamed K. Hassanein, Yosuke Osawa, Masahito Nagaki, Hisataka Moriwaki, Shigetoyo Saji, Robert M. HoffmanAbstract:The tumor microenvironment (TME) has an important influence on tumor progression. For example, we have discovered that passenger stromal cells are necessary for metastasis. In this report, we describe six different Cyan Fluorescent Protein (CFP) multicolor TME nude mouse models. The six different implantation models were used to image the TME using multiple colors of Fluorescent Proteins: I) Red Fluorescent Protein (RFP)- or green Fluorescent Protein (GFP)-expressing HCT-116 human colon cancer cells were implanted subcutaneously in the CFP-expressing nude mice. CFP stromal elements from the subcutaneous TME were visualized interacting with the RFP- or GFP-expressing tumors. II) RFP-expressing HCT-116 cells were transplanted into the spleen of CFP nude mice, and experimental metastases were then formed in the liver. CFP stromal elements from the liver TME were visualized interacting with the RFP-expressing tumor. III) RFP-expressing HCT-116 cancer cells were transplanted in the tail vein of CFP- expressing nude mice, forming experimental metastases in the lung. CFP stromal elements from the lung were visualized interacting with the RFP-expressing tumor. IV) In order to visualize two different tumors in the TME, GFP-expressing and RFP-expressing HCT-116 cancer cells were co-implanted subcutaneously in CFP-expressing nude mice. A 3-color TME was formed subcutaneously in the CFP mouse, and CFP stromal elements were visualized interacting with the RFP- and GFP-expressing tumors. V) In order to have two different colors of stromal elements, GFP-expressing HCT-116 cells were initially injected subcutaneously in RFP-expressing nude mice. After 14 days, the tumor, which consisted of GFP cancer cells and RFP stromal cells derived from the RFP nude mouse, was harvested and transplanted into the CFP nude mouse. CFP stromal cells invaded the growing transplanted tumor containing GFP cancer cells and RFP stroma. VI) Mouse mammary tumor (MMT) cells expressing GFP in the nucleus and RFP in the cytoplasm were implanted in the spleen of a CFP nude mouse. Cancer cells were imaged in the liver 3 days
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development of the transgenic Cyan Fluorescent Protein cfp expressing nude mouse for technicolor cancer imaging
Journal of Cellular Biochemistry, 2009Co-Authors: Hop Tran S Cao, Meng Yang, Jose Reynoso, Robert M. Hoffman, Hiroaki Kimura, Sharmeela Kaushal, Cynthia S Snyder, Michael BouvetAbstract:A major goal for in vivo biology is to develop models which can express multiple colors of Fluorescent Proteins in order to image multiple processes simultaneously in real time. Towards this goal, the CyanoFluorescent Protein (CFP) nude mouse was developed by crossing non-transgenic nude mice with the transgenic CK/ECFP mouse in which the β-actin promoter drives expression of CFP in almost all tissues. In crosses between nu/nu CFP male mice and nu/+ CFP female mice, approximately 50% of the embryos fluoresced blue. In the CFP nude mice, the pancreas and reproductive organs displayed the strongest Fluorescent signals of all internal organs which vary in intensity. Orthotopic implantation of XPA-1 human pancreatic cancer cells expressing red Fluorescent Protein (RFP) or green Fluorescent Protein (GFP) in the nucleus and RFP in the cytoplasm was performed in female nude CFP mice. Color-coded fluorescence imaging of these human pancreatic cancer cells implanted into bright flue Fluorescent pancreas of the CFP nude mouse afforded novel insight into the interaction of the pancreatic tumor and the normal pancreas, in particular the strong desmoplastic reaction of the tumor. The naturally enhanced blue fluorescence of the pancreas in the CFP mouse serves as an ideal background for color-coded imaging of the interaction of implanted cancer cells and the host. The CFP nude mouse will provide unique understanding of the critical interplay between the cancer cells and their microenvironment.
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development of the transgenic Cyan Fluorescent Protein cfp expressing nude mouse for technicolor cancer imaging
Journal of Cellular Biochemistry, 2009Co-Authors: Hop Tran S Cao, Meng Yang, Jose Reynoso, Robert M. Hoffman, Hiroaki Kimura, Sharmeela Kaushal, Cynthia S Snyder, Michael BouvetAbstract:A major goal for in vivo biology is to develop models which can express multiple colors of Fluorescent Proteins in order to image many processes simultaneously in real time. Towards this goal, the Cyan Fluorescent Protein (CFP) nude mouse was developed by crossing non-transgenic nude mice with the transgenic CK/ECFP mouse in which the beta-actin promoter drives expression of CFP in almost all tissues. In crosses between nu/nu CFP male mice and nu/+ CFP female mice, approximately 50% of the embryos fluoresced blue. In the CFP nude mice, the pancreas and reproductive organs displayed the strongest Fluorescent signals of all internal organs which vary in intensity. Orthotopic implantation of XPA-1 human pancreatic cancer cells expressing red Fluorescent Protein (RFP); or green Fluorescent Protein (GFP) in the nucleus and RFP in the cytoplasm, was performed in female nude CFP mice. Color-coded fluorescence imaging of these human pancreatic cancer cells implanted into the bright blue Fluorescent pancreas of the CFP nude mouse afforded novel insight into the interaction of the pancreatic tumor and the normal pancreas, in particular the strong desmoplastic reaction of the tumor. The naturally enhanced blue fluorescence of the pancreas in the CFP mouse serves as an ideal background for color-coded imaging of the interaction of implanted cancer cells and the host. The CFP nude mouse will provide unique understanding of the critical interplay between the cancer cells and their microenvironment.
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The Cyan Fluorescent Protein (CFP) Transgenic Mouse as a Model for Imaging Pancreatic Exocrine Cells
JOP : Journal of the pancreas, 2009Co-Authors: Hop S. Tran Cao, Jose Reynoso, Robert M. Hoffman, Hiroaki Kimura, Sharmeela Kaushal, Cynthia S Snyder, Michael BouvetAbstract:Context The use of Fluorescent Proteins for in vivo imaging has opened many new areas of research. Among the important advances in the field have been the development of transgenic mice expressing various Fluorescent Proteins. Objective To report whole-body and organ-specific fluorescence imaging to characterize the transgenic Cyan Fluorescent Protein mouse. Design Mice were imaged using two devices. Brightfield images were obtained with the OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan). Fluorescence imaging was performed under the Cyan Fluorescent Protein filter using the iBox Small Animal Imaging System (UVP, Upland, CA, USA). Intervention All animals were sacrificed immediately before imaging. They were imaged before and throughout multiple steps of a complete necropsy. Harvested organs were also imaged with both devices. Selected organs were then frozen and processed for histology, fluorescence microscopy, and HE most notably, the pancreas stands out from the rest of the gastrointestinal tract, displaying the strongest fluorescence of all organs in the mouse. Fluorescence microscopy demonstrated that the Cyan Fluorescent Protein fluorescence resided in the acinar cells of the pancreas and not the islet cells. Conclusions The Cyan Fluorescent Protein mouse should lead to a deeper understanding of pancreatic function and pathology, including cancer.
Marie Erard - One of the best experts on this subject based on the ideXlab platform.
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The enhanced Cyan Fluorescent Protein: a sensitive pH sensor for fluorescence lifetime imaging
Analytical and Bioanalytical Chemistry, 2013Co-Authors: Sandrine Poëa-guyon, Fabienne Merola, Hélène Pasquier, Nicolas Morel, Marie ErardAbstract:pH is an important parameter that affects many functions of live cells, from Protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive Fluorescent Proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow Fluorescent Proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced Cyan Fluorescent Protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.
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Cyan Fluorescent Protein carries a constitutive mutation that prevents its dimerization.
Biochemistry, 2010Co-Authors: Agathe Espagne, Marie Erard, Hélène Pasquier, Bernard I Levy, Karine Madiona, Valerie Derrien, Gabriella Jonasson, Ronald Melki, Fabienne MerolaAbstract:The tendency of GFP-like Fluorescent Proteins to dimerize in vitro is a permanent concern as it may lead to artifacts in FRET imaging applications. However, we have found recently that CFP and YFP (the couple of GFP variants mostly used in FRET studies) show no trace of association in the cytosol of living cells up to millimolar concentrations. In this study, we investigated the oligomerization properties of purified CFP, by fluorescence anisotropy and sedimentation velocity. Surprisingly, we found that CFP has a much weaker homoaffinity than other Fluorescent Proteins (K(d) ≥ 3 × 10(-3) M), and that this is due to the constitutive N146I mutation, originally introduced into CFP to improve its brightness.
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Are the Fluorescent properties of the Cyan Fluorescent Protein sensitive to conditions of oxidative stress
Photochemistry and photobiology, 2009Co-Authors: Luis Alvarez, Fabienne Merola, Filippo Rusconi, Chantal Houée Levin, Tania Bizouarn, Hélène Pasquier, Laura Baciou, Marie ErardAbstract:The modifications induced by reactive oxygen species (ROS) on Fluorescent Proteins (FPs) may have important implications for live cell fluorescence imaging. Using quantitative γ-radiolysis, we have studied the ROS-induced biochemical and photophysical perturbations on recombinant Cyan Fluorescent Protein (CFP). After oxidation by the ˙OH radical, the Protein displays a modified RP-HPLC elution profile, while the CFP fluorescence undergoes pronounced decreases in intensity and lifetime, without changes in its excitation and emission spectra. Meanwhile, the Forster resonant energy transfer (FRET) between the single W57 and the chromophore remains unperturbed. These results rule out a direct oxidation of the CFP chromophore and of W57 as well as major changes in the Protein 3D structure, but show that new Fluorescent forms associated to a higher level of dynamic quenching have been generated. Thus, strict in situ controls are required when CFP is to be used for FRET studies in situations of oxidative activity, or under strong illumination.
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Effects Of Reactive Oxygen Species On Cyan Fluorescent Protein
Biophysical Journal, 2009Co-Authors: Luis Alvarez, Fabienne Merola, Chantal Houée-levin, Filippo Rusconi, Marie ErardAbstract:Recent advances in microscopy techniques and the development of many different colour variants of the GFP family of Proteins allow for a more direct analysis of Protein function in live cells. The advantage of genetically coded Fluorescent Protein probes is often offset by their photophysical properties which usually make them very sensitive to cellular environmental changes.Among these, reactive oxygen species (ROS) are an essential part of key cellular processes (mitochondria respiration, apoptosis) and are also involved in the pathogenesis of various diseases (cancer, atherosclerosis, Alzheimer's disease, etc.). We studied the effects of ROS on the Cyan Fluorescent Protein (CFP) in vitro, as this Fluorescent Protein is currently one of the most widely used in Protein interaction studies. We studied the fluorescence and absorption changes of recombinant CFP Protein using γ-radiolysis for ROS production. γ-radiolysis ROS production allowed us to have an exact control over the radical concentrations delivered unto the Protein samples. The radicals used in this study were OH°, O2− or a mixture of OH° and O2−. We also determined the chemical modifications that take place upon ROS induced Protein oxidation by mass spectrometry. We show that the targets of oxidation are one tyrosine and four methionine residues located on the Protein surface and that the chromophore is not likely modified through these oxidation processes.
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Complex fluorescence of the Cyan Fluorescent Protein: comparisons with the H148D variant and consequences for quantitative cell imaging.
Biochemistry, 2008Co-Authors: Aude Villoing, Luis Alvarez, Fabienne Merola, Marie Erard, Germain Vallverdu, Regis Grailhe, Myriam Ridhoir, Bertrand Cinquin, Pascal Pernot, Hélène PasquierAbstract:We have studied the fluorescence decays of the purified enhanced Cyan Fluorescent Protein (ECFP, with chromophore sequence Thr-Trp-Gly) and of its variant carrying the single H148D mutation characteristic of the brighter form Cerulean. Both Proteins exhibit highly complex fluorescence decays showing strong temperature and pH dependences. At neutral pH, the H148D mutation leads (i) to a general increase in all fluorescence lifetimes and (ii) to the disappearance of a subpopulation, estimated to be more than 25% of the total ECFP molecules, characterized by a quenched and red-shifted fluorescence. The fluorescence lifetime distributions of ECFP and its H148D mutant remain otherwise very similar, indicating a high degree of structural and dynamic similarity of the two Proteins in their major form. From thermodynamic analysis, we conclude that the multiexponential decay of ECFP cannot be simply ascribed, as is generally admitted, to the slow conformational exchange characterized by NMR and X-ray crystallographic studies [Seifert, M. H., et al. (2002) J. Am. Chem. Soc. 124, 7932-7942; Bae, J. H., et al. (2003) J. Mol. Biol. 328, 1071-1081]. Parallel measurements in living cells show that these fluorescence properties in neutral solution are very similar to those of cytosolic ECFP.
