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Mammer Bouhou Widad - One of the best experts on this subject based on the ideXlab platform.
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Optimizando la purificación de ornitina transcarbamilasa humana (OTC) producida recombinantemente
'Universitat Politecnica de Valencia', 2019Co-Authors: Mammer Bouhou WidadAbstract:[ES] La ornitina transcarbamilasa (OTC) es un enzima mitocondrial que cataliza el segundo paso del ciclo de la urea, la reacción entre carbamil fosfato y ornitina para formar citrulina y fosfato. Esta ruta metabólica convierte el amonio derivado del catabolismo proteico, que es altamente tóxico, en urea, molécula inocua que se elimina en la orina. [1] Mutaciones en el gen para OTC (Xp21.1) causan la deficiencia de OTC (OTCD), el error más frecuente del ciclo de la urea. Los síntomas de OTCD se deben a la toxicidad del amonio acumulado y son variables dependiendo de la mutación presente en hemicigosis en los pacientes. Su forma más severa, neonatal temprana, generalmente afecta a varones, manifestándose con irritabilidad, falta de energía y apetito, letargia, convulsiones y coma. Aunque tratable, la OTCD puede conducir a la muerte o al retraso del desarrollo, discapacidad intelectual y daño hepático. Con menos frecuencia, la OTCD se manifiesta tardíamente, generalmente en presentaciones menos graves, tanto en hombres como en mujeres. Sus síntomas pueden incluir episodios de delirio, nivel reducido de conciencia, dolores de cabeza, vómitos, convulsiones y pérdidas prolongadas de conciencia en situaciones de estrés catabólico (parto, accidentes, cirugía, etc). [1] Para entender el funcionamiento de la OTC, establecer correlaciones genotipo-fenotipo y determinar el impacto de las mutaciones descritas (la mayoría de ellas sustituciones de aminoácido [2]) sobre la estabilidad, la actividad y los parámetros cinéticos, es clave producir OTC humana recombinante. En el laboratorio esto se consigue utilizando un sistema de expresión en E. coli [3] en el que la forma madura de OTC humana (es decir, sin secuencia señal de internalización mitocondrial) se coexpresa en presencia de chaperonas sobreexpresadas que favorecen su correcto plegamiento [3]. El procedimiento de purificación empleado (modificado de [4]) es laborioso, implica cinco etapas de purificación, incluyendo precipitación con sulfato amónico, diálisis y cromatografia de intercambio iónico. La purificación por afinidad utilizando como ligando N-5-fosfonacetil-L-ornitina (PALO) [5] es una alternativa muy útil, pero requiere sintetizar PALO, pues este compuesto no está disponible comercialmente [5]. Por ello planteamos como objetivo de este trabajo optimizar la producción de OTC humana mediante la introducción de una etiqueta de polihistidinas que facilite la purificación por cromatografía de afinidad metálica con resina comercial. He utilizado dos vectores de expresión y sistemas de clonación independientes de ligasa: pLIC-SGC1 (ver en Addgene, https://www.Addgene.org/39187/) para añadir una etiqueta de polihistidinas en el extremo N-terminal; y popinE/In-fusion [6], para añadir la etiqueta en el extremo C-terminal. Estas dos alternativas se plantearon por el hecho de desconocer el impacto que la extensión de polihistidinas pudiera tener sobre el plegamiento o la actividad enzimática. A partir de estas dos construcciones hemos producido las formas correspondientes de OTC humana recombinante, que hemos purificado utilizando columnas de níquel y un sistema de cromatografía automatizada rápida de media presión (FLPC). Habiendo comprobado que ambas formas con etiquetas de polihistidinas presentan actividad catalítica, el objetivo es ahora comparar estas actividades así como los parámetros cinéticos y la estabilidad enzimática con los del enzima carente de etiquetas que se venía produciendo previamente en el laboratorio, con el objetivo último de establecer un protocolo alternativo de obtención de OTC humana, que permita abordar el estudio facil de nuevos mutantes descritos en pacientes con OTCD tras su producción y purificación rápida. [1]. Häberle J et al. Suggested guidelines for the diagnosis and management of urea cycle disord[EN] Ornithine transcarbamylase (OTC) is a mitochondrial enzyme that catalyzes the second step of the urea cycle, the reaction between carbamyl phosphate and ornithine to form citrulline and phosphate. This metabolic pathway converts ammonium derived from protein catabolism, which is highly toxic, into urea, an innocuous molecule that is eliminated in the urine. [1] Mutations in the gene for OTC (Xp21.1) cause OTC deficiency (OTCD), the most frequent error of the urea cycle. The symptoms of OTCD are due to the toxicity of the accumulated ammonium and are variable depending on the mutation present in hemizigosis in patients. It s more severe, early neonatal form usually affects males, festering with irritability, lack of energy and appetite, lethargy, seizures and coma. Although treatable, OTCD can lead to death or developmental delay, intellectual disability and liver damage. Less frequently, OTCD manifests itself late, usually in less severe presentations, in both men and women. Its symptoms may include episodes of delirium, reduced level of consciousness, headaches, vomiting, seizures and prolonged loss of consciousness in situations of catabolic stress (childbirth, accidents, surgery, etc). [1] To understand the functioning of OTC, establish genotype-phenotype correlations and determine the impact of the described mutations (most of them amino acid substitutions [2]) on stability, activity and kinetic parameters, it is key to produce recombinant human OTC . In the laboratory this is achieved using an expression system in E. coli [3] in which the mature form of human OTC (ie, without a mitochondrial internalization signal sequence) is co-expressed in the presence of overexpressed chaperones that favor its correct folding [3]. The purification procedure employed (modified from [4]) is laborious, involving five purification steps, including ammonium sulfate precipitation, dialysis and ion exchange chromatography. Affinity purification using as ligand N-5-phosphonacetyl-L-ornithine (PALO) [5] is a very useful alternative, but requires synthesizing PALO, as this compound is not commercially available [5]. Therefore, we propose as the goal of this work to optimize the production of human OTC by introducing a polyhistidine label that facilitates the purification by metal affinity chromatography with commercial resin. I have used two expression vectors and ligase-independent cloning systems: pLIC-SGC1 (see Addgene, https://www.Addgene.org/39187/) to add a polyhistidine tag at the N-terminus; and popinE / In-fusion [6], to add the label at the C-terminal end. These two alternatives were raised by the fact of not knowing the impact that the extension of polyhistidines could have on the folding or the enzymatic activity. From these two constructs we have produced the corresponding forms of recombinant human OTC, which we have purified using nickel columns and a rapid medium pressure automated chromatography system (FLPC). Having verified that both forms with polyhistidine labels present catalytic activity, the objective is now to compare these activities as well as the kinetic parameters and the enzymatic stability with those of the enzyme lacking labels that had previously been produced in the laboratory, with the ultimate goal of establish an alternative protocol for obtaining human OTC, which allows easy study of new mutants described in patients with OTCD after its production and rapid purification. [1]. Häberle J et al. Suggested guidelines for the diagnosis and management of urea cycle disorders: first revision. J Inherit Metab Dis. 2019. doi10.1002/jimd.12100. [2]. Arranz JA, Riudor E, Marco-Marín C, Rubio V. Estimation of the total number of disease-causing mutations in ornithine transcarbamylase (OTC) deficiency. Value of the OTC structure in predicting a mutation pathogenic potential. J Inherit Metab Dis. 2007;30:217-26. [3]. Morizono H et al. Expression, pMammer Bouhou, W. (2019). Optimizando la purificación de ornitina transcarbamilasa humana (OTC) producida recombinantemente. http://hdl.handle.net/10251/127721TFG
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Optimizando la purificación de ornitina transcarbamilasa humana (OTC) producida recombinantemente
'Universitat Politecnica de Valencia', 2019Co-Authors: Mammer Bouhou WidadAbstract:[ES] La ornitina transcarbamilasa (OTC) es un enzima mitocondrial que cataliza el segundo paso del ciclo de la urea, la reacción entre carbamil fosfato y ornitina para formar citrulina y fosfato. Esta ruta metabólica convierte el amonio derivado del catabolismo proteico, que es altamente tóxico, en urea, molécula inocua que se elimina en la orina. [1] Mutaciones en el gen para OTC (Xp21.1) causan la deficiencia de OTC (OTCD), el error más frecuente del ciclo de la urea. Los síntomas de OTCD se deben a la toxicidad del amonio acumulado y son variables dependiendo de la mutación presente en hemicigosis en los pacientes. Su forma más severa, neonatal temprana, generalmente afecta a varones, manifestándose con irritabilidad, falta de energía y apetito, letargia, convulsiones y coma. Aunque tratable, la OTCD puede conducir a la muerte o al retraso del desarrollo, discapacidad intelectual y daño hepático. Con menos frecuencia, la OTCD se manifiesta tardíamente, generalmente en presentaciones menos graves, tanto en hombres como en mujeres. Sus síntomas pueden incluir episodios de delirio, nivel reducido de conciencia, dolores de cabeza, vómitos, convulsiones y pérdidas prolongadas de conciencia en situaciones de estrés catabólico (parto, accidentes, cirugía, etc). [1] Para entender el funcionamiento de la OTC, establecer correlaciones genotipo-fenotipo y determinar el impacto de las mutaciones descritas (la mayoría de ellas sustituciones de aminoácido [2]) sobre la estabilidad, la actividad y los parámetros cinéticos, es clave producir OTC humana recombinante. En el laboratorio esto se consigue utilizando un sistema de expresión en E. coli [3] en el que la forma madura de OTC humana (es decir, sin secuencia señal de internalización mitocondrial) se coexpresa en presencia de chaperonas sobreexpresadas que favorecen su correcto plegamiento [3]. El procedimiento de purificación empleado (modificado de [4]) es laborioso, implica cinco etapas de purificación, incluyendo precipitación con sulfato amónico, diálisis y cromatografia de intercambio iónico. La purificación por afinidad utilizando como ligando N-5-fosfonacetil-L-ornitina (PALO) [5] es una alternativa muy útil, pero requiere sintetizar PALO, pues este compuesto no está disponible comercialmente [5]. Por ello planteamos como objetivo de este trabajo optimizar la producción de OTC humana mediante la introducción de una etiqueta de polihistidinas que facilite la purificación por cromatografía de afinidad metálica con resina comercial. He utilizado dos vectores de expresión y sistemas de clonación independientes de ligasa: pLIC-SGC1 (ver en Addgene, https://www.Addgene.org/39187/) para añadir una etiqueta de polihistidinas en el extremo N-terminal; y popinE/In-fusion [6], para añadir la etiqueta en el extremo C-terminal. Estas dos alternativas se plantearon por el hecho de desconocer el impacto que la extensión de polihistidinas pudiera tener sobre el plegamiento o la actividad enzimática. A partir de estas dos construcciones hemos producido las formas correspondientes de OTC humana recombinante, que hemos purificado utilizando columnas de níquel y un sistema de cromatografía automatizada rápida de media presión (FLPC). Habiendo comprobado que ambas formas con etiquetas de polihistidinas presentan actividad catalítica, el objetivo es ahora comparar estas actividades así como los parámetros cinéticos y la estabilidad enzimática con los del enzima carente de etiquetas que se venía produciendo previamente en el laboratorio, con el objetivo último de establecer un protocolo alternativo de obtención de OTC humana, que permita abordar el estudio facil de nuevos mutantes descritos en pacientes con OTCD tras su producción y purificación rápida. [1]. Häberle J et al. Suggested guidelines for the diagnosis and management of urea cycle disord[EN] Ornithine transcarbamylase (OTC) is a mitochondrial enzyme that catalyzes the second step of the urea cycle, the reaction between carbamyl phosphate and ornithine to form citrulline and phosphate. This metabolic pathway converts ammonium derived from protein catabolism, which is highly toxic, into urea, an innocuous molecule that is eliminated in the urine. [1] Mutations in the gene for OTC (Xp21.1) cause OTC deficiency (OTCD), the most frequent error of the urea cycle. The symptoms of OTCD are due to the toxicity of the accumulated ammonium and are variable depending on the mutation present in hemizigosis in patients. It s more severe, early neonatal form usually affects males, festering with irritability, lack of energy and appetite, lethargy, seizures and coma. Although treatable, OTCD can lead to death or developmental delay, intellectual disability and liver damage. Less frequently, OTCD manifests itself late, usually in less severe presentations, in both men and women. Its symptoms may include episodes of delirium, reduced level of consciousness, headaches, vomiting, seizures and prolonged loss of consciousness in situations of catabolic stress (childbirth, accidents, surgery, etc). [1] To understand the functioning of OTC, establish genotype-phenotype correlations and determine the impact of the described mutations (most of them amino acid substitutions [2]) on stability, activity and kinetic parameters, it is key to produce recombinant human OTC . In the laboratory this is achieved using an expression system in E. coli [3] in which the mature form of human OTC (ie, without a mitochondrial internalization signal sequence) is co-expressed in the presence of overexpressed chaperones that favor its correct folding [3]. The purification procedure employed (modified from [4]) is laborious, involving five purification steps, including ammonium sulfate precipitation, dialysis and ion exchange chromatography. Affinity purification using as ligand N-5-phosphonacetyl-L-ornithine (PALO) [5]) is laborious, involving five purification steps, including ammonium sulfate precipitation, dialysis and ion exchange chromatography. Affinity purification using as ligand N-5-phosphonacetyl-L-ornithine (PALO) [5 is a very useful alternative, but requires synthesizing PALO, as this compound is not commercially available [5]. Therefore, we propose as the goal of this work to optimize the production of human OTC by introducing a polyhistidine label that facilitates the purification by metal affinity chromatography with commercial resin. I have used two expression vectors and ligase-independent cloning systems: pLIC-SGC1 (see Addgene, https://www.Addgene.org/39187/) to add a polyhistidine tag at the N-terminus; and popinE / In-fusion [6], to add the label at the C-terminal end. These two alternatives were raised by the fact of not knowing the impact that the extension of polyhistidines could have on the folding or the enzymatic activity. From these two constructs we have produced the corresponding forms of recombinant human OTC, which we have purified using nickel columns and a rapid medium pressure automated chromatography system (FLPC). Having verified that both forms with polyhistidine labels present catalytic activity, the objective is now to compare these activities as well as the kinetic parameters and the enzymatic stability with those of the enzyme lacking labels that had previously been produced in the laboratory, with the ultimate goal of establish an alternative protocol for obtaining human OTC, which allows easy study of new mutants described in patients with OTCD after its production and rapid purification. [1]. Häberle J et al. Suggested guidelines for the diagnosis and management of urea cycle disorders: first revision. J Inherit Metab Dis. 2019. doi10.1002/jimd.12100. [2]. Arranz JA, Riudor E, Marco-Marín C, Rubio V. Estimation of the total number of disease-causing mutations in ornithine transcarbamylase (OTC) deficiency. Value of the OTC structure in predicting a mutation pathogenic potential. J Inherit Metab Dis. 2007;30:217-26. [3]. Morizono H et al. Expression, pMammer Bouhou, W. (2019). Optimizando la purificación de ornitina transcarbamilasa humana (OTC) producida recombinantemente. http://hdl.handle.net/10251/127721TFG
Osorio J. S. - One of the best experts on this subject based on the ideXlab platform.
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A fluorescence resonance energy transfer approach to determine intracellular bioavailability of zinc in blood samples using a bovine mammary epithelial cell model
Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange, 2020Co-Authors: Mohan R., Rosa F., Osorio J. S.Abstract:This study evaluated the intracellular bioavailability of Zn in blood plasma and serum from dairy cows using a Zn fluorescent resonance energy transfer (FRET) system. Bovine mammary alveolar epithelial cells (MacT) were incubated with either serum or plasma at 5, 10, 25, 50, 75, and 100% diluted with reduced serum medium (OptiMEM). Before transfection, MacT cells were cultivated in high glucose Dulbecco modified Eagle’s medium (DMEM) with sodium pyruvate and supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin and Fungizone Antimycotic. The plasmid used in this study was the peZinCh-NB (Addgene) designed to detect intracellular Zn through FRET technology. Cells were seeded 24 h before transfection at 30,000 cells/well in a 96-well plate. Cells were transfected with Lipofectamine 3000 at 0.3 uL/ well and at 150 ng/well of plasmid in OptiMEM. Free intracellular Zn can be depleted by chelating agents such as TPEN (N,N,N’,N’-Tetrakis (2-pyridylmethyl) ethylenediamine). Hence, additional treatments included 5 µM of TPEN(TPEN), 50μM of Zn (Zn), and 50μM of Zn+5 µM of TPEN (Zn+TPEN). An inverted fluorescent microscope for live imagining (EVOS FL Auto) equipped with an environment-controlled chamber at 37°C and 5.0% of CO2 was used to take 4 pictures/well at 4x mag- J. Dairy Sci. 103 (Suppl. 1) 233 nification at 0 and 24 h post-treatment. Quantification of FRET signal and cell viability were assessed using the CellProfiler software. Data were analyzed using the PROC MIXED of SAS. A treatment effect (P \u3c 0.01) in FRET signal was observed in serum and a trend (P = 0.11) in plasma samples. There was an evident decline in FRET signal as serum % was increased with lower (P \u3c 0.01) FRET at 100% serum, this suggests an increase in intracellular Zn. There were no differences in FRET across plasma %. The cell viability at 24h was similar across serum (P ≥ 0.54) and plasma (P ≥ 0.34) treatments. These preliminary data indicate that the available Zn in serum can be detected via FRET in bovine cells. To expand on these effects, gene expression and Zn concentration will be analyzed
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The effects of zinc chelator TPEN in bovine mammary epithelial cells evaluated via a fluorescence resonance energy transfer approach
Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange, 2020Co-Authors: Mohan R., Osorio J. S.Abstract:Zinc plays important roles in many cellular functions. The free intracellular Zn is sensitive to chelating agents like TPEN (N,N,N’,N’-Tetrakis (2-pyridylmethyl) ethylenediamine). Therefore, we evaluated the effect of TPEN on changes in the intracellular Zn in bovine mammary epithelial alveolar cells (MacT). Before transfection, MacT cells were cultivated in high glucose Dulbecco modified Eagle’s medium (DMEM) with sodium pyruvate and supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin, and Fungizone Antimycotic. The plasmid used in this study was the peZinCh-NB (Addgene) designed to detect intracellular Zn through a fluorescence resonance energy transfer (FRET) technology. Cells were seeded 24h before transfection at 30,000 cells/well in a 96-well plate. Cells were transfected with Lipofectamine 3000 at 0.3 µL/well and 150 ng/well of plasmid in reduced serum medium (OptiMEM). The transfected cells were treated in triplicates with 5 µM of TPEN (TPEN), 50 μM of Zn (Zn), and 50 μM of Zn + 5 µM of TPEN (Zn+TPEN). An inverted fluorescent microscope for live imagining (EVOS FL Auto) equipped with an environment-controlled chamber at 37°C and 5.0% of CO2 was used to take 4 pictures/well at 4x magnification at 0 and 24 h post-treatment. Quantification of fluorescence resonance energy transfer (FRET) signal and cell viability were assessed using the CellProfiler software. Data were analyzed using the PROC MIXED of SAS. A treatment × time effect (P \u3c 0.01) was observed in FRET signal, where FRET was similar (P ≥ 0.18) across treatments at 0h but resulted in greater (P \u3c 0.01) FRET in TPEN in comparison to Zn and Zn+TPEN at 24h. This suggests lower intracellular Zn concentration in TPEN than Zn and Zn+TPEN at 24h. The cell viability at 24h was similar (P ≥ 0.68) across treatments. These preliminary data indicate that TPEN is a viable model to study molecular alterations in Zn-depleted bovine cells and allow to evaluate additional conditions such as inflammation. FRET technology was sensitive to TPEN effects on intracellular Zn. To expand on these effects, gene expression and Zn concentration will be analyze
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A fluorescence resonance energy transfer approach to determine intracellular zinc bioavailability in bovine mammary epithelial cells
Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange, 2019Co-Authors: Mohan R., Rosa F., Osorio J. S.Abstract:Zinc is a key micronutrient involved in many cellular processes and biological pathways including oxidative stress and inflammation. Therefore, we evaluated the effect of the intracellular bioavailability of Zn in bovine mammary epithelial alveolar cells (MacT) incubated at 0, 10, and 50 µM concentrations of Zn. Before transfection, MacT cells were cultivated in high glucose Dulbecco modified Eagle’s medium (DMEM) with sodium pyruvate and supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin and Fungizone Antimycotic. The plasmid used in this study was the peZinCh-NB (Addgene) designed to detect intracellular Zn through a fluorescence resonance energy transfer (FRET) technology. Cells were seeded 24 h before transfection at 30,000 cells/ well in a 96-well plate. Additional cells were seeded at 300,000 cells/ well in a 6-well plate for gene expression analysis. Cells were transfected with the transfection reagent Lipofectamine 3000 at 0.3 μL/well and at 150 ng/well of plasmid in a reduced serum medium (OptiMEM) deprived of FBS. Transfected cells were treated for 24h in triplicates with 0, 10, and 50 μM Zn. An inverted fluorescent microscope for live imagining (EVOS FL Auto) equipped with a motorized scanning stage, and an environment-controlled chamber at 37°C and 5.0% of CO2 was used to take 4 pictures/well at 4× magnification at 0, 12, and 24 h posttreatment. Quantification of Zn and cell viability were assessed using the CellProfiler software. Data were analyzed using the PROC MIXED of SAS. Overall intracellular availability of Zn increased (P \u3c 0.01) in cells incubated with both 10 µM and 50 µM of Zn as early as 12 h post-treatment. At 12 and 24 h post-treatment, the greatest (P \u3c 0.01) Zn intracellular bioavailability was observed with 50 µM of Zn, when compared with 10 µM of Zn and control. The cell viability at 24 h was similar (P ≥ 0.26) across treatments with 81.59, 80.92, and 75.06% for control, 10 µM and 50 µM of Zn, respectively. These preliminary data indicate that intracellular Zn can be detected via a fluorescent protein system in real-time in bovine cells. To expand on these effects, gene expression analysis will be performed
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Intracellular zinc bioavailability in bovine mammary epithelial cells is modulated by lipopolysaccharide: A fluorescence resonance energy transfer approach
Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange, 2019Co-Authors: Mohan R., Rosa F., Osorio J. S.Abstract:Zinc is an important trace element which is involved in many intracellular pathways including cell growth, differentiation, and inflammation. Therefore, we evaluated the effect of the intracellular bioavailability of Zn in bovine mammary epithelial cells (MacT) in response to an LPS 287J. Dairy Sci. Vol. 102, Suppl. 1 (Escherichia coli O111:B4, Sigma-Aldrich) challenge. To test this, MacT cells were seeded 24h before transfection at 30,000 cells/well in a 96-well plate. Additional cells were seeded at 300,000 cells/well in a 6-well plate for gene expression analysis. The plasmid used in this study was the peZinCh-NB (Addgene) designed to detect Zn through fluorescence resonance energy transfer (FRET) technology. Cells were transfected with the transfection reagent Lipofectamine 3000 at 0.3 μL/ well and at 150 ng/well of plasmid in a reduced serum medium deprived of FBS. Transfected cells were treated with 50 μM concentration of Zn, and challenged at non-clinical (0 µg of LPS/mL) and clinical levels (250 µg of LPS/mL) for 2 h at 0, 6, and 12 h after the treatment. An inverted fluorescent microscope for live imagining equipped with scanning stage, and an environment-controlled chamber at 37°C and 5.0% of CO2 was used to take 4 pictures/well at 4× magnification. Quantification of Zn and cell viability were assessed using the CellProfiler software. Data were analyzed using the PROC MIXED of SAS. Overall intracellular availability of Zn was not affected by LPS in MacT cells incubated with 50 µM Zn at 0 and 12 h post-treatment. However, at 6 h post Zn treatment, there was a significant increase (P \u3c 0.01) in intracellular bioavailability of Zn compared with the control over time during LPS challenge. Within 1 h post LPS challenge, the Zn intracellular bioavailability was greater (P = 0.05) in the challenged cells than the control. Similarly, at 1.5 and 2 h post LPS challenge, the Zn intracellular bioavailability was greater (P \u3c 0.001) in the challenged cells than the control. These preliminary data indicate that FRET technology is sensitive enough to detect changes in intracellular Zn content mediate through an LPS model. To expand on these effects, gene expression analysis will be performed
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The potential role of choline to alter histone methylation status revealed through a fluorescent protein system in bovine mammary epithelial cells.
Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange, 2018Co-Authors: Rosa F., Osorio J. S.Abstract:Dietary choline can be a source of methyl groups for histone methylation (HM), which can affect gene expression and consequently milk biosynthesis. Therefore, we evaluated the effect of choline on histone methylation in bovine mammary epithelial alveolar cells (MacT). Prior to transfection, cells were cultivated in high glucose Dulbecco modified Eagle’s medium (DMEM) with sodium pyruvate and supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin and Fungizone antimycotic. The plasmids used in this study were the pcDNA3-K9 and pcDNA3-K27 (Addgene) for analysis of HM through fluorescence resonance energy transfer (FRET) technology. Cells were seeded 24h before transfection at 30,000 cells/well in a 96-well plate. Cells were transfected with Lipofectamine 3000 at 0.3 uL/well and at 50 ng/well of plasmid in a reduced serum medium (OptiMEM) deprived of FBS. Transfected cells were treated for 24h in triplicates with 0, 200, 400, and 800 ug/mL of choline. An inverted fluorescent microscope for live imagining (EVOS FL Auto) equipped with a motorized scanning stage, and an environment-controlled chamber at 37°C and 5.0% of CO2 was used to take 4 pictures/well at 4x magnification 0, 12, and 24h posttreatment. Transfection efficiency, viability, and quantification of HM were assessed using the CellProfiler software. Data were analyzed using the PROC MIXED of SAS and significance was declared at P ≤ 0.05 and tendencies at P ≤ 0.15. Overall HM tend to decrease in K9 (P = 0.13) and increased in K27 (P ≤ 0.01) during the 24h treatment. In K9 transfected cells, the 400 and 800 choline treatments maintain a HM status, whereas HM decreased (P \u3c 0.01) over time in the control and 200 choline. In K27 transfected cells, the 200 choline treatment produced the greatest (P \u3c 0.01) HM by 24h post-treatment. To expand on these effects, global DNA methylation and gene expression analysis will be performed. Our results indicate that choline can affect the HM status of histone tail residues (K9 and K27) differently, and this may be reflected in transcriptional changes and consequently in milk biosynthesis
Adam J Bogdanove - One of the best experts on this subject based on the ideXlab platform.
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efficient design and assembly of custom talen and other tal effector based constructs for dna targeting
Nucleic Acids Research, 2011Co-Authors: Erin L Doyle, Clarice Schmidt, Michelle Christian, Yong Zhang, Lili Wang, Joshua A Baller, Nikunj V Somia, Adam J BogdanoveAbstract:TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository Addgene
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efficient design and assembly of custom talen and other tal effector based constructs for dna targeting
Nucleic Acids Research, 2011Co-Authors: Erin L Doyle, Clarice Schmidt, Michelle Christian, Yong Zhang, Lili Wang, Joshua A Baller, Nikunj V Somia, Adam J BogdanoveAbstract:TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository Addgene
Roel Nusse - One of the best experts on this subject based on the ideXlab platform.
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Lentiviral vectors to probe and manipulate the Wnt signaling pathway.
PLOS ONE, 2010Co-Authors: Christophe Fuerer, Roel NusseAbstract:Background The Wnt signaling pathway plays key roles in development, adult tissue homeostasis and stem cell maintenance. Further understanding of the function of Wnt signaling in specific cell types could benefit from lentiviral vectors expressing reporters for the Wnt pathway or vectors interfering with signaling. Methodology/Principal Findings We have developed a set of fluorescent and luminescent lentiviral vectors that report Wnt signaling activity and discriminate between negative and uninfected cells. These vectors possess a 7xTcf-eGFP or 7xTcf-FFluc (Firefly Luciferase) reporter cassette followed by either an SV40-mCherry or SV40-PuroR (puromycin N-acetyltransferase) selection cassette. We have also constructed a vector that allows drug-based selection of cells with activated Wnt signaling by placing PuroR under the control of the 7xTcf promoter. Lastly, we have expressed dominant-negative Tcf4 (dnTcf4) or constitutively active beta-catenin (β-catenin4A) from the hEF1α promoter in a SV40-PuroR or SV40-mCherry backbone to create vectors that inhibit or activate the Wnt signaling pathway. These vectors will be made available to the scientific community through Addgene. Conclusions These novel lentiviruses are efficient tools to probe and manipulate Wnt signaling. The use of a selection cassette in Wnt-reporter viruses enables discriminating between uninfected and non-responsive cells, an important requirement for experiments where selection of clones is not possible. The use of a chemiluminescent readout enables quantification of signaling. Finally, selectable vectors can be used to either inhibit or activate the Wnt signaling pathway. Altogether, these vectors can probe and modulate the Wnt signaling pathway in experimental settings where persistence of the transgene or gene transfer cannot be accomplished by non-viral techniques.
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Lentiviral Vectors to Probe and Manipulate the Wnt Signaling Pathway
2010Co-Authors: Christophe Fuerer, Roel NusseAbstract:Background: The Wnt signaling pathway plays key roles in development, adult tissue homeostasis and stem cell maintenance. Further understanding of the function of Wnt signaling in specific cell types could benefit from lentiviral vectors expressing reporters for the Wnt pathway or vectors interfering with signaling. Methodology/Principal Findings: We have developed a set of fluorescent and luminescent lentiviral vectors that report Wnt signaling activity and discriminate between negative and uninfected cells. These vectors possess a 7xTcf-eGFP or 7xTcf-FFluc (Firefly Luciferase) reporter cassette followed by either an SV40-mCherry or SV40-Puro R (puromycin N-acetyltransferase) selection cassette. We have also constructed a vector that allows drug-based selection of cells with activated Wnt signaling by placing Puro R under the control of the 7xTcf promoter. Lastly, we have expressed dominantnegative Tcf4 (dnTcf4) or constitutively active beta-catenin (b-catenin 4A) from the hEF1a promoter in a SV40-Puro R or SV40mCherry backbone to create vectors that inhibit or activate the Wnt signaling pathway. These vectors will be made available to the scientific community through Addgene. Conclusions: These novel lentiviruses are efficient tools to probe and manipulate Wnt signaling. The use of a selection cassette in Wnt-reporter viruses enables discriminating between uninfected and non-responsive cells, an important requirement for experiments where selection of clones is not possible. The use of a chemiluminescent readout enable
Gaetan Burgio - One of the best experts on this subject based on the ideXlab platform.
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no evidence for genome editing in mouse zygotes and hek293t human cell line using the dna guided natronobacterium gregoryi argonaute ngago
bioRxiv, 2017Co-Authors: Nay Chi Khin, Jenna Lowe, Lora M Jensen, Gaetan BurgioAbstract:Abstract A recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci (Sptb, Tet-1, Tet-2 and Tet-3) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5’- phosphorylated guide DNA (gDNA) from 2.5 ng/μl to 50 ng/μl and cultured to blastocyst stage of development. The presence of indels was verified using T7 endonuclease 1 assay (T7E1) and Sanger sequencing. We reported no evidence of successful editing of the mouse genome. We then assessed the lack of editing efficiency in HEK293T cell line for the EMX1 endogenous locus by monitoring the NgAgo protein expression level and the editing efficiency by T7E1 assay and Sanger sequencing. We reported that the NgAgo protein was expressed from 8 hours to a maximum expression at 48 hours post-transfection, confirming the efficient delivery of the plasmid and the gDNA but no evidence of successful editing of EMX1 target in all transfected samples. Together our findings indicate that we failed to edit using NgAgo.
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no evidence for genome editing of the endogenous dna in mouse zygotes and hek293t human cell line using the dna guided natronobacterium gregoryi argonaute ngago
bioRxiv, 2016Co-Authors: Nay Chi Khin, Jenna Lowe, Lora M Jensen, Gaetan BurgioAbstract:A recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci (Sptb, Tet-1, Tet-2 and Tet-3) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5′-phosphorylated guide DNA (gDNA) from 2.5 ng/μl to 50 ng/μl and cultured to blastocyst stage of development. The presence of indels was verified using T7 endonuclease 1 assay (T7E1) and Sanger sequencing. We reported no evidence of successful editing of the mouse genome. We then assessed the lack of editing efficiency in HEK293T cell line for the EMX1 endogenous locus by monitoring the NgAgo protein expression level and the editing efficiency by T7E1 assay and Sanger sequencing. We reported that the NgAgo protein was expressed from 8 hours to a maximum expression at 48 hours post-transfection, confirming the efficient delivery of the plasmid and the gDNA but no evidence of successful editing of EMX1 target in all transfected samples. Together our findings indicate that NgAgo is unsuitable as a genome editing tool.