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Gary Clark - One of the best experts on this subject based on the ideXlab platform.

  • using Inducible Expression Vector technology to create stable cell lines expressing kcnq2 3 kcnq4 and kcnq3 5 currents suitable for automated electrophysiology platforms
    Biophysical Journal, 2009
    Co-Authors: Andrew Southan, Scott A. Maidment, Simon Dowler, Matthew Gardener, Anthony Lawrence, Omar Aziz, Tristana Von Will, Gary Clark
    Abstract:

    The KCNQ (Kv7) family of voltage gated ion channels conduct a number of hyperpolarising currents in various tissue types, including the heteromultimeric KCNQ2/3 M-current found in sensory neurones. Cell lines constructed using constitutive Expression Vectors to stably transfect KCNQ2/3, KCNQ4, and KCNQ3/5 genes gave acceptable performance when using rubidium efflux methodology. However, Expression levels within the cell population were found to be variable when assessed using conventional electrophysiology. Cell morphology changed during passage and the cell lines were unsuitable for automated electrophysiology recording. Using RheoSwitch™ Inducible Vector technology we have created new stable cell lines where the production of ion channel can be closely controlled by addition of an inducer agent. Putative clones were screened using IonWorks® Quattro™ recording in single hole PatchPlate™ mode. For each of the three cell lines, clones were identified displaying more than 60% of the cells having greater than 0.5nA of current. The performance of the clones in single hole mode was suitable for progression to Population Patch Clamp™ (PPC) mode recording. Each cell line displayed acceptable seal properties and current amplitudes, KCNQ2/3 26±5 MΩ, 0.77±0.19 nA (n=250); KCNQ4 112±49 MΩ, 0.44±0.07 nA (n=372) and KCNQ3/5 159±44 MΩ, 0.84±0.50 nA (n=124). In addition, each cell line each cell line displayed the appropriate pharmacology for regitabine, linopridine, XE991, TEA and bepridil. The cell lines are suitable for compound screening and selectivity profiling using automated and conventional electrophysiology.

  • Using Inducible Expression Vector Technology To Create Stable Cell Lines Expressing KCNQ2/3, KCNQ4, And KCNQ3/5 Currents Suitable For Automated Electrophysiology Platforms
    Biophysical Journal, 2009
    Co-Authors: Andrew Southan, Scott A. Maidment, Simon Dowler, Matthew Gardener, Anthony Lawrence, Omar Aziz, Tristana Von Will, Gary Clark
    Abstract:

    The KCNQ (Kv7) family of voltage gated ion channels conduct a number of hyperpolarising currents in various tissue types, including the heteromultimeric KCNQ2/3 M-current found in sensory neurones. Cell lines constructed using constitutive Expression Vectors to stably transfect KCNQ2/3, KCNQ4, and KCNQ3/5 genes gave acceptable performance when using rubidium efflux methodology. However, Expression levels within the cell population were found to be variable when assessed using conventional electrophysiology. Cell morphology changed during passage and the cell lines were unsuitable for automated electrophysiology recording. Using RheoSwitch™ Inducible Vector technology we have created new stable cell lines where the production of ion channel can be closely controlled by addition of an inducer agent. Putative clones were screened using IonWorks® Quattro™ recording in single hole PatchPlate™ mode. For each of the three cell lines, clones were identified displaying more than 60% of the cells having greater than 0.5nA of current. The performance of the clones in single hole mode was suitable for progression to Population Patch Clamp™ (PPC) mode recording. Each cell line displayed acceptable seal properties and current amplitudes, KCNQ2/3 26±5 MΩ, 0.77±0.19 nA (n=250); KCNQ4 112±49 MΩ, 0.44±0.07 nA (n=372) and KCNQ3/5 159±44 MΩ, 0.84±0.50 nA (n=124). In addition, each cell line each cell line displayed the appropriate pharmacology for regitabine, linopridine, XE991, TEA and bepridil. The cell lines are suitable for compound screening and selectivity profiling using automated and conventional electrophysiology.

Josef Altenbuchner - One of the best experts on this subject based on the ideXlab platform.

  • Optimization of an E. coli L-rhamnose-Inducible Expression Vector: test of various genetic module combinations
    BMC Biotechnology, 2008
    Co-Authors: Angelika Wegerer, Josef Altenbuchner
    Abstract:

    Background A capable Expression Vector is mainly characterized by its production efficiency, stability and induction response. These features can be influenced by a variation of modifications and versatile genetic modules. Results We examined miscellaneous variations of a rhaP _ BAD Expression Vector. The introduction of a stem loop into the translation initiation region of the rhaP _ BAD promoter resulted in the most significant improvement of eGFP Expression. Starting from this plasmid, we constructed a set of Expression Vectors bearing different genetic modules like rop , ccdAB , cer and combinations thereof, and tested the efficiency of Expression and plasmid stability. The plasmid pWA21, containing the stem loop, one cer site and rop , attained high Expression levels accompanied by a good stability, and on that score seems to be a well-balanced choice. Conclusion We report the generation of variations of the rhaP _ BAD Expression Vector and characterization hereof. The genetic modules showed a complex interplay, therefore two positive effects combined sometimes resulted in a disadvantage.

  • optimization of an e coli l rhamnose Inducible Expression Vector test of various genetic module combinations
    BMC Biotechnology, 2008
    Co-Authors: Angelika Wegerer, Tianqi Sun, Josef Altenbuchner
    Abstract:

    A capable Expression Vector is mainly characterized by its production efficiency, stability and induction response. These features can be influenced by a variation of modifications and versatile genetic modules. We examined miscellaneous variations of a rhaP BAD Expression Vector. The introduction of a stem loop into the translation initiation region of the rhaP BAD promoter resulted in the most significant improvement of eGFP Expression. Starting from this plasmid, we constructed a set of Expression Vectors bearing different genetic modules like rop, ccdAB, cer and combinations thereof, and tested the efficiency of Expression and plasmid stability. The plasmid pWA21, containing the stem loop, one cer site and rop, attained high Expression levels accompanied by a good stability, and on that score seems to be a well-balanced choice. We report the generation of variations of the rhaP BAD Expression Vector and characterization hereof. The genetic modules showed a complex interplay, therefore two positive effects combined sometimes resulted in a disadvantage.

Andrew Southan - One of the best experts on this subject based on the ideXlab platform.

  • using Inducible Expression Vector technology to create stable cell lines expressing kcnq2 3 kcnq4 and kcnq3 5 currents suitable for automated electrophysiology platforms
    Biophysical Journal, 2009
    Co-Authors: Andrew Southan, Scott A. Maidment, Simon Dowler, Matthew Gardener, Anthony Lawrence, Omar Aziz, Tristana Von Will, Gary Clark
    Abstract:

    The KCNQ (Kv7) family of voltage gated ion channels conduct a number of hyperpolarising currents in various tissue types, including the heteromultimeric KCNQ2/3 M-current found in sensory neurones. Cell lines constructed using constitutive Expression Vectors to stably transfect KCNQ2/3, KCNQ4, and KCNQ3/5 genes gave acceptable performance when using rubidium efflux methodology. However, Expression levels within the cell population were found to be variable when assessed using conventional electrophysiology. Cell morphology changed during passage and the cell lines were unsuitable for automated electrophysiology recording. Using RheoSwitch™ Inducible Vector technology we have created new stable cell lines where the production of ion channel can be closely controlled by addition of an inducer agent. Putative clones were screened using IonWorks® Quattro™ recording in single hole PatchPlate™ mode. For each of the three cell lines, clones were identified displaying more than 60% of the cells having greater than 0.5nA of current. The performance of the clones in single hole mode was suitable for progression to Population Patch Clamp™ (PPC) mode recording. Each cell line displayed acceptable seal properties and current amplitudes, KCNQ2/3 26±5 MΩ, 0.77±0.19 nA (n=250); KCNQ4 112±49 MΩ, 0.44±0.07 nA (n=372) and KCNQ3/5 159±44 MΩ, 0.84±0.50 nA (n=124). In addition, each cell line each cell line displayed the appropriate pharmacology for regitabine, linopridine, XE991, TEA and bepridil. The cell lines are suitable for compound screening and selectivity profiling using automated and conventional electrophysiology.

  • Using Inducible Expression Vector Technology To Create Stable Cell Lines Expressing KCNQ2/3, KCNQ4, And KCNQ3/5 Currents Suitable For Automated Electrophysiology Platforms
    Biophysical Journal, 2009
    Co-Authors: Andrew Southan, Scott A. Maidment, Simon Dowler, Matthew Gardener, Anthony Lawrence, Omar Aziz, Tristana Von Will, Gary Clark
    Abstract:

    The KCNQ (Kv7) family of voltage gated ion channels conduct a number of hyperpolarising currents in various tissue types, including the heteromultimeric KCNQ2/3 M-current found in sensory neurones. Cell lines constructed using constitutive Expression Vectors to stably transfect KCNQ2/3, KCNQ4, and KCNQ3/5 genes gave acceptable performance when using rubidium efflux methodology. However, Expression levels within the cell population were found to be variable when assessed using conventional electrophysiology. Cell morphology changed during passage and the cell lines were unsuitable for automated electrophysiology recording. Using RheoSwitch™ Inducible Vector technology we have created new stable cell lines where the production of ion channel can be closely controlled by addition of an inducer agent. Putative clones were screened using IonWorks® Quattro™ recording in single hole PatchPlate™ mode. For each of the three cell lines, clones were identified displaying more than 60% of the cells having greater than 0.5nA of current. The performance of the clones in single hole mode was suitable for progression to Population Patch Clamp™ (PPC) mode recording. Each cell line displayed acceptable seal properties and current amplitudes, KCNQ2/3 26±5 MΩ, 0.77±0.19 nA (n=250); KCNQ4 112±49 MΩ, 0.44±0.07 nA (n=372) and KCNQ3/5 159±44 MΩ, 0.84±0.50 nA (n=124). In addition, each cell line each cell line displayed the appropriate pharmacology for regitabine, linopridine, XE991, TEA and bepridil. The cell lines are suitable for compound screening and selectivity profiling using automated and conventional electrophysiology.

Angelika Wegerer - One of the best experts on this subject based on the ideXlab platform.

  • Optimization of an E. coli L-rhamnose-Inducible Expression Vector: test of various genetic module combinations
    BMC Biotechnology, 2008
    Co-Authors: Angelika Wegerer, Josef Altenbuchner
    Abstract:

    Background A capable Expression Vector is mainly characterized by its production efficiency, stability and induction response. These features can be influenced by a variation of modifications and versatile genetic modules. Results We examined miscellaneous variations of a rhaP _ BAD Expression Vector. The introduction of a stem loop into the translation initiation region of the rhaP _ BAD promoter resulted in the most significant improvement of eGFP Expression. Starting from this plasmid, we constructed a set of Expression Vectors bearing different genetic modules like rop , ccdAB , cer and combinations thereof, and tested the efficiency of Expression and plasmid stability. The plasmid pWA21, containing the stem loop, one cer site and rop , attained high Expression levels accompanied by a good stability, and on that score seems to be a well-balanced choice. Conclusion We report the generation of variations of the rhaP _ BAD Expression Vector and characterization hereof. The genetic modules showed a complex interplay, therefore two positive effects combined sometimes resulted in a disadvantage.

  • optimization of an e coli l rhamnose Inducible Expression Vector test of various genetic module combinations
    BMC Biotechnology, 2008
    Co-Authors: Angelika Wegerer, Tianqi Sun, Josef Altenbuchner
    Abstract:

    A capable Expression Vector is mainly characterized by its production efficiency, stability and induction response. These features can be influenced by a variation of modifications and versatile genetic modules. We examined miscellaneous variations of a rhaP BAD Expression Vector. The introduction of a stem loop into the translation initiation region of the rhaP BAD promoter resulted in the most significant improvement of eGFP Expression. Starting from this plasmid, we constructed a set of Expression Vectors bearing different genetic modules like rop, ccdAB, cer and combinations thereof, and tested the efficiency of Expression and plasmid stability. The plasmid pWA21, containing the stem loop, one cer site and rop, attained high Expression levels accompanied by a good stability, and on that score seems to be a well-balanced choice. We report the generation of variations of the rhaP BAD Expression Vector and characterization hereof. The genetic modules showed a complex interplay, therefore two positive effects combined sometimes resulted in a disadvantage.

Simon Dowler - One of the best experts on this subject based on the ideXlab platform.

  • using Inducible Expression Vector technology to create stable cell lines expressing kcnq2 3 kcnq4 and kcnq3 5 currents suitable for automated electrophysiology platforms
    Biophysical Journal, 2009
    Co-Authors: Andrew Southan, Scott A. Maidment, Simon Dowler, Matthew Gardener, Anthony Lawrence, Omar Aziz, Tristana Von Will, Gary Clark
    Abstract:

    The KCNQ (Kv7) family of voltage gated ion channels conduct a number of hyperpolarising currents in various tissue types, including the heteromultimeric KCNQ2/3 M-current found in sensory neurones. Cell lines constructed using constitutive Expression Vectors to stably transfect KCNQ2/3, KCNQ4, and KCNQ3/5 genes gave acceptable performance when using rubidium efflux methodology. However, Expression levels within the cell population were found to be variable when assessed using conventional electrophysiology. Cell morphology changed during passage and the cell lines were unsuitable for automated electrophysiology recording. Using RheoSwitch™ Inducible Vector technology we have created new stable cell lines where the production of ion channel can be closely controlled by addition of an inducer agent. Putative clones were screened using IonWorks® Quattro™ recording in single hole PatchPlate™ mode. For each of the three cell lines, clones were identified displaying more than 60% of the cells having greater than 0.5nA of current. The performance of the clones in single hole mode was suitable for progression to Population Patch Clamp™ (PPC) mode recording. Each cell line displayed acceptable seal properties and current amplitudes, KCNQ2/3 26±5 MΩ, 0.77±0.19 nA (n=250); KCNQ4 112±49 MΩ, 0.44±0.07 nA (n=372) and KCNQ3/5 159±44 MΩ, 0.84±0.50 nA (n=124). In addition, each cell line each cell line displayed the appropriate pharmacology for regitabine, linopridine, XE991, TEA and bepridil. The cell lines are suitable for compound screening and selectivity profiling using automated and conventional electrophysiology.

  • Using Inducible Expression Vector Technology To Create Stable Cell Lines Expressing KCNQ2/3, KCNQ4, And KCNQ3/5 Currents Suitable For Automated Electrophysiology Platforms
    Biophysical Journal, 2009
    Co-Authors: Andrew Southan, Scott A. Maidment, Simon Dowler, Matthew Gardener, Anthony Lawrence, Omar Aziz, Tristana Von Will, Gary Clark
    Abstract:

    The KCNQ (Kv7) family of voltage gated ion channels conduct a number of hyperpolarising currents in various tissue types, including the heteromultimeric KCNQ2/3 M-current found in sensory neurones. Cell lines constructed using constitutive Expression Vectors to stably transfect KCNQ2/3, KCNQ4, and KCNQ3/5 genes gave acceptable performance when using rubidium efflux methodology. However, Expression levels within the cell population were found to be variable when assessed using conventional electrophysiology. Cell morphology changed during passage and the cell lines were unsuitable for automated electrophysiology recording. Using RheoSwitch™ Inducible Vector technology we have created new stable cell lines where the production of ion channel can be closely controlled by addition of an inducer agent. Putative clones were screened using IonWorks® Quattro™ recording in single hole PatchPlate™ mode. For each of the three cell lines, clones were identified displaying more than 60% of the cells having greater than 0.5nA of current. The performance of the clones in single hole mode was suitable for progression to Population Patch Clamp™ (PPC) mode recording. Each cell line displayed acceptable seal properties and current amplitudes, KCNQ2/3 26±5 MΩ, 0.77±0.19 nA (n=250); KCNQ4 112±49 MΩ, 0.44±0.07 nA (n=372) and KCNQ3/5 159±44 MΩ, 0.84±0.50 nA (n=124). In addition, each cell line each cell line displayed the appropriate pharmacology for regitabine, linopridine, XE991, TEA and bepridil. The cell lines are suitable for compound screening and selectivity profiling using automated and conventional electrophysiology.