Breeding of AC knockout mice with or without cardiomyocyte expression of a cAMP sensor called pmEpac (copy)
Project objective: To breed and sustain colonies of mice for ex vivo experimentation that have selective knockout of adenylyl cyclase (AC) type 5 or type 6 with or without expression of a cAMP sensor at the cardiomyocyte membrane called pmEpac (FOTS protocol 10308).
Harm: Prior breeding of these mice gave rise to a viable phenotype (normal) without any observable differnces from wild type.
Predicted benefit: These mice will allow us to investigate receptor systems activating the cAMP signalling pathway without the need to virally transfect cAMP sensors into cardiomyocytes in vitro. These mice also offer the advantage of localizing the sensor to the plasma membrane where AC is located increasing sensitivity of the signal. Utilizing these mice we will be able to measure in real time beta-adrenergic receptor signaling (amongst other receptors activating AC) and its regulation by inhibitory G protein, phosphodiesterases and other scaffolding or regulatory proteins. These studies will contribute to our understanding how beta-adrenergic receptor signaling is regulated by multiple proteins which may prove beneficial in heart failure, thus results of these studies may lead to new treatment for heart failure.
Numbers and types: 96 mice of each group 1)C57/BL6 Wild type-pmEpac, 2)C57/BL6 AC5KO-pmEpac, 3) C57/BL6 AC6KO-pmEpac: 160 mice of each group 4) C57/BL6 Wild type, 5) C57/BL6 AC5KO, 6) C57/BL6 AC6KO.
Accomplish the requirement of 3Rs: Performing parts of our investigation in cells (primary cells)
Performing some preliminary experiments to optimize the system and avoid repetitive experimentation.
Performing many measurements in each animal whenever possible (multiple muscle strips from one mouse) and accepted for the wellbeing of the animal. This will help to reduce the total number of animals for the study.
The animals will be kept in the required cages by the KPM personnel.
Based on the above points, we are seeking permission to conduct only ex vivo studies utilizing the heart or isolated heart cells.
Harm: Prior breeding of these mice gave rise to a viable phenotype (normal) without any observable differnces from wild type.
Predicted benefit: These mice will allow us to investigate receptor systems activating the cAMP signalling pathway without the need to virally transfect cAMP sensors into cardiomyocytes in vitro. These mice also offer the advantage of localizing the sensor to the plasma membrane where AC is located increasing sensitivity of the signal. Utilizing these mice we will be able to measure in real time beta-adrenergic receptor signaling (amongst other receptors activating AC) and its regulation by inhibitory G protein, phosphodiesterases and other scaffolding or regulatory proteins. These studies will contribute to our understanding how beta-adrenergic receptor signaling is regulated by multiple proteins which may prove beneficial in heart failure, thus results of these studies may lead to new treatment for heart failure.
Numbers and types: 96 mice of each group 1)C57/BL6 Wild type-pmEpac, 2)C57/BL6 AC5KO-pmEpac, 3) C57/BL6 AC6KO-pmEpac: 160 mice of each group 4) C57/BL6 Wild type, 5) C57/BL6 AC5KO, 6) C57/BL6 AC6KO.
Accomplish the requirement of 3Rs: Performing parts of our investigation in cells (primary cells)
Performing some preliminary experiments to optimize the system and avoid repetitive experimentation.
Performing many measurements in each animal whenever possible (multiple muscle strips from one mouse) and accepted for the wellbeing of the animal. This will help to reduce the total number of animals for the study.
The animals will be kept in the required cages by the KPM personnel.
Based on the above points, we are seeking permission to conduct only ex vivo studies utilizing the heart or isolated heart cells.