Genetically engineered mice for combinatorial cardiovascular optobiology
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Curated by eLife
Evaluation Summary:
The manuscript by Lee and co-workers describes the development of 21 unique transgenic mouse lines that express optogenetic sensors and effectors in a cell lineage-specific fashion. The knock-in approach allows the sensors and effectors to be rapidly combined or moved to different backgrounds, such as genetic disease models. Such manipulations are often impractical when using a Cre-based system. This constitutes a vital advantage for many studies. The new mice described here will be very powerful tools to study physiology and alteration in disease models.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca 2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP 3 (optoα1AR) and cAMP (optoß2AR), or Ca 2+ -permeant membrane channels (CatCh2) in smooth muscle ( Acta2 ) and endothelium ( Cdh5 ). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca 2+ imaging in Hcn4 BAC -CatCh2/ Myh6 -GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.
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Evaluation Summary:
The manuscript by Lee and co-workers describes the development of 21 unique transgenic mouse lines that express optogenetic sensors and effectors in a cell lineage-specific fashion. The knock-in approach allows the sensors and effectors to be rapidly combined or moved to different backgrounds, such as genetic disease models. Such manipulations are often impractical when using a Cre-based system. This constitutes a vital advantage for many studies. The new mice described here will be very powerful tools to study physiology and alteration in disease models.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript is clearly written and was a pleasure to read. Rigorous example data from several of the mouse lines were presented, nicely illustrating their utility. The manuscript could be improved by an open discussion of potential problems/limitations with using the transgenic mice described here.
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Reviewer #2 (Public Review):
This paper is well-written and discusses an important and efficient approach to generating optogenetic mice. The major strength of the paper is the combination of optogenetic activators and sensors that are expressed in complex tissue networks. The ease of breeding to generate these mice is an important step that will allow testing of the interactions between cells in complex networks. Generation of lines that enable ratiometric measurements of Ca2+ are also important development that should allow better assessment of intracellular Ca2+ levels.
A weakness of the paper is that it is essentially a methods paper that describes the characteristics of the mice and gives proof-of-principal examples of various combinations of activators and sensors in cardiac, smooth muscle and endothelial cells. The paper lacks …
Reviewer #2 (Public Review):
This paper is well-written and discusses an important and efficient approach to generating optogenetic mice. The major strength of the paper is the combination of optogenetic activators and sensors that are expressed in complex tissue networks. The ease of breeding to generate these mice is an important step that will allow testing of the interactions between cells in complex networks. Generation of lines that enable ratiometric measurements of Ca2+ are also important development that should allow better assessment of intracellular Ca2+ levels.
A weakness of the paper is that it is essentially a methods paper that describes the characteristics of the mice and gives proof-of-principal examples of various combinations of activators and sensors in cardiac, smooth muscle and endothelial cells. The paper lacks applications that test novel hypotheses and demonstrate new findings. That being said, it is likely that the mice described and available to all investigators, will provide myriad new experimental opportunities. It will be up to the creativity of investigators at large to devise these applications.
While the breeding tactics to generate experimentally useful chimeras are clear, it would also be helpful to compare relative expression/signal strengths for Cre recombinase driver mice vs. the monoallelic mice promoted in the present study. Comments about breeding success/difficulties would also be useful for those planning experiments using combinations of the mice from CHROMus.
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Reviewer #3 (Public Review):
The authors describe the generation of a battery of monoallelic homozygous transgenic mice expressing optogenetic effectors and sensors in specific cells primarily in the cardiovascular system. The paper potentially provides a relatively simple framework for expressing optogenetic reporters and effectors in any desired cellular system and/or disease model. An important advantage of this approach is that a simple breeding strategy can be used to produce animals expressing both sensor and effector in the desired cell-type. The paper provides convincing direct evidence of the utility of the resulting effector/sensor pairs in a variety of cell types. The development and demonstration of utility of animals expressing "ratiometric" cytoplasmic Ca2+ probes is particularly impressive. These probes are demonstrated …
Reviewer #3 (Public Review):
The authors describe the generation of a battery of monoallelic homozygous transgenic mice expressing optogenetic effectors and sensors in specific cells primarily in the cardiovascular system. The paper potentially provides a relatively simple framework for expressing optogenetic reporters and effectors in any desired cellular system and/or disease model. An important advantage of this approach is that a simple breeding strategy can be used to produce animals expressing both sensor and effector in the desired cell-type. The paper provides convincing direct evidence of the utility of the resulting effector/sensor pairs in a variety of cell types. The development and demonstration of utility of animals expressing "ratiometric" cytoplasmic Ca2+ probes is particularly impressive. These probes are demonstrated to have advantages for challenging in vivo imaging where movement artifacts can confound interpretation. These mice are freely available and will be a powerful and valuable resource to the community.
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