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T. Wei, K. Koeppen, T. Ott, N. Rieger, B. Baumann, T. Euler, O. Griesbeck, T. Ladewig, B. Wissinger; Generation of a Transgenic Mouse Expressing a Ca2+ Biosensor to Investigate Cone Photoreceptor Ca2+ Homeostasis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1897.
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© ARVO (1962-2015); The Authors (2016-present)
Ca2+ plays a crucial role in modulating the photoresponse as well as in glutamate release at the photoreceptor synapse and there is growing evidence that Ca2+ is involved in photoreceptor apoptosis in hereditary retinal dystrophies. To monitor [Ca2+] fluctuations underlying those processes in mammalian cone photoreceptors we generated a transgenic mouse line that express a Ca2+ biosensor specifically in cones.
We generated a plasmid construct containing the sequence of the Ca2+ biosensor TN-XL under the control of the human red opsin promoter. The linearized construct was injected into the pronuclei of fertilized oocytes, which were then implanted into pseudo-pregnant mice. The presence and copy number of the transgene were determined by PCR. The retinal expression of the biosensor was studied using confocal microscopy and immunohistochemistry, and the functionality of the biosensor was tested using confocal Ca2+ imaging.
In the founder mice the copy number of the transgene ranged from 1 to 45. In their progeny we identified one mouse line that expressed the biosensor in a subset of retinal photoreceptors, immunostaining with antibodies against L/M opsins confirmed that the biosensor is expressed in cones. We performed initial Ca2+ imaging experiments, in which changing the external [Ca2+] in the presence of ionomycin led to fluorescence changes that were small but showed the expected polarity.
We generated a transgenic mouse line that selectively expresses a Ca2+ biosensor in cones, allowing us to address questions such as the role of internal [Ca2+] changes in cone degeneration using mice models for cone dystrophy in humans. Preliminary Ca2+ imaging data suggest that the biosensor is functional in cone photoreceptors.
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