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Transgenic mice encoding modern imaging probes: Properties and applications.

ABSTRACT: Modern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

SUBMITTER: Kasatkina LA

PROVIDER: S-EPMC9183799 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Transgenic mice encoding modern imaging probes: Properties and applications.

Kasatkina Ludmila A LA Verkhusha Vladislav V VV

Cell reports 20220501 8

Modern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pa ...[more]

PMID: 35613592

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Project description:RationaleCyclic GMP (cGMP) is an important intracellular signaling molecule in the cardiovascular system, but its spatiotemporal dynamics in vivo is largely unknown.ObjectiveTo generate and characterize transgenic mice expressing the fluorescence resonance energy transfer-based ratiometric cGMP sensor, cGMP indicator with an EC50 of 500 nmol/L (cGi500), in cardiovascular tissues.Methods and resultsMouse lines with smooth muscle-specific or ubiquitous expression of cGi500 were generated by random transgenesis using an SM22α promoter fragment or by targeted integration of a Cre recombinase-activatable expression cassette driven by the cytomegalovirus early enhancer/chicken β-actin/β-globin promoter into the Rosa26 locus, respectively. Primary smooth muscle cells isolated from aorta, bladder, and colon of cGi500 mice showed strong sensor fluorescence. Basal cGMP concentrations were < 100 nmol/L, whereas stimulation with cGMP-elevating agents such as 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO) or the natriuretic peptides, atrial natriuretic peptide, and C-type natriuretic peptide evoked fluorescence resonance energy transfer changes corresponding to cGMP peak concentrations of ≈ 3 µmol/L. However, different types of smooth muscle cells had different sensitivities of their cGMP responses to DEA/NO, atrial natriuretic peptide, and C-type natriuretic peptide. Robust nitric oxide-induced cGMP transients with peak concentrations of ≈ 1 to > 3 µmol/L could also be monitored in blood vessels of the isolated retina and in the cremaster microcirculation of anesthetized mice. Moreover, with the use of a dorsal skinfold chamber model and multiphoton fluorescence resonance energy transfer microscopy, nitric oxide-stimulated vascular cGMP signals associated with vasodilation were detected in vivo in an acutely untouched preparation.ConclusionsThese cGi500 transgenic mice permit the visualization of cardiovascular cGMP signals in live cells, tissues, and mice under normal and pathological conditions or during pharmacotherapy with cGMP-elevating drugs.

Project description:Two probes, AIE-1 and AIE-2, were synthesized to investigate the effect of substitutional functional group on aggregation (aggregation-caused quenching (ACQ) or aggregation-induced emission (AIE)) and intramolecular charge transfer (ICT) behavior as well as on the cell imaging aspect. The yellow-color non-substituted probe AIE-1 showed weak charge-transfer absorption and an emission band at 377 nm and 432 nm, whereas the yellowish-orange color substituted probe AIE-2 showed a strong charge-transfer absorption and an emission band at 424 nm and 477 nm in THF solvent. The UV-Vis studies of AIE-1 and AIE-2 in THF and THF with different water fractions showed huge absorption changes in AIE-2 with high water fractions due to its strong aggregation behavior, but no such noticeable absorption changes were observed for AIE-1. Interestingly, the fluorescence intensity of AIE-1 at 432 nm gradually decreased with increasing water fractions and became almost non-emissive at 90% water. However, the monomer-type emission of AIE-2 at 477 nm was shifted to 584 nm with a 6-fold increase in fluorescence intensity in THF-H2O (1:9, v/v) solvent mixtures due to the restriction of intramolecular rotation on aggregation in high water fractions. This result indicates that the probe AIE-1 shows ACQ and probe AIE-2 shows AIE behaviors in THF-H2O solvent mixtures. Furthermore, the emission spectra of AIE-1 and AIE-2 were carried out in different solvent and with different concentrations to see the solvent- or concentration-dependent aggregation behavior. Scanning electron microscope (SEM) and dynamic light scattering (DLS) experiments were also conducted to assess the morphology and particle size of two probes before and after aggregation. Both of the probes, AIE-1 and AIE-2, showed less toxicity on HeLa cells and were suitable for cell imaging studies. Density functional theory (DFT) calculation was also carried out to confirm the ICT process from an electron-rich indole moiety to an electron-deficient cyano-phenyl ring of AIE-1 or AIE-2.

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Transgenic mice encoding modern imaging probes: Properties and applications.

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Transgenic mice encoding modern imaging probes: Properties and applications.

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