Three-Dimensional X-ray Imaging of β-Galactosidase Reporter Activity by Micro-CT: Implication for Quantitative Analysis of Gene Expression

Ermakova, Olga; Orsini, Tiziana; Fruscoloni, Paolo; Chiani, Francesco; Gambadoro, Alessia; Putti, Sabrina; Cirilli, Maurizio; Mezzi, Alessio; Kaciulis, Saulius; Pasquini, Miriam; Raspa, Marcello; Scavizzi, Ferdinando; Tocchini-Valentini, Glauco P.

doi:10.3390/brainsci11060746

Open AccessArticle

Three-Dimensional X-ray Imaging of β-Galactosidase Reporter Activity by Micro-CT: Implication for Quantitative Analysis of Gene Expression

by

Olga Ermakova

^1,2,*,†,

Tiziana Orsini

^1,2,†

,

Paolo Fruscoloni

^1,2

,

Francesco Chiani

^1,2,

Alessia Gambadoro

^1,2,

Sabrina Putti

^1,2,

Maurizio Cirilli

²,

Alessio Mezzi

³

,

Saulius Kaciulis

³

,

Miriam Pasquini

^1,2,

Marcello Raspa

^1,2,

Ferdinando Scavizzi

^1,2 and

Glauco P. Tocchini-Valentini

¹

Adriano Buzzati-Traverso Campus, European Mouse Mutant Archive (EMMA), INFRAFRONTIER, Monterotondo Mouse Clinic (MMC), Italian National Research Council (CNR), Via Ramarini, 32, Monterotondo, 00015 Rome, Italy

²

Adriano Buzzati-Traverso Campus, Institute of Biochemistry and Cell Biology (IBBC), Italian National Research Council (CNR), Via Ramarini, 32, Monterotondo Scalo, 00015 Rome, Italy

³

Institute for the Study of Nanostructured Materials, ISMN–CNR, Monterotondo Staz., 00015 Rome, Italy

^*

Author to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Brain Sci. 2021, 11(6), 746; https://doi.org/10.3390/brainsci11060746

Submission received: 5 April 2021 / Revised: 24 May 2021 / Accepted: 27 May 2021 / Published: 4 June 2021

(This article belongs to the Special Issue Imaging of Neuronal Environments)

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Abstract

Acquisition of detailed anatomical and molecular knowledge from intact biological samples while preserving their native three-dimensional structure is still a challenging issue for imaging studies aiming to unravel a system’s functions. Three-dimensional micro-CT X-ray imaging with a high spatial resolution in minimally perturbed naive non-transparent samples has recently gained increased popularity and broad application in biomedical research. Here, we describe a novel X-ray-based methodology for analysis of β-galactosidase (lacZ) reporter-driven gene expression in an intact murine brain ex vivo by micro-CT. The method relies on detection of bromine molecules in the product of the enzymatic β-galactosidase reaction. Enhancement of the X-ray signal is observed specifically in the regions of the murine brain where expression of the lacZ reporter gene is also detected histologically. We performed quantitative analysis of the expression levels of lacZ reporter activity by relative radiodensity estimation of the β-galactosidase/X-gal precipitate in situ. To demonstrate the feasibility of the method, we performed expression analysis of the Tsen54-lacZ reporter gene in the murine brain in a semi-quantitative manner. Human mutations in the Tsen54 gene cause pontocerebellar hypoplasia (PCH), a group of severe neurodegenerative disorders with both mental and motor deficits. Comparing relative levels of Tsen54 gene expression, we demonstrate that the highest Tsen54 expression is observed in anatomical brain substructures important for the normal motor and memory functions in mice.

Keywords: mouse brain; gene expression; lacZ reporter; X-ray imaging; pontocerebellar hypoplesia; Tsen54; mouse phenotyping

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MDPI and ACS Style

Ermakova, O.; Orsini, T.; Fruscoloni, P.; Chiani, F.; Gambadoro, A.; Putti, S.; Cirilli, M.; Mezzi, A.; Kaciulis, S.; Pasquini, M.; et al. Three-Dimensional X-ray Imaging of β-Galactosidase Reporter Activity by Micro-CT: Implication for Quantitative Analysis of Gene Expression. Brain Sci. 2021, 11, 746. https://doi.org/10.3390/brainsci11060746

AMA Style

Ermakova O, Orsini T, Fruscoloni P, Chiani F, Gambadoro A, Putti S, Cirilli M, Mezzi A, Kaciulis S, Pasquini M, et al. Three-Dimensional X-ray Imaging of β-Galactosidase Reporter Activity by Micro-CT: Implication for Quantitative Analysis of Gene Expression. Brain Sciences. 2021; 11(6):746. https://doi.org/10.3390/brainsci11060746

Chicago/Turabian Style

Ermakova, Olga, Tiziana Orsini, Paolo Fruscoloni, Francesco Chiani, Alessia Gambadoro, Sabrina Putti, Maurizio Cirilli, Alessio Mezzi, Saulius Kaciulis, Miriam Pasquini, and et al. 2021. "Three-Dimensional X-ray Imaging of β-Galactosidase Reporter Activity by Micro-CT: Implication for Quantitative Analysis of Gene Expression" Brain Sciences 11, no. 6: 746. https://doi.org/10.3390/brainsci11060746

APA Style

Ermakova, O., Orsini, T., Fruscoloni, P., Chiani, F., Gambadoro, A., Putti, S., Cirilli, M., Mezzi, A., Kaciulis, S., Pasquini, M., Raspa, M., Scavizzi, F., & Tocchini-Valentini, G. P. (2021). Three-Dimensional X-ray Imaging of β-Galactosidase Reporter Activity by Micro-CT: Implication for Quantitative Analysis of Gene Expression. Brain Sciences, 11(6), 746. https://doi.org/10.3390/brainsci11060746

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Three-Dimensional X-ray Imaging of β-Galactosidase Reporter Activity by Micro-CT: Implication for Quantitative Analysis of Gene Expression

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