Three-Dimensional Imaging of the Intracellular Fate of Plasmid DNA and Transgene Expression: ZsGreen1 and Tissue Clearing Method CUBIC Are an Optimal Combination for Multicolor Deep Imaging in Murine Tissues

Abstract

Evaluation methods for determining the distribution of transgene expression in the body and the in vivo fate of viral and non-viral vectors are necessary for successful development of in vivo gene delivery systems. Here, we evaluated the spatial distribution of transgene expression using tissue clearing methods. After hydrodynamic injection of plasmid DNA into mice, whole tissues were subjected to tissue clearing. Tissue clearing followed by confocal laser scanning microscopy enabled evaluation of the three-dimensional distribution of transgene expression without preparation of tissue sections. Among the tested clearing methods (ClearT2, SeeDB, and CUBIC), CUBIC was the most suitable method for determining the spatial distribution of transgene expression in not only the liver but also other tissues such as the kidney and lung. In terms of the type of fluorescent protein, the observable depth for green fluorescent protein ZsGreen1 was slightly greater than that for red fluorescent protein tdTomato. We observed a depth of ~1.5 mm for the liver and 500 μm for other tissues without preparation of tissue sections. Furthermore, we succeeded in multicolor deep imaging of the intracellular fate of plasmid DNA in the murine liver. Thus, tissue clearing would be a powerful approach for determining the spatial distribution of plasmid DNA and transgene expression in various murine tissues.

Effective intraperitoneal gene transfection system using nanobubbles and ultrasound irradiation

“…We previously assessed spatial distribution of transgene expression by a tissue-clearing method without sectioning (Fumoto et al, 2016). Transgene expression was observed to depths of ca.…”

Nanobubble Mediated Gene Delivery in Conjunction With a Hand-Held Ultrasound Scanner

“…Raw values were corrected with RLU value of blank wells with bis-CTZ solution only. For evaluation of luciferase in the mice, the luciferase activity was measured as described previously (Fumoto et al, 2016). The liver was excised after 6 h from sonoporation.…”

Advances in Ex Situ Tissue Optical Clearing

“…Although BABB clearing of liver can encouragingly be achieved within 1 day, no further data about its applications, microscopic imaging depth, and possible limitations were presented. Fumoto et al . compared liver transparency in CUBIC, SeeDB, and Clear T2 ‐treated samples to evaluate compatibility of organ TOC with gene delivery systems (Figure c).…”

“…c) Bright‐field images of liver lobes in: 1) PBS and cleared with 2) Clear T2 , 3) SeeDB, and 4) CUBIC (scale bar: 1 mm), with the latter method allowing for deep, up to 1500 µm, imaging. c) Reproduced under the terms of the CC‐BY Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/) . Copyright 2016, The Authors.…”