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.

Fig 1. Comparison of tissue clearing methods.

(A, E) PBS, (B, F) Clear^T2, (C, G) SeeDB, and (D, H) CUBIC. ZsGreen1 expression in the liver was detected by CLSM. (A–D) Depth coding. The color chart indicates depth on the Z-axis. (E–H) Maximum intensity projection (X–Z plane). Scale bar: 100 μm. Acquisition conditions: lens, 20× dry; laser, 488 nm; output, 5%; emission, 492–540 nm; master gain, (A, E) 432–1200, (B, F) 433–1129, (C, G) 405–1129, and (D, H) 492–1115.

Fig 2. Transmission color images of the liver after tissue clearing.

(A) PBS, (B) Clear^T2, (C) SeeDB, and (D) CUBIC. Each lattice indicates 4×4 mm.

Fig 3. Comparison of the observable depth with fluorescent proteins ZsGreen1 (green) and tdTomato (red) in the liver treated with CUBIC.

(A) Maximum intensity projections (X–Z plane). Scale bar: 100 μm. (B) Single plane images at the indicated depth (μm) from the liver surface. Scale bar: 50 μm. Acquisition conditions: lens, 20× dry; laser, 488 nm (output, 1%–12%; emission, 492–540 nm; master gain, 480–766) and 543 nm (output, 5%–100%; emission, 550–670 nm; master gain, 600–906).

Fig 4. Transmission color images of each tissue treated with CUBIC.

Each lattice indicates 4×4 mm.

Fig 5. Observations of ZsGreen1 expression in various tissues treated with CUBIC.

Three-dimensional maximum intensity projection. Acquisition conditions: lens, 20× dry (A, C, E, H, and J) or 40× oil-immersion (B, D, F, G, and I); laser, 488 nm; output, 5%; emission, 493–589 nm; master gain, (A) 951–1130, (B) 1081–1110, (C) 887–1152, (D) 732, (E) 823–977, (F) 732, (G) 1081–1110, (H) 1081–1110, (I) 1081–1110, and (J) 978–1146.

Fig 6. Intracellular fate of plasmid DNA in transgene expression-positive cells of the liver treated with CUBIC after hydrodynamic injection.

Three-dimensional maximum intensity projections and enlarged planes. Scale bar: 5 μm. Nuclei (blue), ZsGreen1 (cyan), Cy5-plasmid DNA (red), (A) TMR-dextran (green), and (B) TMR-plasmid DNA (green). Acquisition conditions: (A) lens, 63× oil-immersion; laser, 405 nm (output, 12.5%; emission, 425–460 nm; master gain, 781–862), 488 nm (output, 0.5%; emission, 503–533 nm; master gain, 580), 543 nm (output, 3.0%; emission, 548–627 nm; master gain, 1153) and 633 nm (output, 4.0%; emission, 639–748 nm; master gain, 1150); (B) lens, 63× oil-immersion; laser, 405 nm (output, 12.5%; emission, 414–480 nm; master gain, 752–815), 488 nm (output, 0.4%; emission, 525–531 nm; master gain, 525–531), 543 nm (output, 5.0%; emission, 585–647 nm; master gain, 1150), and 633 nm (output, 4.0%; emission, 639–690 nm; master gain, 1132).