Simultaneous expression of different transgenes in neurons and glia by combining in utero electroporation with the Tol2 transposon-mediated gene transfer system

Abstract

In utero electroporation is widely used to study neuronal development and function by introducing plasmid DNA into neural progenitors during embryogenesis. This is an effective and convenient method of introducing plasmid DNA into neural precursors and is suitable for manipulating gene expression in cells of the CNS. However, the applicability of this technique is comparatively limited to neuronal research, as the plasmid DNA introduced into neural progenitors during embryogenesis is diluted by cell proliferation and is not stably maintained in glial cells generated around and after birth. To overcome this limitation, we applied the Tol2 transposon system, which integrates a transgene into the genome of the host cell, to in utero electroporation. With this system, we confirmed that the transgene was effectively maintained in the progeny of embryonic neural precursors, astrocytes and oligodendrocytes. Using the glial promoters GFAP and S100beta, targeted and stable expressions of transgenes in glia were obtained, which enabled the expression of different transgenes simultaneously in neurons and glia. Glia-targeted expression of the transgene that causes neuronal migration defect was achieved without the defect. Thus, use of the Tol2 transposon system in combination with in utero electroporation is a powerful method for studying glia-neuron interactions in vivo.

Figure 1

Persistent expression of Tol2-flanked EGFP in proliferative cells during neural development. (A) Schematic representation of plasmids introduced into the dorsolateral telencephalon of mouse embryos. (B–D) Coronal vibratome sections showing cerebral cortex electroporated at E11.5 and fixed at E16.5. (B) Without transposase. (C,D) With transposase. High-magnification view of VZ is shown in (D). Radial glia-like processes are indicated by arrows. (E,F) Expressions of EGFP introduced at E12.5 were observed after 4 days (E16.5) and some were positive for radial glial marker (nestin, E) or mitosis marker (PH3, F). Views from XY, XZ and YZ are shown in the right lower panels. (G) Relationship between the relative efficiency of electroporation and the retention of Tol2-flanked EGFP with or without T2TP. The relative efficiency of electroporation is represented as the relative intensity of DsRed1 in CP, and the retention of Tol2-flanked EGFP was assessed by evaluating the relative intensity of EGFP in the SVZ/VZ. (H,I) Retention of Tol2-flanked EGFP in late-born neurons. (H–H′′) Coronal cryosection of the cerebral cortex electroporated at E12.5 with T2TP and fixed at P8. The pial surface is indicated by the white dotted line. (I) The numbers of neurons expressing Tol2-flanked EGFP and/or TagRFP. Nucleus was counterstained with HOECHST 33342 (B–D,H). Scale bars, 250 μm in (B,C,H,H′) and 25 μm in (D–F).

Figure 2

Tol2-flanked transgene was inherited by glial cells generated postnatally. (A) Emergence of non-neuronal cells labeled by Tol2-flanked EGFP (right, arrows). The dorsal telencephalon was electroporated to introduce pT2K-CAGGS-EGFP at E14.5 without (left) or with (right) pCAGGS-T2TP. The brain was analyzed at P16–17. (B,C) Higher magnification of EGFP-positive cells with non-neuronal morphology showing fibrous astrocytes in the marginal zone (B) and protoplasmic astrocytes in the cortical plate (C). (D–G) Immunostaining of vibratome sections against cell-type-specific markers. Higher magnification view of the insets in 1 is shown in 2–4. EGFP-expressing cells were positive for GFAP (D), S100 (E), or Olig2 (G) and negative for the neuronal marker, NeuN (F). Merged image of EGFP and cell type markers is shown in 2. Scale bars, 250 μm (A,D1,E1,F1,G1) and 25 μm (B,C,D2–4,E2–4, F2–4,G2–4). The pial surface is indicated by the white dotted line.

Figure 3

The Tol2 system resulted in postnatal expression of the transgene by mitotic cells. (A) Relationship between the relative efficiency of electroporation and the number of labeled glial cells at P16–17. (B) Retention of the transgene by mitotic cells at P5 after in utero electroporation at E14.5. Some EGFP-positive cells (green) were also positive for BrdU (magenta). A higher magnification view of the inset is shown. Scale bars, 50 μm (left panel) and 10 μm (right three panels). The pial surface is indicated by the white dotted line. (C) Schematic image of the Tol2 transposon system combined with the in utero electroporation method. (1) Without transposase, the plasmid introduced into progenitor cells was diluted following cell proliferation, and the expression of the transgene was restricted to cells born and becoming postmitotic soon after electroporation. (2) With the Tol2 transposase, a transgene flanked by the Tol2 cis-sequences was integrated into the genomic DNA of progenitor cells and inherited by its descendants, including neurons and glia.

Figure 4

The mouse GFAP promoter and S100β promoter drove targeted expression of the transgene in glial cells. (A–G) Astrocyte-targeted expression of EGFP driven by the mouse GFAP promoter or S100β promoter. The E14.5 telencephalon was electroporated to introduce pT2K-GFAP-EGFP (A,B) or pT2K-S100β-EGFP (C,D) along with pCAX2-TagRFP and pCAGGS-T2TP and analyzed at P10–12. TagRFP was expressed in pyramidal neurons (A,C). Higher magnification views of double-labeled cells with EGFP by each promoter and cell type marker are shown in (A2–7) (GFAP) or (C2–4) (S100) (white arrows). The pial surface is indicated by the white dotted line. (B,D) Percentages of GFAP- or S100-positive cells among EGFP-positive cells. (B: 124 cells/4 brains, D: 632 cells/5 brains) (E) Distribution of pT2K-GFAP-EGFP- or pT2K-S100β-EGFP-positive cells throughout the cortex. Vertical lines indicate the ratio of EGFP-positive cells in each layer against total EGFP-positive cells in the brain. Each line indicates an individual brain (GFAP; n=4, S100β; n=5). (F) Cells exhibiting typical morphology of oligodendrocytes. (G) Dual differential labeling of astrocytes and neurons by pT2K-S100β-EGFP and pCAX2-TagRFP, respectively. Scale bars, 250 μm (A,C, left), 50 μm (A, right six panels, F), 100 μm (C, right three panels) and 25 μm (G).

Figure 5

Targeted expression of constitutively active Rac1 (Rac1CA) under the control of the mouse GFAP or S100β promoter did not affect neuronal migration. (A,B) Expression of pT2K-GFAP- or pT2K-S100β-EGFP detected by anti-GFP immunostaining at E18.5. Some cells expressing EGFP were positive for the radial glial marker, nestin (arrows). (C–G) Distributions of cells expressing pT2K-CAG-EGFP-Rac1CA (C), pT2K-GFAP-EGFP-Rac1CA (D), pT2K-S100β-EGFP-Rac1CA (E), pT2K-GFAP-EGFP (F) or pT2K-S100β-EGFP (G). Glial-targeted expressions were visualized by anti-GFP immunostaining (D–G). Lateral neocortex electroporated at E14.5 was examined at P16 (C) or P10 (D–G). Scale bars, 50 μm (A,B), 250 μm (C, left panel, D–G) and 100 μm (C, three rightmost panels). The pial surface or the border between the ventricle and SVZ is indicated by a white or cyan dotted line, respectively.