Efficient in vivo electroporation of the postnatal rodent forebrain

Abstract

Functional gene analysis in vivo represents still a major challenge in biomedical research. Here we present a new method for the efficient introduction of nucleic acids into the postnatal mouse forebrain. We show that intraventricular injection of DNA followed by electroporation induces strong expression of transgenes in radial glia, neuronal precursors and neurons of the olfactory system. We present two proof-of-principle experiments to validate our approach. First, we show that expression of a human isoform of the neural cell adhesion molecule (hNCAM-140) in radial glia cells induces their differentiation into cells showing a neural precursor phenotype. Second, we demonstrate that p21 acts as a cell cycle inhibitor for postnatal neural stem cells. This approach will represent an important tool for future studies of postnatal neurogenesis and of neural development in general.

Figure 1. Electroporation of the postnatal forebrain.

(a) A virtual line (red) connecting the right eye to the craniometrical landmark lambda served as positional marker for DNA injection. The incision point is indicated as a dot. Lateral bars indicate the position of the electrodes. (b) Table recapitulating the tested voltages, survival after electric shocks and the success rate concerning GFP-expression. (c) Example of a coronal section through a GFP-electroporated forebrain at the level of the lateral ventricle (LV) at 2dpe. A section containing relatively few positive cells was selected to simplify identification of the different cell types. The section was counterstained with Hoechst 33258 to facilitate orientation. Strongly GFP positive cells with the morphology of radial glia are visible in the ventricular zone (arrowheads), while cells with generally lower levels of GFP expression are organized mainly parallel to the ventricular surface (see high magnification of the boxed area in the insert). Direction of processes is suggestive of migration towards the dorso-lateral edge of the ventricle (arrow). (d) Evaluation of electroporation efficiency. Histological sections were grouped in bins representing sections containing more or less than 200 cells. 75.8% of the sections were classed in the higher group. ST: striatum. Scale bar: 40 µm; 20 µm in the insert.

Figure 2. Characterization of the electroporated cells and their offspring.

(a) Eight hours post electroporation (8hpe), cells with the typical morphology of radial glia exhibit strong GFP expression in the somata and processes. (b) At 2dpe radial glia cells are generally surrounded by cells showing weaker and variable GFP expression. These have no contact with the LV and show a generally tangential orientation (arrowheads). (c) Labelling of GFP positive cells in the ventricular zone (c) with anti-RC2 antibody (c') verifies their radial glia identity (merged image in c”). Mark that weaker GFP expressing neuronal precursors are not labelled for RC2, as expected. (d) a sub-population of GFP+ radial glia cell (d) expresses the mitotic marker PH3 (d'; 1dpe), merge in (d”) and are therefore proliferating. (e) Nuclear RFP-expression (in red) in combination with PSA-NCAM staining (green, antibody MenB) in the RMS at 4dpe identifies offspring of the electroporated cells as migratory neuronal precursors. (f) At 4dpe large numbers of GFP expressing cells arrive in the OB via tangential migration in the RMS. (g) At 6dpe GFP positive cells switch to radial migration and invade the granule cell layer (GCL). (h) After 15dpe large amounts of GFP expressing cells with complex morphologies can be identified in the OB. Higher magnification shows that these cells have neuronal morphology of the granule (i) and periglomerular (j) type. RMS: rostral migratory stream; GL: glomerular layer; LV: lateral ventricle. Scale bar: 20 µm in a,b,e; 15 µm in c,d; 300 µm in f,g; 100 µm in g; 30 µm in i,j.

Figure 3. Expression of hNCAM140 and p21 in the postnatal forebrain.

(a–h) Expression of human NCAM 140 in wt (a–d) and NCAM deficient (e–h) mice. Co-electroporation of GFP (a) with a hNCAM-140 expression plasmid (b, hNCAM recognized by specific antibody 123C3) in the wildtype context led to slightly enhanced PSA expression in the PSA positive SVZ (c). In contrast, hNCAM140 expression in the NCAM-deficient context (f) induced specific expression of PSA (g, merge in h) in the transfected cells. (i–k) Phenotypic consequences of hNCAM140 expression in wt mice. Co-electroporation of hNCAM140 and GFP (j) induced an increase in cells with the characteristics of migratory neuronal precursors (arrowheads) in relation to radial glia cells. (k) Quantification, open bars: radial glia; black bars: neuronal precursors – control: radial glia 12.23 +/− 0.22 cells per section, neuronal precursors 8.8 +/− 0.24 cells per section, at least 3 sections per animal, n = 6; hNCAM140: radial glia 6.59 +/− 0.06 cells per section, p<0.0001, neuronal precursors 25.53+/− 0.36 cells per section, p<0.0001, at least 3 sections per animal, n = 7). (l) Overexpression of p21 via postnatal electroporation induced a significant reduction of proliferation in the affected cells as measured by BrdU incorporation (control: 25.66 +/− 2.02%, at least 4 sections per animal, n = 7; p21: 7.79 +/− 2.15%, at least 4 sections per animal, n = 5; p<0.001). Scale bar: 10 µm a–h, 30 µm in i,j.