Fig. 3. Viral transduction of spinal cord. (A) Transduction of adult mouse spinal cord with pONY8.0cRARß2. This culture was analyzed 8 days post-transduction with a FITC-labelled anti-Flag antibody (green). The area of green fluorescence shows a high level of transduction within the cultured cord. (B) The effect of transducing adult mouse spinal cord with pONY8.0cRARß2. Neurites, stained with an antibody against NF-200 and visualized with diaminobenzidine can be seen growing out from the explant (ex) after 8 days. (C) Light cycler PCR analysis for RARß2 expression in three individual RARß2 transduced explants (lanes 1-3) and three individual lacZ control transduced explants (lanes 4-6) cultured for 8 days. The levels of RARß2 expression relative to GAPDH are recorded in the blue columns. Explants 1-3 show between a 100-fold and a 300-fold induction of RARß2, whereas explants 4-6 show no expression. (D) Double-labelled adult mouse cord explant following transduction with pONY8.0cRARß2. Neurites were stained with NF-200 and visualized with a Texas Red-labelled secondary antibody and can be seen emerging from the lower part of the explant. Transduced cells were visualized with a FITC-labelled anti-Flag antibody (green). Double-labelled cells (yellow) are transduced cells that are also NF-200 positive. (E) Close-up of the explant in D showing green RARß2-transduced cells, red neurofilament-positive cells and yellow double-stained cells. (F) Adult mouse cord transduced with the control virus (pONY8Z) containing the lacZ marker gene. After X-gal staining, a good percentage of transduced cells (mostly neurons as judged by their size) is obtained. (G) The effect of transducing adult mouse cord with a herpes virus containing the RARß4 gene (pHSVRARß4). No neurites are induced. (H) The effect of transducing adult cord with a herpes virus containing the RARß2 gene (pHSVRARß2). In contrast to G, neurites are induced in this case, as visualized with NF-200 and diaminobenzidine. (I) RT-PCR analysis of adult mouse cords transduced with either the RARß2 gene (pHSVRARß2) or the RARß4 gene (pHSVRARß4 — lanes 5-7) and analyzed after 4 days. Lanes 1-3, RARß2 analysis: lane 1, no virus; lane 2, pHSVRARß2 transduction showing the presence of RARß2; lane 3, pHSVRARß4 transduction showing no RARß2 expression. Lanes 5-7, RARß4 analysis: lane 5, no virus; lane 6, pHSVRARß2 transduction showing the absence of RARß2; lane 7, pHSVRARß4 transduction showing the presence of RARß4 expression. Arrows to the right mark the position of RARß2, RARß4 and GAPDH.

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