To over-express miR-124, we cloned the pre-miRNA sequence of miR-124 into a plasmid. To determine cells expressing this miR-124 encoding plasmid, we added a GFP-coding sequence to the construct under the control of an IRES. Thus, miR-124 over-expressing cells also express GFP. To inhibit synaptopodin expression, we cloned a ShRNA sequence directed against synaptopodin into a plasmid. To determine cells expressing this ShRNA, we added a GFP-coding sequence to the construct under the control of an IRES. Thus, ShRNA expressing cells also expressed GFP. Two micrograms of these plasmids or the corresponding controls, were solubilized in 10 µl of i-Fect reagent (Neuromics, Edina, USA), and injected intrathecally between the L5 and L6 lumbar vertebrae every two days for a total of 3 injections, according to the manufacturer’s instructions and previously published experiments42 (link), 66 (link)–68 .
To test the therapeutic potential of miRNAs, miR-124 and C. elegans-specific Cel-miR-67 purified mature mimics were obtained from Eurogentec. Two micrograms of these miRNA mimics were solubilized in 10 µl of i-Fect reagent and injected intrathecally between the L5 and L6 lumbar vertebrae every 2 days for 14 days.
To test the therapeutic potential of miRNAs, miR-124 and C. elegans-specific Cel-miR-67 purified mature mimics were obtained from Eurogentec. Two micrograms of these miRNA mimics were solubilized in 10 µl of i-Fect reagent and injected intrathecally between the L5 and L6 lumbar vertebrae every 2 days for 14 days.