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. 2016 Jun;22(6):883–895. doi: 10.1261/rna.053272.115

FIGURE 7.

FIGURE 7.

The 3′UTR of TH mRNA is sufficient to target reporter mRNA into distal axons. (A) Representative DIC and fluorescent images of reporter (tdTomato) mRNA in the cell bodies and distal axons of SCG neurons as visualized by in situ hybridization. tdTomato-specific fluorescent in situ hybridization signal appears as punctate mRNA granules in the distal axons. A significant amount of staining was detected in the axons of cell bodies transfected with the tdTomato construct containing the TH Full 3′UTR as compared to the control. The axons labeled with a negative control probe DapB (i.e., Bacillus subtilis dihydrodipicolinate reductase gene) produced little or no fluorescence under identical hybridization and wash conditions (data not shown). This probe was used as a negative control because it is a bacterial transcript not present in rat tissue. Scale bar, 200 µm. (B,C) Quantification of reporter mRNA fluorescence in situ hybridization signal in cell bodies (B) and distal axons (C) of SCG transfected neurons. Note that the signal intensity in distal axons of neurons transfected with Full 3′UTR [1–262 bp] is about five times that of distal axons of neurons transfected with the control plasmid, whereas the signal in transfected cell bodies do not differ. (D) tdTomato mRNA levels in the cell bodies of SCG neurons determined by qRT-PCR 72 h after DNA transfection. GAPDH served as an internal control. (E) tdTomato mRNA enrichment levels in the distal axons of SCG neurons 72 h after plasmid DNA transfection. GAPDH served as an internal control. All experiments were repeated three times with similar results. Histograms shown are representative of one of these experiments. Cell soma and axon tdTomato mRNA levels were visualized by fluorescence microscopy and quantified using Image J. Data are normalized to the fluorescence observed in nontransfected neurons. Each experiment was repeated three or more times with similar results. Error bars represent the standard error of the mean. Two-tailed Student's t-test (***) P ≤ 0.0001, (**) P ≤ 0.001. NT-SCG, nontransfected SCGs.