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. 2014 Dec 10;34(50):16594–16604. doi: 10.1523/JNEUROSCI.2585-14.2014

Figure 2.

Figure 2.

Neuronal expression of dnSNARE and 2 reporter genes LacZ and EGFP in dnSNARE mice. A, Top, Schematic outlining a collection of cortical neurons from 8-d old C57BL/6 WT or dnSNARE mice under Off-Dox condition using density gradient-based dissociation method and FACS. Neurons were enriched and separated from myelin debris by collecting Fraction 2 and 3 before FACS. Bottom, Under Off-Dox condition, human GFAP promoter drives the expression of 3 reporter units, dnSNARE, LacZ, and EGFP, in dnSNARE mice. B, qPCR analysis of markers expression in purified mouse PSA-NCAM+ population. Neuronal markers; RNA binding protein fox-1 homolog 3 (Rbfox3), Tubulin, beta 3 (Tubb3), Neurofilament, light polypeptide (Nefl), Neurofilament, medium polypeptide (Nefm), Glutamic acid decarboxylase 1 (Gad1), Glutamic acid decarboxylase 2 (Gad2), Choline acetyltransferase (Chat) and Solute carrier family 6, member 4 (Slc6a4). Astrocyte markers; Aquaporin 4 (Aqp4), Glial fibrillary acidic protein (Gfap), Excitatory amino acid transporter 2 (Slc1a2) and aldehyde dehydrogenase family 1, member L1 (Aldh1l1). Oligodendrocyte precursor marker, platelet derived growth factor receptor, α polypeptide (Pdgfra). Oligodendrocyte marker, myelin basic protein (Mbp). Microglial marker, Integrin α M (Itgam). n = 7 biological replicates. C, Immunolabeling analysis of neuronal marker expression in purified mouse PSA-NCAM+ population. Neuronal marker NeuN is highly enriched in cortical PSA-NCAM+ populations compared with PSA-NCAM− populations in which the astrocyte marker Aqp4 is highly expressed. Scale bar, 100 μm. D, qPCR analysis of PSA-NCAM+ and PSA-NCAM− populations showed neuronal expression of dnSNARE and reporter transgenes (LacZ and EGFP) in dnSNARE mice (n = 4 biological samples) and no expression in both populations isolated from WT mice (n = 3 biological samples). E, Expression of the tTA transgene in PSA-NCAM+ and PSA-NCAM− populations in dnSNARE mice but not in WT mice (n = 4 each). F, Relative expression of transgenes (dnSNARE, LacZ, and EGFP) in cerebral cortex in adult Off-Dox dnSNARE mice compared with On-Dox condition (n = 4). G, Enrichment of neuronal marker gene Rbfox3 expression in neurons isolated from adult dnSNARE mice compared with tissue (n = 3). H, Neuronal expression of transgenes (dnSNARE, LacZ, and EGFP) in adult dnSNARE mice under Off-Dox condition compared with On-Dox condition (n = 3). Error bars indicate SEM in B and D–H.