Fig. 4

Fig. 4 A distal enhancer element directs glial ctgfa expression after SCI. (A) Sequences within a 1 Kb enhancer element located 3-4 Kb upstream of the ctgfa transcriptional start site were combined with a 96 bp mouse Fos minimal promoter and subcloned upstream of an EGFP-expressing cassette. The clone was co-injected into one-cell-stage wild-type embryos, and three founders were isolated for propagation. (B) EGFP and Gfap immunostaining was used to assess reporter expression at 10 dpi and in uninjured tissue. SC tissues from three independent lines of 1Kb-ctgfa:EGFP transgenic animals (L1, L2 and L3) are shown. Cross-sections are shown at 150 (proximal) and 450 (distal) µm rostral to the lesion. Dashed lines delineate central canal edges. Arrows point to EGFP expression in ventral ependymal progenitors. (C) Quantification of EGFP fluorescence in −5.5Kb-ctgfa:EGFP and 1Kb-ctgfa:EGFP-L1 transgenic animals. SC cross-sections 150 µm (proximal) and 450 µm (distal) rostral to the lesion were quantified. Dots indicate individual animals and sample sizes are indicated in parentheses. Error bars represent s.e.m. ns, not significant (P>0.05; unpaired t-test with Welch's correction). (D) Schematic of wild-type ctgfa locus (ctgfa^WT allele) and the targeting strategy adopted to delete sequences within the 1 Kb enhancer element upstream of ctgfa (ctgfa^stl683 allele). (E) Quantitative RT-PCR for ctgfa in ctgfa^{stl683/ stl683} relative to ctgfa^WT. eef1a1l1 was used as a loading control. Relative expression was normalized to eef1a1l1 expression and to expression levels in ctgfa^WT siblings. SC tissues from four animals were pooled into a single biological replicate. Four biological replicates were used. Dots represent biological replicate pools. Error bars represent s.e.m. *P<0.05 (unpaired t-test with Welch's correction). Scale bar: 50 µm.