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. 2013 Mar 6;33(10):4570–4583. doi: 10.1523/JNEUROSCI.5481-12.2013

Figure 1.

Figure 1.

Design and validation of Egr3-Cre knockin mice to trace historical Egr3 expression in tissues. A, Gene targeting in ES cells was used to replace both exons and the intron of Egr3 with the open reading frame encoding nuclear localized Cre-recombinase (nlsCre). The Egr3-Cre (Egr3+/Cre) allele was generated by removing the frt-site flanked Neo-positive selection cassette from the germline using CAG-FlpE mice. B, Left, Electroporated G418 selected ES cell clones were screened using long-range PCR to amplify across the 3′ recombination arm (primers: OT1182 and OT1114). Right, PCR confirmed ES cell clones were screened by Southern blotting using a 713 nt probe located upstream of the 5′ recombination arm (5′ ext), which was hybridized to BglII restricted genomic DNA. C, To validate the appropriate expression and function of Cre-recombinase, Egr3+/Cre mice were mated to Ai14 Cre-recombinase reporter mice that irreversibly express fluorescent tdTomato protein after Cre-recombinase mediated loxP-site recombination (Madisen et al., 2010). Dentate gyrus granule neurons expressed Egr3 protein in (left) Egr3+/Cre; Ai14+/f and Egr3+/+; Ai14+/f mice, but only in (right) Egr3+/Cre; Ai14+/f mice did they also express the tdTomato reporter. D, In Egr3+/Cre; Ai14+/f double heterozygous mice, Egr3 reporter (T) was observed in arteries along which sympathetic nerves travel (splenic artery shown here), which colocalized in endothelial cells with the vascular endothelial marker PECAM (arrowheads). The tdTomato reporter is localized primarily in the endothelial nucleus whereas the PECAM protein is expressed on the endothelial membrane surface. E, Left, Sympathetic nerves labeled with TH and T (data not shown) were observed coursing along the adventitia of arteries (arrowheads). Right, Within nerves (sciatic nerve shown here) T was colocalized in S100+ Schwann cells (data not shown) and a fraction (28%) of TH+ unmyelinated axons was closely associated with T+ Schwann cells (TH+; T+) in sectioned nerves. Scale bars: C, 200 μm; D, E, left, 50 μm; E, right, 5 μm.

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