Figure 3.

Design and validation of Egr3-flx conditional knock-out and sympathetic neuron Cre-driver mice. A, The mouse Egr3 genomic locus was modified to contain forward-oriented loxP sites in intron 1 and downstream of the stop codon using homologous recombination in E. coli. A frt-flanked Neo selection cassette was inserted upstream of the 3′ loxP site and the Egr3-flx (Egr3^+/f) allele was generated by removing the Neo selection cassette from the germline using CAG-FlpE mice. B, Left, Electroporated G418 selected ES cell clones were screened using long-range PCR with primers that flanked the 3′ recombination arm (primers: OT1111 and OT1114). Right, PCR confirmed ES cell clones were screened by Southern blotting analysis using a 713 nt probe (5′ ext) located upstream of the 5′ recombination arm that was hybridized to BglII restricted genomic DNA. C, To validate the conditional knock-out allele, CaMKII-iCre transgenic mice (Casanova et al., 2001) were mated to Egr3^+/f mice to ablate Egr3 in postnatal forebrain and hippocampal dentate gyrus neurons. As expected, Egr3 protein was robustly expressed in CaMKII-iCre⁻; Egr3^f/f (left; Ctl) dentate gyrus neurons but was completely absent in CaMKII-iCre⁺; Egr3^f/f (right; cKO) neurons. D, The human DβH promoter was used to generate transgenic mice that express a Cre-recombinase and axon localized β-galactosidase (τlacZ) bicistronic message in noradrenergic neurons (designated DCτlZ transgenic mice). E, Cre-recombinase and lacZ expression were detectable in all TH+ sympathetic neurons. F, To validate the efficiency of DCτlZ mice to recombine loxP sites in sympathetic neurons, they were mated to Rosa-eGFP reporter mice (Belteki et al., 2005). DCτlZ⁻; Rosa-eGFP^+/f mice showed no expression of eGFP, whereas DCτlZ⁺; Rosa-eGFP^+/f mice showed expression of eGFP in all sympathetic neurons (SCG shown here). Scale bars: C, 200 μm; E, F, 100 μm.