Figure 10.

Silencing of a single Nmnat2 allele is sufficient to induce neurite degeneration in primary SCG neuron cultures. A, Schematic showing how sequential exposure to FLPe and Cre recombinases allows restoration of expression from the targeted Nmnat2 allele, and subsequent resilencing, through successive inversion of the gene trap facilitated by pairs of inversely orientated heterotypic FLPe and Cre recombinase target sequences (colored triangles) (Schnutgen et al., 2005). B, C, Representative RT-PCR (B) and representative immunoblot (C) showing Nmnat2 mRNA and NMNAT2 protein levels in brains of wild-type, Nmnat2^+/gt(inv), and Nmnat2^{gt(inv)/gt(inv)} P1 pups. Actb and βIII-Tubulin represent sample references. D, Survival of DsRed2-labeled distal neurites (top) and neuron viability (bottom) for wild-type (+/+), Nmnat2^+/gt(inv), and Nmnat2^{gt(inv)/gt(inv)} SCG neurons injected with a Cre-EGFP expression vector and pDsRed2 (both at 10 ng/μl). The mean percentage (± SEM) of healthy DsRed2-labeled neurites or viable cell bodies remaining each day after injection, of those (both healthy and abnormal) present in the same field at 2 d after injection, is plotted. Neurite data are from multiple fields in five independent injection experiments for each genotype. Injected cell bodies with normal gross morphology were classed as viable. We previously demonstrated that morphology revealed by fluorescent proteins is a reliable indicator of neuronal viability (Gilley and Coleman, 2010). p values were calculated from individual experiment averages using two-way repeated-measures ANOVA with Bonferroni post hoc correction. We also saw no increase in neurite degeneration (relative to wild-type) for Nmnat2^+/gt(inv) and Nmnat2^{gt(inv)/gt(inv)} SCG neurons injected with a control EGFP expression vector, rather than Cre-EGFP (data not shown). E, Representative fields of DsRed2-labeled distal neurites from experiments quantified in D at 2 and 6 d after injection.