Figure 2. In vitro–targeted mutagenesis in neurons isolated from HSV-infected mice.
(A) Schematic of neuronal culture generation. Five mice were infected with 2 × 105 PFU of HSV-1(F) in the right eye following corneal scarification for each time point and left for 7, 14, or 32 days prior to collection of the right TGs. After tissue dissociation by enzymatic digestion, neuronal cultures were established, and cells were cultured in medium supplemented with 100 μM ACV. (B) Experimental timeline. Cells were cotransduced in quadruplicate with either scAAV8-sCMV-HSV1m5 and scAAV8-sCMV-Trex2 or scAAV8-sCMV-HSV1m8 and scAAV8-sCMV-Trex2 at an MOI of 1 × 106 vg/AAV/neuron for 3 days prior to analysis. (C) Timeline for the evaluation of the effect of TSA. Nine mice were infected with 2 × 105 PFU in the right eye following corneal scarification and, 7 days later, the right TGs were collected. After tissue dissociation by enzymatic digestion, neuronal cultures were established and cells were cultured in medium supplemented with 100 μM ACV. Eight wells per condition were cotransduced with scAAV8 vectors expressing HSV1m5 and Trex2, HSV1m8 and Trex2, NV1 and Trex2, or GFP and mCherry at an MOI of 1 × 106 vg/AAV/neuron for 3 days, after which 4 wells were left untreated while the other 4 were treated with 300 nM TSA for 1 day prior to analysis. (D) Mutagenic event detection by T7E1 assay. The HSV regions containing the target site were PCR amplified from total genomic DNA obtained from pooled quadruplicate wells, subjected to T7E1 digest, and separated on a 3% agarose gel. mw, molecular weight size ladder; red asterisks indicate cleavage products. G, GFP ; NV, NV1; mC, mCherry; T, Trex2; M8, HSV1m8, m5, HSV1m5; T7, T7E1. (E) HSV target sequences from enzyme-treated neurons with and without TSA. ACV, Acyclovir.