Figure 4.

Use of 2A strategy to express other activators, fluorescent proteins, and secreted proteins. A, Diagram of the rAAVs for dual virus activator-responder approach to test for itTA and KO coexpression by 2A self-processing. B, Western blots of protein extracts from rAAV-Syn-itTA2A-KO-infected dissociated hippocampal neuron in culture at DPI 10. The 30.5 kDa itTA2A fusion protein was detected and 55.4 kDa full-length protein was faintly visible. I+II, Coinfected neurons. Ctrl, Noninfected neurons. C, Confocal images of a brain section from a mouse coinjected with rAAV-Syn-itTA2A-KO and the responder virus rAAV-Ptetbi-iCre-Venus 14 d after injection. Cells expressing itTA are labeled with the coexpressed red fluorescent protein KO. The itTA-driven Venus expression from the responder virus was detected by green fluorescence. Scale bars, 200 μm. D, At higher magnification, many neurons were fluorescently labeled in the infected cortical area, and most of them showed coexpression of KO and Venus as expected. Scale bars, 50 μm. E, Left, Dissociated primary hippocampal neurons at DPI 10 with rAAV-Syn-Noggin2A-Venus. Right, Western blots from rAAV-Syn-Noggin2A-Venus-infected primary hippocampal cultures at DPI 10. Three different forms of Noggin were detected in hippocampal neuron lysate, and only the 32 kDa secreted form was detected in the culture medium. Ctrl, Noninfected neurons.