Figure 3.

iBAC-MAPT transgene expression in mouse primary neurons is regulated by endogenous developmental cues. (a) Vector backbone-encoded GFP expression 48 hours post-transduction confirms highly efficient delivery of iBAC-MAPT (MOI = 1) to primary mouse neuronal cultures prepared from P0 newborn pups. (b) Species-specific RT-PCR was performed on RNA extracted from transduced cells 6, 12, and 24 hours post-transduction. MAPT transgene expression is first seen 6 hours post-transduction. RNA extracted from human G16-9 glioblastoma cells is a positive control for MAPT expression. (c) P0 mouse primary cultures were transduced with iBAC-MAPT after 6 days in culture. RNA was extracted 1, 2, 3, or 7 days post-transduction and species-specific RT-PCR was performed. Relative expression of the exon 10⁺(4R) splice forms of both MAPT and Mapt increases with developmental maturation of neurons in culture. (d) P0 mouse primary cultures were maintained in vitro for either 5 or 13 days before transduction with iBAC-MAPT (MOI = 1). RNA was extracted 2 days post-transduction and species-specific RT-PCR was performed. The relative expression of both MAPT and Mapt exon 10⁺(4R) increased over time. Measuring relative band strength by quantifying pixel intensity showed the exon 10⁺(4R) form represents 100% of Mapt and ~20% of MAPT expression after 15 days in culture. Primer pairs: Hx10: human exon 10; Mx10: mouse exon 10.