We focus on temporarily knocking down a protein called PTEN in injured neurons. While permanent PTEN deletion can drive axonal regeneration of the corticospinal tract (CST) , it also risks unwanted side effects, like uncontrolled scratching. By incorporating a doxycycline (dox)-inducible shRNA system, we can switch PTEN knockdown “on” to boost axonal growth and then turn it “off” to minimize long-term complications. We also track transgene expression in vivo through bioluminescence imaging, ensuring precise timing for safe and effective recovery
Right: PTEN knockdown in corticomotor neurons (CMNs) activates the mTOR (mammalian target of rapamycin) pathway and promotes axonal regeneration. Middle: AAV-retro-TetON/shPTEN is injected a cervical level 5 (C5) in the spinal cord enabling transduction of CMNs. Left (top): Dox administration in drinking water enables shPTEN and luciferase expression in CMNs. Left (bottom): In vivo transgene expression assessed by the in vivo imaging system (IVIS), allowing longitudinal measures in the same animal and precise assessment of transgene expression.
IVIS readings of healthy mice (no spinal cord injury) with intraspinal AAV-retro-TetOn-shPTEN injections.
Left: Mice received Dox (2mg/ml) two days after injection. Transgene expression was measured 5 minutes after luciferin injections over two weeks. Mice without Dox (-Dox) did not show bioluminescence (BLI), while mice with Dox (+Dox) showed BLI in the motor cortex.
Right: Brain sections stained for PTEN (green) and phosphorylated ribosomal protein S6 (pS6, red), indicating mTOR pathway activation. Dox treatment (+Dox) enhances PTEN knockdown, seen as “ghost cells,” and increases pS6 immunofluorescence, signaling mTOR activation.
These results demonstrate the precise regulation of gene expression in the presence of Dox, indicating that the AAV-retro-TetOn-shPTEN inducible promoter platform works as intended with the administration of Dox.
