Abstract:

Objective ·To develop an in vivo genetic manipulation strategy based on the replication-competent avian sarcoma/leukosis virus (RCAS)‒tumor virus receptor A (TVA) gene delivery system to specifically target oligodendrocyte lineage cells (OLCs), enabling efficient labeling and targeted modulation of these cells. Methods ·Using RCAS-EGFP (enhanced green fluorescent protein) as the backbone vector, shRNA sequences targeting G protein-coupled receptor 17 (GPR17) or a non-targeting Scramble control sequence were inserted. Following viral packaging, viral titers were determined using the fluorescent focus units (FFU). In Olig2-TVA-IRES-Cre transgenic mice, which allow specific RCAS infection of OLCs, stereotactic injections of either the RCAS-shRNA(Scramble)-EGFP (control group) or the RCAS-shRNA(GPR17)-EGFP (knockdown group) were performed into the hippocampus (HPC) or the arcuate nucleus (ARC), respectively. Immunofluorescence staining was conducted for the oligodendrocyte lineage transcription factor 2 (Olig2), the neuronal marker neuronal nuclei (NeuN), and the astrocytic marker glial fibrillary acidic protein (GFAP) to validate viral transduction specificity. To validate knockdown efficiency of the virus, immunofluorescence staining for the target protein GPR17 was performed in both the control group and the knockdown group. The expression levels of GPR17 protein in the ARC were measured by Western blotting before and after knockdown. To evaluate the impact of GPR17 knockdown on cellular differentiation, immunofluorescence staining for the oligodendrocyte precursor cell marker platelet-derived growth factor receptor α (PDGFRα) and the mature oligodendrocyte marker CC1 was performed in both groups. Results ·The packaged viral titer was 5×10⁷ PFU/mL. Immunofluorescence analysis revealed high specificity of infection, with 77.99% and 75.02% of GFP-positive cells co-localizing with Olig2 in the HPC and ARC, respectively. Additionally, co-localization of GFP with NeuN or GFAP was scarcely detected. The knockdown efficiency was confirmed by a significant reduction in GPR17-positive cells among GFP-positive populations. In the HPC, the percentage of GPR17⁺/GFP⁺ cells decreased from 71.85% in the control group to 34.34% in the knockdown group (P<0.001), indicating a marked reduction in GPR17 expression. In the ARC, the proportions of GPR17⁺/GFP⁺ double-positive cells were 78.12% in the control group and 31.72% in the knockdown group. Western blotting results showed that the expression level of GPR17 protein in the ARC was significantly reduced following viral knockdown. In the HPC, CC1⁺ mature oligodendrocytes exhibited an increasing trend after GPR17 knockdown, though the difference was not statistically significant. However, PDGFRα⁺ oligodendrocyte precursor cells decreased significantly (P<0.001). In the ARC, knockdown of GPR17 significantly increased CC1⁺ mature oligodendrocytes and reduced PDGFRα⁺ oligodendrocyte precursor cells (both P<0.001). Conclusion ·The RCAS‒TVA gene delivery system enables efficient in vivo labeling and targeted knockdown of genes in oligodendrocyte lineage cells.

Key words: oligodendrocyte lineage cell, replication-competent avian sarcoma/leukosis virus (RCAS), tumor virus receptor A (TVA), G protein-coupled receptor 17 (GPR17)