Characterization of NeuN expression in the mouse neuronal NSC-34 cell line using RT-qPCR, immunological staining and siRNA-mediated gene suppression

• Main Author: Hallgren, Henrik
• Format: Others
• Language: English
• Published: Uppsala universitet, Institutionen för kvinnors och barns hälsa 2019
• Subjects: Rbfox-3; Neuronal Nucleus; motor neuron; differentiation; secondary injury; Biomedical Laboratory Science/Technology; Biomedicinsk laboratorievetenskap/teknologi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-389757

Background: Acute spinal trauma is followed by a secondary injury that causes additional damage to the tissue. The mouse neuronal hybrid cell line NSC-34 is planned for studies regarding this process, wherefore the cell line needed to be established in the laboratory and a proof-of-concept study needed to be performed. A suitable target gene for this study was Neuronal Nucleus (NeuN), a neuronal marker expressed in nearly all neuronal cells although not yet studied in NSC-34. Aim: The aim of this project was to characterize the expression of NeuN in differentiated and undifferentiated NSC-34 cells and silence gene expression by using siRNA. Methods: RT-qPCR was used to measure NeuN expression during passages 5 to 15 and a comparison was performed between one early and one late passage. Lipofectamine® RNAiMAX was used for siRNA-treatment in different concentrations and several different medium compositions were tested as differentiation media. Results: NeuN was expressed in passages 5 to 15, with decreased expression levels in passage 13 (ΔCt 15.36 ± 0.16) compared to passage 5 (ΔCt 15.09 ± 0.16), p < 0.05. The expression levels did not change after differentiation. siRNA-treatment yielded knockdown when using  high concentrations of the reagent (p < 0.05). Conclusion: NeuN was expressed in a stable, low level throughout passages 5 to 15 with a slightly decreased expression during later passages and no change after differentiation. The siRNA-treatment suppressed gene expression, although further optimization is needed to increase the suppression.