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.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-389757 |
| Date | January 2019 |
| Creators | Hallgren, Henrik |
| Publisher | Uppsala universitet, Institutionen för kvinnors och barns hälsa |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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