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Characterization of NeuN expression in the mouse neuronal NSC-34 cell line using RT-qPCR, immunological staining and siRNA-mediated gene suppression
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
2019 (English)Independent thesis Basic level (professional degree), 10 credits / 15 HE creditsStudent thesis
Abstract [en]

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.

Place, publisher, year, edition, pages
2019. , p. 26
Keywords [en]
Rbfox-3, Neuronal Nucleus, motor neuron, differentiation, secondary injury
National Category
Biomedical Laboratory Science/Technology
Identifiers
URN: urn:nbn:se:uu:diva-389757OAI: oai:DiVA.org:uu-389757DiVA, id: diva2:1338665
Educational program
Biomedical Laboratory Science Programme; Biomedical Laboratory Science Programme
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Examiners
Available from: 2019-08-21 Created: 2019-07-23 Last updated: 2019-08-21Bibliographically approved

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