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Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death
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Programmed cell death 4 in bacterially-challenged Apostichopus japonicus: Molecular cloning, expression analysis and
Zhimeng Lv1, Chenghua Li1, Yina Shao1
1School of Marine Sciences, Ningbo University, Ningbo, Zhejiang 315211, PR China.
Molecular Immunology
|June 6, 2016
Summary
A novel sea cucumber gene, AjPDCD4, is involved in cellular defense. Knocking down AjPDCD4 reduces UV-induced DNA damage and reactive oxygen species (ROS) production in sea cucumber immune cells.
Area of Science:
- Marine biology
- Molecular biology
- Immunology
Background:
- Programmed cell death 4 (PDCD4) is a key regulator of cellular signaling and immune responses, often involving TOLL pathways.
- Understanding PDCD4 homologs in invertebrates can provide insights into conserved cellular defense mechanisms.
Purpose of the Study:
- To clone and characterize a novel PDCD4 homologue gene (AjPDCD4) from the sea cucumber Apostichopus japonicus.
- To investigate the role of AjPDCD4 in cellular responses to stress, specifically UV-induced damage and reactive oxygen species (ROS) production.
Main Methods:
- Rapid amplification of cDNA ends (RACE) technique was used for gene cloning.
- Phylogenetic analysis was performed to determine evolutionary relationships.
- Spatial expression analysis and quantitative real-time PCR were employed to assess gene expression levels.
- RNA interference (RNAi) was used to knockdown AjPDCD4 expression in coelomocytes.
Main Results:
- A full-length AjPDCD4 cDNA was successfully cloned, encoding a 450-amino acid protein with two MA3 domains.
- AjPDCD4 mRNA was highly expressed in tentacles and lowly in muscle tissue, with lower expression in coelomocytes.
- Expression of AjPDCD4 was significantly downregulated by Vibrio splendidus challenge and LPS exposure.
- Knockdown of AjPDCD4 repressed UV-induced ROS production and DNA damage, accompanied by upregulation of NF-kappa B homologue p105.
Conclusions:
- AjPDCD4 is a functional PDCD4 homologue in Apostichopus japonicus.
- AjPDCD4 plays a role in modulating DNA damage and ROS production in sea cucumber coelomocytes.
- The findings suggest AjPDCD4 may influence the TLR pathway in response to cellular stress.
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