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Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing
Published on: January 20, 2018
Identification and expression analyses of poly [I:C]-stimulated genes in channel catfish (Ictalurus punctatus)
Ivanka Milev-Milovanovic1, Sai Majji, Venkata Thodima
1Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216, USA.
Insights
Channel catfish immune responses to viral infections were studied using poly [I:C] stimulation. This research identified over two dozen catfish genes, including interferon-stimulated genes (ISGs), that are upregulated during antiviral responses.
Area of Science:
- Immunology
- Comparative genomics
- Virology
Background:
- Channel catfish (Ictalurus punctatus) serve as a valuable model for studying lower vertebrate immune systems.
- Previous research identified antiviral antibodies, cytotoxic cells, and type I and II interferons (IFN) in catfish, indicating a robust antiviral response.
Purpose of the Study:
- To investigate additional components of the catfish antiviral immune response.
- To identify genes upregulated in catfish cells following double-stranded RNA (dsRNA) stimulation.
Main Methods:
- Catfish cells were treated with poly [I:C], a synthetic dsRNA analog.
- Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect specific gene homologs.
- Microarray analysis was employed to identify a broader range of upregulated genes in B cells and fibroblasts.
Main Results:
- Poly [I:C] induced type I interferon within 2 hours and interferon-stimulated genes (ISGs) within 6-12 hours.
- Detected ISG homologs included ISG15, Mx1, IFN regulatory factor 1 (IRF-1), inhibitor of apoptosis protein-1 (IAP-1), and CXCL10.
- Microarray analysis revealed upregulation of 13 genes in B cells and 24 genes in fibroblasts, with some identified as mammalian ISGs.
Conclusions:
- dsRNA, directly or via IFN induction, upregulates catfish gene products involved in antiviral defense.
- These upregulated genes contribute to inhibiting viral replication through individual or collective action.
- The findings highlight conserved and novel mechanisms in vertebrate antiviral immunity.
Abstract:
Channel catfish (Ictalurus punctatus) have proven to be an excellent model with which to study immune responses of lower vertebrates. Identification of anti-viral antibodies and cytotoxic cells, as well as both type I and II interferon (IFN), demonstrates that catfish likely mount a vigorous anti-viral immune response. In this report, we focus on other elements of the anti-viral response, and identify more than two dozen genes that are induced following treatment of catfish cells with poly [I:C]. We showed that poly [I:C] induced type I interferon within 2 h of treatment, and that characteristic interferon stimulated genes (ISGs) appeared 6-12 h after exposure. Among the ISGs detected by RT-PCR assay were homologs of ISG15, Mx1, IFN regulatory factor 1 (IRF-1), inhibitor of apoptosis protein-1 (IAP-1) and the chemokine CXCL10. Microarray analyses showed that 13 and 24 cellular genes, respectively, were upregulated in poly [I:C]-treated B cell and fibroblast cultures. Although many of these genes were novel and did not fit the profile of mammalian ISGs, there were several (ISG-15, ubiquitin-conjugating enzyme E2G1, integrin-linked kinase, and clathrin-associated protein 47) that were identified as ISGs in mammalian systems. Taken together, these results suggest that dsRNA, either directly or through the prior induction of IFN, upregulates catfish gene products that function individually and/or collectively to inhibit virus replication.
