[논문]Long Double-stranded RNA Induces Sequence-specific RNA Interference and Type I Interferon Responses in Rock Bream (Oplegnathus fasciatus)

Zenke, Kosuke; Kim, Ki-Hong

doi:10.5657/fas.2010.13.1.056

Long Double-stranded RNA Induces Sequence-specific RNA Interference and Type I Interferon Responses in Rock Bream (Oplegnathus fasciatus) 원문보기

Fisheries and aquatic sciences, v.13 no.1, 2010년, pp.56 - 62

Zenke, Kosuke (Department of Aquatic Life Medicine, Pukyong National University) , Kim, Ki-Hong (Department of Aquatic Life Medicine, Pukyong National University)

Abstract ▼ AI-Helper

To determine whether long double-stranded RNA (dsRNA) induces RNA interference and type I interferon (IFN) responses in fish, long dsRNAs encoding enhanced green fluorescent protein (EGFP), GFPuv, and polyinosinic-polycytidylic acid sequences were co-injected with an EGFP expressing plasmid, into rock bream (Oplegnathus fasciatus). We investigated the EGFP mRNA and protein levels, and the transcriptional responses of dsRNA-dependent protein kinase and Mx1 genes. Long dsRNAs were strong inducers of a type I IFN response in rock bream, resulting in nonspecific suppression of exogenous gene expression. Furthermore, sequence-specific knockdown of exogenous gene expression at the mRNA level was detected at an early phase (24 h). These results suggested that long dsRNA may inhibit exogenous gene expression through an early mRNA interference response and a later type I IFN response in fish.

주제어

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제안 방법

5 μg pPA-EGFP plasmid alone (positive control) or mixed with 5 μg of dsRNA-EGFP, dsRNA- GFPuv, or poly I:C; or with PBS alone (negative control). At 24 and 72 h after injection, the five groups were sampled, and a portion of muscle (about 5x5x5 mm) near the injection site was excised from the fish. For fluorescence quantification, half of the excised tissue was homogenized in 0.

대상 데이터

PCR amplicons were electrophoresed in a 1% agarose gel and visualized using ethidium bromide staining. Images of the stained gels were imported into Quantity-One image analysis software using a Gel Doc XR imaging device (Bio-Rad). Band intensities of the target genes were normalized to that of P-actin to estimate the relative mRNA levels.

데이터처리

One-way ANOVA followed by Turkey's HSD test was used for statistical analysis.

이론/모형

The lysate was centrifuged at 8, 000 xg for 10 min, and the supernatant containing the soluble fraction was collected. The quantity of total protein in the supernatant was measured using the BCA method. Fluorescence was measured using a Polarion fluorescence plate reader (Tecan).

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