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Next generation sequencing establishes the differentially expression profiles of transgenic rainbow trout bearing cecropin P1 transgene

ABSTRACT: Purpose: mRNA-Seq to reveal the differentially expression pattern of genes in innate/adaptive immune relevant pathways, and combining to the previous DNA microarray analysis to address the immunomodulatory effects of cecropin P1 transgene in rainbow trout. Methods: Total RNA were isolated from three tissues, spleen, liver and kidney, from two individuals of two families of transgenic and one family of non-transgenic fish. The sequencing was conducted by Illumina HiSeqTM 2000 followed by Trinity de novo assembly into unigenes (the reference library constructed by non-trasngenic control fish), and, finally, been normalized to RPKM to represent the expression levels. Results: By sorting the RPKM ratio among transgenic and non-transgenic with threshold > two fold, DEGs can be defined. By RT-qPCR analysis with random selected DEGs (n=36), the reliability of RNA-Seq was comfirmed with high linear correlation (R square= 0.81). By the enrichment analysis via GeneCodis and pathway analyses via customized pathway editor tool, the functional perturbed immune relevant pathway can be determined in tissue specific manner. Conclusion: By combining the results of RNA-Seq and previous DNA microarray, the immunomodulatory effects of cecropin P1 transgene were estalished in the host, i.e. rainbow trout. By combining the direct eliminating pathogens ability and indirect immunomodulatory stimulating host immune responces of cecropin P1, the host elevates its disease resistance to pathogenic infections.

ORGANISM(S): Oncorhynchus mykiss

PROVIDER: GSE111373 | GEO | 2018/03/03

REPOSITORIES: GEO

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Project description:Abstract. The molecular pathways in embryonic vertebrates leading to gonad formation in each sex are incompletely understood. The purpose of this study was to identify novel genes that could be associated with sex-specific gonadal differentiation in a fish, the rainbow trout (Oncorhynchus mykiss). This study was facilitated by a custom microarray based on 7,681 genes derived from embryonic rainbow trout gonad cDNA libraries and public databases. Gonad samples for total RNA isolation were obtained from pvasa-green fluorescent protein (pvasa-GFP) transgenic rainbow between 300 and 700 degree days of development post-fertilization. The transgenic fish permitted the collection of gonads from embryonic rainbow trout during the period of molecular sex differentiation in advance of any morphologically distinguishable characteristics of sex. A bioinformatic method was used with the microarray data that looked for strong associations in gene expression patterns between known sex differentiation genes (the target genes) and novel genes (the target-associated genes) previously not allied with sex differentiation in fishes. The expression patterns of representative targets genes from both sexes and their target-associated genes were independently confirmed by real-time reverse transcription-polymerase chain reaction to support the validity of the bioinformatics method employed. Numerous, novel genes were identified in the gonads of embryonic female and male rainbow trout that could be involved in sex-specific differentiation pathways in this fish. Embryonic gonads were removed by dissection from known genetic female (XX) and male (XY) rainbow trout (Oncorhynchus mykiss) from a transgenic population where expression of GFP is controlled by vasa-gene regulatory elements (Yoshizaki et al. 2000; Takeuchi et al. 2002). These fish were maintained in incubators containing flowing freshwater (10Â°C) at the Ooizumi Research Station, Yamanashi, Japan. Ten female or male transgenic rainbow trout were randomly selected every 5 days, beginning at 30 days (i.e., 300 degree days = incubation temperature in Â°C x number of days) post fertilization through until 70 days (i.e., 700 degree days) post fertilization. The gonads from each sex, at each sampling time, were pooled and immediately frozen for subsequent total RNA isolation.

Project description:We have constructed a rainbow trout high-density oligonucleotide microarray by using all the available tentative consensus (TC) sequences from the Rainbow Trout Gene Index database (The Computational Biology and Functional Genomics Lab., Dana Farber Cancer Institute and Harvard School of Public Health). The Rainbow Trout Gene Index integrates research data from all available international rainbow trout genomic research projects. The newly designed microarray incorporates 37,394 unique transcript-specific oligonucleotide probes, 60-mer long each. The microarray was printed according to our design by Agilent Technologies using the 4 X 44-design format and contains 1417 Agilent control spots. The performance of the new microarray platform was evaluated by analyzing gene expression associated with the rainbow trout vitellogenesis-induced muscle atrophy. These chips can be ordered from Agilent using design number 016320. This microarray is anticipated to open new avenues of research that will aid in the development of novel strategies to enhance growth efficiency and quality in salmonid species. Keywords: Development of an oligo-array for rainbow trout The performance of the new microarray platform was evaluated by analyzing transcriptome response associated with the rainbow trout vitellogenesis-induced muscle atrophy. Severe muscle deterioration accompanies the physiological responses of the energetic demands of the rainbow trout spawning/vitellogenesis. Atrophying muscle of fertile fish had 11% less extractable muscle (g/bw) and 11% less protein content compared to non-atrophying muscle of sterile fish (p<0.01). The rainbow trout was used to profile changes in gene expression of atrophying muscles. Gene expression levels were determined by comparing the amount of mRNA transcript present in the experimental sample (fertile fish) to the control (sterile fish). RNAs isolated from each experimental fish were run on separate microarrays in independent experiments, with no pooling. A total of 8 fish were used in the microarray experiments (4 replicates x 2 groups). Fluorophors (Cy3 and Cy5) were randomly assigned to RNA from each of the atrophying and nonatrophying muscles to limit the dye effect.

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Next generation sequencing establishes the differentially expression profiles of transgenic rainbow trout bearing cecropin P1 transgene

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