Production of Homozygous Transgenic Rainbow Trout with Enhanced Disease Resistance

Previous studies conducted in our laboratory showed that transgenic medaka expressing cecropin B transgenes exhibited resistant characteristic to fish bacterial pathogens, Pseudomonas fluorescens and Vibrio anguillarum. To confirm whether antimicrobial peptide gene will also exhibit anti-bacterial and anti-viral characteristics in aquaculture important fish species, we produced transgenic rainbow trout expressing cecropin P1 or a synthetic cecropin B analog, CF-17, transgene by sperm-mediated gene transfer method. About 30 % of fish recovered from electroporation were shown to carry the transgene as determined by polymerase chain reaction (PCR) amplification assay. Positive P(1) transgenic fish were crossed to non-transgenic fish to establish F(1) transgenic founder families, and subsequently generating F(2), and F(3) progeny. Expression of cecropin P1 and CF-17 transgenes was detected in transgenic fish by reverse transcription (RT)-PCR analysis. The distribution of body sizes among F(1) transgenic fish were not significantly different from those of non-transgenic fish. Results of challenge studies revealed that many families of F(2) and F(3) transgenic fish exhibited resistance to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). All-male homozygous cecropin P1 transgenic families were produced by androgenesis from sperm of F(3) heterozygous transgenic fish in one generation. The resistant characteristic to A. salmonicida was confirmed in progeny derived from the outcross of all-male fish to non-transgenic females. Results of our current studies confirmed the possibility of producing disease-resistant homozygous rainbow trout strains by transgenesis of cecropin P1 or CF-17 gene and followed by androgenesis

RNA-Seq analysis of differentially expressed genes relevant to innate and adaptive immunity in cecropin P1 transgenic rainbow trout (Oncorhynchus mykiss)

Abstract Background In the past years, our laboratory successfully generated transgenic rainbow trout bearing cecropin P1 transgene. These fish exhibited resistant characteristic to infection by Aeromonas salmonicida, Infectious Hematopoietic Necrosis Virus (IHNV) and Ceratomyxa shasta (a parasitic pathogen). Previously, treating rainbow trout macrophage cells (RTS-11) with cecropin B, pleurocidin and CF17, respectively, resulted in elevated expression of two pro-inflammatory genes, e.g. cyclooxygenase-2 (cox-2) and interleukin-1β (il-1β). In addition, a profiling of global gene expression by 44 k salmonid microarray analysis was conducted, and the results showed that immune relevant processes have been perturbed in cecopin P1 transgenic rainbow trout. Therefore, we hypothesized that cecropin P1 may not only eliminate pathogens directly, but also modulate the host immune systems, leading to increased resistance against pathogen infections. To confirm this hypothesis, we performed de novo mRNA deep sequencing (RNA-Seq) to analyze the transcriptomic expression profiles in three immune competent tissues of cecropin P1 transgenic rainbow trout. Results De novo sequencing of mRNA of the rainbow trout spleen, liver and kidney tissues were conducted by second-generation Illumina system, followed by Trinity assembly. Tissue specific unigenes were obtained, and annotated according to the Gene Ontology (GO) and the Nucleotide Basic Local Alignment Search Tool (BLAST). Over 2000 differentially expressed genes (DEGs) were determined by normalized ratio of Reads Per Kilobase of transcript per million mapped reads (RPKM) among the transgenic and non-transgenic fish in a tissue specific manner, and there were 82 DEGs in common among the three tissues. In addition, the enrichment analysis according to Gene Ontology Biological Process (GO:BP), and Kyoto Encyclopedia of Genes and Genomes (KEGG) based pathway analysis associated with innate/adaptive immunity of fish were also performed to illustrate the altered immune-related functions in each tissue. Conclusions According to the RNA-Seq data, the correlations between alteration of gene expression profiles and the functional perturbations of the host immune processes were revealed. In comparison with the results of cDNA microarray analysis conducted by Lo et al., the overall results supported our hypothesis that the gene product of cecropin P1 transgene may not only directly eliminate pathogens, but also modulate the host immune system. Results of this study present valuable genetic information for Oncorhynchus mykiss, and will benefit future studies on the immunology of this fish species

Additional file 6: of RNA-Seq analysis of differentially expressed genes relevant to innate and adaptive immunity in cecropin P1 transgenic rainbow trout (Oncorhynchus mykiss)

Summarizing of software versions, databases and parameters for Bioinformatics analyses. (XLSX 9 kb

Additional file 1: of RNA-Seq analysis of differentially expressed genes relevant to innate and adaptive immunity in cecropin P1 transgenic rainbow trout (Oncorhynchus mykiss)

Supplemental figures for pie charts of qualities of clean reads and results of sequencing saturation analyses to show quality of aligning, and supplemental table to show overall mapping results (to reference genes) per each sample. (PDF 206 kb

Additional file 3: of RNA-Seq analysis of differentially expressed genes relevant to innate and adaptive immunity in cecropin P1 transgenic rainbow trout (Oncorhynchus mykiss)

Supplement figures for pathways generated from custom-made visualization tool. (PDF 743 kb

Additional file 5: of RNA-Seq analysis of differentially expressed genes relevant to innate and adaptive immunity in cecropin P1 transgenic rainbow trout (Oncorhynchus mykiss)

Primers for RT-qPCR analysis. (XLSX 11 kb

Additional file 4: of RNA-Seq analysis of differentially expressed genes relevant to innate and adaptive immunity in cecropin P1 transgenic rainbow trout (Oncorhynchus mykiss)

Supplement figures from KEGG database and sprayed by the DEGs expression profiles. (PDF 240 kb