Altered gene expression patterns of innate and adaptive immunity pathways in transgenic rainbow trout harboring Cecropin P1 transgene

Background

We have recently developed several homozygous families of transgenic rainbow trout harbouring cecropin P1 transgene. These fish exhibit resistance characteristic to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). In our earlier studies we have reported that treatment of a rainbow trout macrophage cell line (RTS11) with a linear cationic α-helical antimicrobial peptide (e.g., cecropin B) resulted in elevated levels of expression of two pro-inflammatory relevant genes (e.g., IL-1β and COX-2). Therefore, we hypothesized that in addition to the direct antimicrobial activity of cecropin P1 in the disease resistant transgenic rainbow trout, this antimicrobial peptide may also affect the expression of immune relevant genes in the host. To confirm this hypothesis, we launched a study to determine the global gene expression profiles in three immune competent organs of cecropin P1 transgenic rainbow trout by using a 44k salmonid microarray.

Results

From the microarray data, a total of 2480 genes in the spleen, 3022 in the kidney, and 2102 in the liver were determined as differentially expressed genes (DEGs) in the cecropin P1 transgenic rainbow trout when compared to the non-transgenics. There were 478 DEGs in common among three tissues. Enrichment analyses conducted by two different bioinformatics tools revealed a tissue specific profile of functional pathway perturbation. Many of them were directly related to innate immune system such as phagocytosis, lysosomal processing, complement activation, antigen processing/presentation, and leukocyte migration. Perturbation of other biological functions that might contribute indirectly to host immunity was also observed.

Conclusions

The gene product of cecropin P1 transgene produced in the disease resistant transgenic rainbow trout not only can kill the pathogens directly but also exert multifaceted immunomodulatory properties to boost host immunity. The identified genes involved in different pathways related to immune function are valuable indicators associated with enhanced host immunity. These genes may serve as markers for selective breeding of rainbow trout or other aquaculture important fish species bearing traits of disease resistance.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-887) contains supplementary material, which is available to authorized users.     

Enhanced Resistance to Phytopathogenic Bacteria in Transgenic Tobacco Plants with Synthetic Gene of Antimicrobial Peptide Cecropin P1

Plasmids with a synthetic gene of the mammalian antimicrobial peptide cecropin P1 (cecP1) controlled by the constitutive promoter 35S RNA of cauliflower mosaic virus were constructed. Agrobacterial transformation of tobacco plants was conducted using the obtained recombinant binary vector. The presence of gene cecP1 in the plant genome was confirmed by PCR. The expression of gene cecP1 in transgenic plants was shown by Northern blot analysis. The obtained transgenic plants exhibit enhanced resistance to phytopathogenic bacteria Pseudomonas syringae, P. marginata, and Erwinia carotovora. The ability of transgenic plants to express cecropin P1 was transmitted to the progeny. F₀ and F₁ plants had the normal phenotype (except for a changed coloration of flowers) and retained the ability to produce normal viable seeds upon self-pollination. Lines of F₁ plants with Mendelian segregation of transgenic traits were selected.     

Gene transfer, expression and inheritance of pRSV-rainbow trout-GH cDNA in the common carp, Cyprinus carpio (Linnaeus)

A recombinant plasmid containing the Rous sarcoma virus-long terminal repeat (RSV-LTR) promoter linked to rainbow trout (Salmo gairdneri) growth hormone (GH) cDNA was microinjected into fertilized carp eggs. Genomic DNA extracted from pectoral fin of individual presumptive transgenic fish was analyzed by dot blot and Southern blot hybridization, using the RSV-LTR and/or the GH cDNA sequences as probes. Out of 365 presumptive transgenic fish analyzed, 20 individuals were found to contain pRSV-rtGH-cDNA sequence in the genomic DNA. Expression of the trout GH polypeptide was detected by immunobinding assay in the red blood cells of nine transgenic fish tested. The level of expression, however, varied among the transgenics and could not be correlated with exogenous DNA copy number. Although there was considerable variation in the sizes of the transgenic fish, those microinjected during the one-cell stage were (P less than 0.05) 22% larger, on the average, than their sibling controls. A randomly selected fraction of the progeny derived from crosses between transgenic males and non-transgenic females inherited the foreign DNA. These transgenic progeny grew faster (P less than 0.05) than their non-transgenic siblings.     

Effect of genetic background on growth of mice hemizygous for wild-type or dwarf mutated bovine growth hormone transgenes

The effects of a high-growth genetic background on the growth of mice hemizygous for one of two growth hormone transgenes were examined. Male mice hemizygous for wild-type (W) and dwarf mutant (M) bovine growth hormone (bGH) transgenes were crossed with females of a high-growth selected (S) and control (C) line as follows: W x S, W x C, M x S and M x C. Body weights of progeny were recorded weekly from 2 to 10 weeks of age. F₁ progeny were classified as carriers (P) or non-carriers (N) of the transgene by assaying tail DNA for bGH using the polymerase chain reaction and agarose gel electrophoresis. A deficiency in the number of f₁ progeny carrying the W (P<0.05) and M (P<0.01) bGH transgene was most likely due to differential prenatal and early postnatal mortality. Bodyweight means of wild-type transgenic mice were larger (P < 0.05) than those of non-transgenic littermates by 3 weeks of age in a C background in contrast to 5 weeks in S. The wild-type bGH transgene increased adult body weights more in the C (155%) than in the S (136%) background, indicating transgene expression by selection background interaction (P < 0.05). However, the growth response to the wild-type transgene in the S background was still large. The dwarf mutant transgene had a greater effect on growth reduction in the S (70%) than in the C (84%) background, thus causing transgene expression by selection background interaction (P < 0.05). Gender by wild-type transgene effect interactions (P < 0.001) for adult body weight were caused by the transgene reducing the gender difference for body weight in C and eliminating it in S. The dwarf mutant caused a larger negative effect on growth in males than in females, resulting in a gender by dwarf mutant transgene interaction (P < 0.001) for adult body weights. Results indicate that the effect of a GH transgene on growth can be affected both by a high-growth genetic background and the gender of progeny.     

Limited fitness advantages of crop-weed hybrid progeny containing insect-resistant transgenes (Bt/CpTI) in transgenic rice field

Background

The spread of insect-resistance transgenes from genetically engineered (GE) rice to its coexisting weedy rice (O. sativa f. spontanea) populations via gene flow creates a major concern for commercial GE rice cultivation. Transgene flow to weedy rice seems unavoidable. Therefore, characterization of potential fitness effect brought by the transgenes is essential to assess environmental consequences caused by crop-weed transgene flow.

Methodology/Principal Findings

Field performance of fitness-related traits was assessed in advanced hybrid progeny of F₄ generation derived from a cross between an insect-resistant transgenic (Bt/CpTI) rice line and a weedy strain. The performance of transgene-positive hybrid progeny was compared with the transgene-negative progeny and weedy parent in pure and mixed planting of transgenic and nontransgenic plants under environmental conditions with natural vs. low insect pressure. Results showed that under natural insect pressure the insect-resistant transgenes could effectively suppress target insects and bring significantly increased fitness to transgenic plants in pure planting, compared with nontransgenic plants (including weedy parent). In contrast, no significant differences in fitness were detected under low insect pressure. However, such increase in fitness was not detected in the mixed planting of transgenic and nontransgenic plants due to significantly reduced insect pressure.

Conclusions/Significance

Insect-resistance transgenes may have limited fitness advantages to hybrid progeny resulted from crop-weed transgene flow owning to the significantly reduced ambient target insect pressure when an insect-resistant GE crop is grown. Given that the extensive cultivation of an insect-resistant GE crop will ultimately reduce the target insect pressure, the rapid spread of insect-resistance transgenes in weedy populations in commercial GE crop fields may be not likely to happen.     

Baltic salmon, Salmo salar, from Swedish river Lule älv is more resistant to furunculosis compared to rainbow trout

Background

Furunculosis, caused by Aeromonas salmonicida, continues to be a major health problem for the growing salmonid aquaculture. Despite effective vaccination programs regular outbreaks occur at the fish farms calling for repeated antibiotic treatment. We hypothesized that a difference in natural susceptibility to this disease might exist between Baltic salmon and the widely used rainbow trout.

Study Design

A cohabitation challenge model was applied to investigate the relative susceptibility to infection with A. salmonicida in rainbow trout and Baltic salmon. The course of infection was monitored daily over a 30-day period post challenge and the results were summarized in mortality curves.

Results

A. salmonicida was recovered from mortalities during the entire test period. At day 30 the survival was 6.2% and 34.0% for rainbow trout and Baltic salmon, respectively. Significant differences in susceptibility to A. salmonicida were demonstrated between the two salmonids and hazard ratio estimation between rainbow trout and Baltic salmon showed a 3.36 higher risk of dying from the infection in the former.

Conclusion

The finding that Baltic salmon carries a high level of natural resistance to furunculosis might raise new possibilities for salmonid aquaculture in terms of minimizing disease outbreaks and the use of antibiotics.     

Effects of genomic position and copy number of Acyl-ACP thioesterase transgenes on the level of the target fatty acids in Brassica napus L.

The effects of genomic position and copy number of acyl-acyl carrier protein (ACP) thioesterase (TE) transgenes on the major target fatty acid, either lauric acid (C12:0) or palmitic acid (C16:0) depending on the TE, in transgenic Brassica napus seed oil were investigated. Four transgenic parental lines, transformed individually with the bay-TE (Uc FatB1), elm-TE (Ua FatB1), nutmeg-TE (Mf FatB1) and Cuphea-TE (Ch FatB1) transgenes, were crossed with the non-transgenic recipient genotypes '212/86' or 'QO4'. Bay-TE and Cuphea-TE F₁ seeds, which carry half the number of the construct copies compared to the self-pollinated seeds of the transgenic parents, showed significantly lower levels of the target fatty acid. Doubled haploid (DH) lines were developed through microspore culture from F₁ hybrids with the elm-TE or the Cuphea-TE transgenes. DH lines carrying one to five copies of the Cuphea-TE transgene displayed a positive correlation between transgene copy number and the target fatty acid C16:0 level (r = 0.77**). DH lines with elm-TE transgene copies at four different loci showed different C16:0 levels, with one of the loci (E-II) leading to significantly higher C16:0 levels. This study supports the importance of the selection of high transgene copy number and/or the optimum genomic integration site in order to achieve maximum expression levels of the target fatty acid in transgenic oil quality modification.     

Spermatogonial transplantation in fish: A novel method for the preservation of genetic resources

Recent progress in genome-based breeding has created various fish strains carrying desirable genetic traits; however, methods for the long-term preservation of their genetic resources have not yet been developed, mainly due to the lack of cryopreservation techniques for fish eggs and embryos. Recently, we established an alternative cryopreservation technique for fish spermatogonia using a slow-freezing method. Furthermore, we developed a transplantation system to produce functional eggs and sperm derived from spermatogonia. Spermatogonia isolated from the testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout (Oncorhynchus mykiss) were transplanted into the peritoneal cavity of triploid masu salmon (Oncorhynchus masou) hatchlings of both genders. The transplanted trout spermatogonia migrated towards the gonadal anlagen of the recipient salmon, into which they were subsequently incorporated. We confirmed that the donor-derived spermatogonia resumed gametogenesis, and produced sperm and eggs in male and female recipient salmon, respectively. Fertilization of the resultant eggs and sperm produced only rainbow trout in the first filial (F₁) generation, suggesting that the sterile triploid recipient salmon produced functional eggs and sperm derived from the trout donors. A combination of spermatogonial transplantation and cryopreservation could be a powerful tool for preserving valuable fish strains with desirable genetic traits and endangered species.     

Stability and inheritance of endosperm-specific expression of two transgenes in progeny from crossing independently transformed barley plants

To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T₈ plant, having uidA (or gus) driven by the barley endosperm-specific B₁-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T₄ plant, having sgfp(S65T) driven by the barley endosperm-specific D-hordein promoter (localized on the subtelomeric region of chromosome 2H). Both lines stably expressed the two transgenes in the generations prior to the cross. Three independently crossed F₁ progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F₂ seeds followed a 3:1 Mendelian segregation ratio and transgenes were localized by FISH to the same location as in the parental plants. FISH was used to screen F₂ plants for homozygosity of both transgenes; four homozygous plants were identified from the two crossed lines tested. FISH results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F₃ and F₄ generations. Thus, even after crossing independently transformed, homozygous parental plants containing a single, stably expressed transgene, progeny were obtained that continued to express multiple transgenes through generation advance. Such stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies.     

Hybridization between genetically modified Atlantic salmon and wild brown trout reveals novel ecological interactions

Interspecific hybridization is a route for transgenes from genetically modified (GM) animals to invade wild populations, yet the ecological effects and potential risks that may emerge from such hybridization are unknown. Through experimental crosses, we demonstrate transmission of a growth hormone transgene via hybridization between a candidate for commercial aquaculture production, GM Atlantic salmon (Salmo salar) and closely related wild brown trout (Salmo trutta). Transgenic hybrids were viable and grew more rapidly than transgenic salmon and other non-transgenic crosses in hatchery-like conditions. In stream mesocosms designed to more closely emulate natural conditions, transgenic hybrids appeared to express competitive dominance and suppressed the growth of transgenic and non-transgenic (wild-type) salmon by 82 and 54 per cent, respectively. To the best of our knowledge, this is the first demonstration of environmental impacts of hybridization between a GM animal and a closely related species. These results provide empirical evidence of the first steps towards introgression of foreign transgenes into the genomes of new species and contribute to the growing evidence that transgenic animals have complex and context-specific interactions with wild populations. We suggest that interspecific hybridization be explicitly considered when assessing the environmental consequences should transgenic animals escape to nature.     

Potential Role of Specific Antibodies as Important Vaccine Induced Protective Mechanism against Aeromonas salmonicida in Rainbow Trout

Furunculosis caused by infection with Aeromonas salmonicida subsp. salmonicida has been a known threat to aquaculture for more than a century. Efficient prophylactic approaches against this disease are essential for continued growth of salmonid aquaculture. Since the introduction of successful oil-adjuvanted vaccines in the early 1990''s, a number of studies have been published on the protective as well as adverse effects of these vaccines. Most studies focus on vaccination of salmon (Salmo salar). However, rainbow trout (Oncorhynchus mykiss) are also very susceptible to infection and are vaccinated accordingly. In this study we have examined the protection against infection with a Danish strain of A. salmonicida in both vaccinated and non-vaccinated rainbow trout. A commercial and an experimental auto-vaccine were tested. The protective effects of the vaccines were evaluated through an A. salmonicida challenge 18 weeks post vaccination. Both vaccines resulted in a significantly increased survival in the vaccinated fish during a 28 day challenge period relative to non-vaccinated fish (P = 0.01 and P = 0.001 for the commercial and experimental vaccine, respectively). Throughout the entire experiment, the presence of specific antibodies in plasma was monitored using ELISA. A significant increase in specific antibody levels was seen in fish vaccinated with both vaccines during the 18 weeks between vaccination and challenge. Within 3 days post challenge, a significant decrease in specific antibodies occurred in vaccinated fish. A positive correlation was found between mean levels of specific antibodies pre challenge and overall survival. This correlation, along with the observed depletion of antibodies during the initial phase of infection, suggests that specific antibodies play an essential role in vaccine mediated protection against A. salmonicida in rainbow trout.     

Electroporation as a new technique for producing transgenic fish

A recombinant plasmid, pMV-GH, containing rainbow trout growth hormone cDNA fused to mouse metallothionein I promoter, was introduced into medaka (Oryzias latipes) by electroporation. Of 3109 fertilized eggs treated with electric pulses (750 V/cm, 50 μs, 5 times), 783 (25%) hatched out. Four percent of the hatchlings were transgenic. To obtain transgenic lines, 180 hatchlings were maintained and 35 of them grew into adult fish. Two of these fish were transgenic. When one transgenic fish was mated with a normal female, the transgene was found in all the F1 offspring assayed. In F2 offspring obtained by mating transgenic F1 fish, 88% were transgenic.     

Detection of QTL in Rainbow Trout Affecting Survival When Challenged with Flavobacterium psychrophilum

Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture. We previously detected genetic variation in survival following challenge with Flavobacterium psychrophilum (Fp), the causative agent of BCWD in rainbow trout (Oncorhynchus mykiss). A family-based selection program to improve resistance was initiated in 2005 at the USDA National Center for Cool and Cold Water Aquaculture. Select crosses were made in 2007 and 2009 to evaluate family-based disease survival using Fp injection challenges. From each putative F₂/BC₁ family generated in 2009, 200–260 fish were challenged in 4–7 replicates per family. Whole genome QTL scans of three F₂/BC₁ families were conducted with about 270 informative microsatellite loci per family spaced at an average interval size of 6 cM throughout the rainbow trout genome. Markers on chromosomes containing QTL were further evaluated in three additional F₂/BC₁ families. The additional F₂/BC₁ families were sire or dam half-sibs (HS) of the initially genome scanned families. Overall, we identified nine major QTL on seven chromosomes that were significant or highly significant with moderate to large effects of at least 13 % of the total phenotypic variance. The largest effect QTL for BCWD resistance explaining up to 40 % of the phenotypic variance was detected on chromosome OMY8 in family 2009070 and in the combined dam HS family 2009069–070. The nine major QTL identified in this study are candidates for fine mapping to identify new markers that are tightly linked to disease resistance loci for using in marker assisted selection strategies.     

Mendelian transmission,structure and expression of transgenes following their injection into the cytoplasm of trout eggs

High frequencies of mosaic transgenic trout are generated when linear DNA fragments are injected into the egg cytoplasm. The fact that these animals transmit the foreign DNA to a minority of their offspring suggests that their germline is also mosaic, but it may also indicate that some transgenes are lost. We have produced seven F₂ families from wild-type females mated with F₁ transgenic males. In all of them, the ratio of transgenics was Mendelian, and classical Southern analyses clearly showed the segregation of one or two foreign loci. They also provide firm arguments for gene integration in families carrying single-copy transgenes. Images obtained by Southern blotting strongly suggest the presence of extrachromosomal linear concatemers in two other families. However, this hypothesis was contradicted by further analyses usingin situ hybridization on chromosomes and with pulse field gel electrophoresis (PFGE). The reconciliation of these apparently contradictory observations is allowed by a model in which integrated concatemers adopt hairpin loop structures at the junction of inversely oriented copies. Further experiments with denaturing electrophoresis, which opens such secondary structures, provides additional support for this model. In this stable gene transfer system accompanied by chromosomal integration, the CAT reporter gene was expressed from the promoter/enhancer of human cytomegalovirus early genes, in all tissues examined in F₀ fishes, as well as in the F₁ offsprings of nine males. This indicates that the palindromic structures of some of the transgenes do not prevent their expression.     

phiC31 Integrase-Mediated Site-Specific Recombination in Barley

The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F₁, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.     

Disease resistance and health parameters of growth-hormone transgenic and wild-type coho salmon,Oncorhynchus kisutch

To extend previous findings regarding fish health and disease susceptibility of growth-enhanced fish, hematological and immunological parameters have been compared between growth hormone (GH) transgenic and wild-type non-transgenic coho salmon (Oncorhynchus kisutch). Compared to non-transgenic coho salmon, transgenic fish had significantly higher hematocrit (Hct), hemoglobin (Hb), mean cellular hemoglobin (MCH), mean cellular volume (MCV), and erythrocyte numbers, and lower white cell numbers. In addition, resistance to the bacterial pathogen Aeromonas salmonicida (causal agent of furunculosis) has been assessed between the strains. Higher susceptibility of transgenic fish to this disease challenge was observed in two separate year classes of fish. The present findings provide fundamental knowledge of the disease resistance on GH enhanced transgenic coho salmon, which is of importance for assessing the fitness of transgenic strains for environmental risk assessments, and for improving our understanding effects of growth modification on basic immune functions.