Transgenic Rainbow Trout Research Articles

Establishment of novel monoclonal antibodies for identification of type A spermatogonia in teleosts†.

We recently established a germ cell transplantation system in salmonids. Donor germ cells transplanted into the body cavity of recipient embryos migrate toward and are incorporated into the recipient gonad, where they undergo gametogenesis. Among the various types of testicular germ cells, only type A spermatogonia (A-SG) can be incorporated into the recipient gonads. Enriching for A-SG is therefore important for improving the efficiency of germ cell transplantation. To enrich for A-SG, an antibody against a cell surface marker is a convenient and powerful approach used in mammals; however, little is known about cell surface markers for A-SG in fish. To that end, we have produced novel monoclonal antibodies (mAbs) against cell-surface molecules of rainbow trout (Oncorhynchus mykiss) A-SG. We inoculated mice with living A-SG isolated from pvasa-GFP transgenic rainbow trout using GFP-dependent flow cytometry. By fusing lymph node cells of the inoculated mice with myeloma cells, we generated 576 hybridomas. To identify hybridomas that produce mAbs capable of labeling A-SG preferentially and effectively, we screened them using cell ELISA, fluorescence microscopy, and flow cytometry. We thereby identified two mAbs that can label A-SG. By using flow cytometry with these two antibodies, we could enrich for A-SG with transplantability to recipient gonads from amongst total testicular cells. Furthermore, one of these mAbs could also label zebrafish (Danio rerio) spermatogonia. Thus, we expect these monoclonal antibodies to be powerful tools for germ cell biology and biotechnology.

A pathway-focused RT-qPCR array study on immune relevant genes in rainbow trout (Oncorhynchus mykiss) harboring cecropin P1 transgene

Recently, our laboratory had produced five families of transgenic rainbow trout harboring cecropin P1 transgene, and via repeated challenge studies these fish exhibited a significant elevation of resistance to infection by microbial pathogens. By cDNA microarray and mRNA deep sequencing (mRNA-seq) analyses on two of the five families of cecropin P1 transgenic fish, differentially expressed genes (DEGs) relevant to the innate and adaptive immune pathways in three different immune-related tissues, (i.e. spleen, kidney and liver) were profiled. These results supported our hypothesis that in addition to its direct microbicidal activity, the transgene product of cecropin P1 induces immunomodulatory activity in the transgenic host. Here, we have adapted the technique of quantitative reverse transcription real time PCR (RT-qPCR) array to analyze the expression of genes relevant to the innate and adaptive immune pathways in the rest three families. A RT-qPCR array was constructed with oligonucleotide primers of fifty-two innate/adaptive immune relevant DEGs shown to be the most perturbed by cecropin P1 transgene product in previous studies. Messenger RNA isolated from the spleen, kidney and liver of transgenic fish and non-transgenic fish control were studied on this array. Results of RT-qPCR array revealed that statistically significant perturbations of gene expression were detected in pathways of cytokine/chemokine signaling, Toll-like receptor signaling, complement cascade, antigen processing/presentation, lysosomal phagocytosis and leukocyte trans-endothelial migration in the transgenic spleen; extracellular matrix (ECM) organization and leukocyte trans-endothelial migration pathways in the transgenic kidney; lysosomal activity pathway in the transgenic liver. Furthermore, genes related to the pathways of the peroxisome proliferator-activated receptors (PPAR) signaling, lipid metabolism process and arachidonic acid metabolism were also impacted in the transgenic liver. Findings of the current study are in good agreement with those discoveries in previous two transgenic families by cDNA microarray and mRNA-seq analyses.

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

BackgroundIn 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.ResultsDe 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.ConclusionsAccording 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.

Predation, metabolic priming and early life-history rearing environment affect the swimming capabilities of growth hormone transgenic rainbow trout.

The period of first feeding, when young salmonid fishes emerge from natal stream beds, is one fraught with predation risk. Experiments conducted in semi-natural stream mesocosms have shown that growth hormone transgenic salmonids are at greater risk of predation than their non-transgenic siblings, due partly to the higher metabolic demands associated with transgenesis, which force risky foraging behaviours. This raises questions as to whether there are differences in the swim-performance of transgenic and non-transgenic fishes surviving predation experiments. We tested this hypothesis in wild-origin rainbow trout (Oncorhynchus mykiss) that were reared from first feeding in semi-natural stream mesocosms characterized by complex hydrodynamics, the presence of predators and oligotrophic conditions. Using an open-flume raceway, we swam fish and measured their capacity for burst-swimming against a sustained flow. We found a significant genotype effect on burst-performance, with transgenic fish sustaining performance longer than their wild-type siblings, both in predator and predator-free stream segments. Importantly, this effect occurred before differences in growth were discernable. We also found that mesocosm-reared fish had greater burst-performance than fish reared in the controlled hatchery environment, despite the latter being unexposed to predators and having abundant food. Our results suggest a potential interaction between predation and metabolic priming, which leads to greater burst capacity in transgenic trout.

Motor neuron scaling and somatic growth in normal and transgenic rainbow trout, Oncorhynchus mykiss

Teleost fishes demonstrate indeterminate growth and a dramatic increase in body mass over their life cycle. Myotonal muscle mass comprises >60% of the adult body mass in active species and effective growth and development of functional capability requires interactions between multuinucleated muscle fibers (myocytes) and motor neurons. Basically, as all organisms grow and develops, the nervous system must compensate for the change in size. While numerous studies have examined myogenesis, muscle fiber recruitment and hypertrophy, and phenotypic plasticity in fishes, our understanding of the myocyte/neuron linkage during development is extremely limited. The goal of this project is to determine how motor neurons in rainbow trout change through development and how this change can be viewed through principles of scaling. Sections of spinal cord were removed at the rostral, medial, and caudal positions from normal and genetically modified rainbow trout to overexpress follistatin, which antagonizes myostatin and increases myotonal muscle. Increased muscling in these transgenic trout is due primarily to myocyte hyperplasia. Spinal cords (n= 35) were sectioned on a microtome and stained with acridine orange. Sections were analyzed to 1) measure the cross sectional area of the spinal cord and ventral horn motor neurons and 2) determine how motor neurons develop to compensate for growth in total muscle mass. The average motor neuron area from normal rainbow trout revealed an unusual allometric relationship compared to endothermic animals. As the mass of the fish increased there was a positive (r2 = 0.961) correlation to the motor neuron area. Compared with normal rainbow trout, transgenic trout had an average neuron area similar to that of the normal trout, but relative to per gram of body mass motor neuron is significantly less, suggesting that the motor unit size – the number of skeletal muscle fibers per motor neuron ‐ increases with an increase in muscle mass. Previous studies suggest that neuron size in endothermic organisms follow a pattern of metabolic allometric scaling, yet motor neuron from ectotherms in this study had scaling coefficient of just 0.204. These findings suggest that as the axial muscle increases mass in normal and transgenic rainbow trout, the peripheral nervous system is not undergoing a similar growth in neuron size.Support or Funding InformationNIH Grant #P20 RR0116454

Hypothermic storage of isolated spermatogonia and oogonia from rainbow trout (Oncorhynchus mykiss)

A growing number of fish species are endangered due to human activities. A short- or long-time preservation of gametes could conserve genetic resources of threatened fish species. The aim of this study was to evaluate a hypothermic condition for short-term preservation of spermatogonia and oogonia cells isolated from immature transgenic rainbow trout, Oncorhynchus mykiss, and to determine the maximum time point for further transplantation. Viability rate of germ cells was investigated after isolation and during storage at 4 °C up to 24 h. Dulbecco's modification of Eagle's medium supplemented with Hepes fetal bovine serum and l-glutamine was used as hypothermic storage media. The results showed that while viability decreased following 24 h storage, the remaining viable cells did not vary morphologically as well as GFP intensity retained similar to those observed in freshly isolated cells. The hypothermal storage study indicated that culture medium is suitable for preserving germ cells in the short periods of time. Simplicity, easily available culture media and low cost provide new insight into hypothermic conditions for preserving and transporting of germ cells for next applied and basic studies.

Early life-history consequences of growth-hormone transgenesis in rainbow trout reared in stream ecosystem mesocosms.

There is persistent commercial interest in the use of growth modified fishes for shortening production cycles and increasing overall food production, but there is concern over the potential impact that transgenic fishes might have if ever released into nature. To explore the ecological consequences of transgenic fish, we performed two experiments in which the early growth and survival of growth-hormone transgenic rainbow trout (Oncorhynchus mykiss) were assessed in naturalized stream mesocosms that either contained predators or were predator-free. We paid special attention to the survival bottleneck that occurs during the early life-history of salmonids, and conducted experiments at two age classes (first-feeding fry and 60 days post-first-feeding) that lie on either side of the bottleneck. In the late summer, the first-feeding transgenic trout could not match the growth potential of their wild-type siblings when reared in a hydrodynamically complex and oligotrophic environment, irrespective of predation pressure. Furthermore, overall survival of transgenic fry was lower than in wild-type (transgenic = 30% without predators, 8% with predators; wild-type = 81% without predators, 31% with predators). In the experiment with 60-day old fry, we explored the effects of the transgene in different genetic backgrounds (wild versus domesticated). We found no difference in overwinter survival but significantly higher growth by transgenic trout, irrespective of genetic background. We conclude that the high mortality of GH-transgenic trout during first-feeding reflects an inability to sustain the basic metabolic requirements necessary for life in complex, stream environments. However, when older, GH-transgenic fish display a competitive advantage over wild-type fry, and show greater growth and equal survival as wild-type. These results demonstrate how developmental age and time of year can influence the response of genotypes to environmental conditions. We therefore urge caution when extrapolating the results of GH-transgenesis risk assessment studies across multiple life-history or developmental stages.

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

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

Production of genetically diversified fish seeds using spermatogonial transplantation

Abstract Large-scale release of hatchery-produced seeds has been conducted in order to restore worldwide fishery production; however, concerns exist regarding the genetic effects of hatchery stock on wild fish populations, due to the reduced genetic variation often associated with hatchery-reared fish. Therefore, it is important that fish seeds used in stock enhancement possess sufficient genetic diversity to mitigate their genetic impact on wild fish populations. To promote genetic diversity of artificial seeds, seed production should be performed using a sufficiently large broodstock; however, maintenance of large broodstocks requires significant investments with respect to space, labor, and other associated costs. In order to circumvent the need for these investments, the present study proposed a means of producing gametes that possess a large amount of genetic diversity using only a small number of surrogate-broodstock through spermatogonial transplantation. We previously established spermatogonial transplantation methodology capable of producing functional eggs and sperm derived from donor-type A spermatogonia (ASGs). It was expected that if ASGs isolated from testes of different donor individuals were mixed prior to their transplantation into the peritoneal cavity of a single recipient, the resulting recipient would produce gametes derived from several donor individuals. To determine the feasibility of this scenario, mixed donor ASGs derived from vasa-GFP, vasa-DsRed and hsc-GFP transgenic rainbow trout (Oncorhynchus mykiss) were transplanted into triploid recipients. At 20 days post-transplantation, approximately 80% of recipients possessed donor ASGs derived from all three strains within their gonads and mean numbers of incorporated ASGs derived from each donor strain were nearly equal. At 100 days post-transplantation, incorporated donor ASGs proliferated within the gonads of both male and female recipients, and some had differentiated into oocytes within recipient ovaries. Furthermore, in order to ascertain whether incorporated donor ASGs derived from the three strains could differentiate into functional gametes, gametes collected from mature male and female recipients were used in artificial insemination with gametes obtained from wild-type fish of the opposite sex. Phenotypic analysis of F1 offspring revealed that functional sperm and eggs derived from three donor strains were successfully produced in the gonads of individual recipients. These results suggested that individual recipients that received ASGs from multiple donors were capable of producing gametes with different genetic backgrounds. Therefore, it was concluded that the method of spermatogonial transplantation outlined in the present study could serve as a novel and efficient method of producing fish seeds with increased genetic diversity for use in stock enhancement.

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 P1 transgenic fish were crossed to non-transgenic fish to establish F1 transgenic founder families, and subsequently generating F2, and F3 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 F1 transgenic fish were not significantly different from those of non-transgenic fish. Results of challenge studies revealed that many families of F2 and F3 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 F3 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.

Muscle growth in teleost fish is regulated by factors utilizing the activin II B receptor

The activin type IIB receptor (Acvr2b) is the cell surface receptor for multiple transforming growth factor β (TGF-β) superfamily ligands, several of which regulate muscle growth in mammals. To investigate the role of the Acvr2b signaling pathway in the growth and development of skeletal muscle in teleost fish, transgenic rainbow trout (RBT; Oncorhynchus mykiss) expressing a truncated form of the acvr2b-2a (acvr2b(▵)) in muscle tissue were produced. High levels of acvr2b(▵) expression were detected in the majority of P1 transgenic fish. Transgenic P1 trout developed enhanced, localized musculature in both the epaxial and hypaxial regions (dubbed 'six pack'). The F1 transgenic offspring did not exhibit localized muscle growth, but rather developed a uniform body morphology with greater girth, condition factor and increased muscle fiber hypertrophy. There was a high degree of variation in the mass of both P1 and F1 transgenic fish, with several fish of each generation exhibiting enhanced growth compared with other transgenic and control siblings. The 'six pack' phenotype observed in P1 transgenic RBT overexpressing acvr2b(▵) and the presence of F1 individuals with altered muscle morphology provides compelling evidence for the importance of TGF-β signaling molecules in regulating muscle growth in teleost fish.

Biological Characteristics of Fish Germ Cells and their Application to Developmental Biotechnology

We have revealed several unique characteristics of germ cell development using rainbow trout, including the fact that spermatogonia transplanted into the peritoneal cavity of newly hatched embryos migrate toward recipient gonads, that spermatogonia transplanted into female recipients start oogenesis and produce functional eggs and that diploid germ cells transplanted into triploid trout can complete gametogenesis. By combining these unique features of fish germ cells, we established allogeneic and xenogeneic transplantation systems for spermatogonia in several fish species. Spermatogonia isolated from the mature testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout were transplanted into the peritoneal cavity of triploid masu salmon newly hatched embryos. These spermatogonia migrated toward recipient salmon genital ridges with extending pseudopodia and were subsequently incorporated into them. We further confirmed that the donor-derived spermatogonia resumed gametogenesis and produced sperm and eggs in male and female salmon recipients, respectively. By inseminating the resulting eggs and sperm, we obtained only rainbow trout offspring in the F1 generation, suggesting that the triploid salmon recipients produced functional gametes derived only from donor trout. We further confirmed that this intra-peritoneal transplantation of germ cells is applicable to several marine fishes, which could be of benefit in the production of bluefin tuna that has a large broodstock (>100 kg) and is difficult to maintain in captivity. Gamete production of bluefin tuna could be more easily achieved by generating a surrogate species, such as mackerel, that can produce tuna gametes.

Non‐sex specific genes associated with the secondary mitotic period of primordial germ cell proliferation in the gonads of embryonic rainbow trout (Oncorhynchus mykiss)

The purposes of this study were to quantify the secondary proliferation of primordial germ cells (PGCs) in both sexes of rainbow trout, determine if a sex difference in the timing of PGC proliferation and eventual pre-meiotic number exists, and use microarray data collected during this period to identify genes that are associated with PGC mitosis. The experiments used vasa-green fluorescent protein (vasa-GFP) transgenic rainbow trout of known genetic sex that allowed for the identification and collection of PGCs in vivo. An increase was observed in the number of PGCs counted in the gonads of both female and male embryonic vasa-GFP rainbow trout, from 300 to 700° days (water temperature in °C × days post-fertilization). For both sexes, a statistically significant (P < 0.05) increase in the PGC number was first noted at either 350 or 400° days of development. By 700° days, a 20-50-fold increase in germ cell number was apparent. No sex-specific differences in the timing of PGC proliferation or number were notable in any of the families until 700° days. In conjunction, a custom microarray based on cDNA libraries from embryonic rainbow trout gonads was used to identify genes involved in PGC mitosis. Five genes were discovered: guanine nucleotide binding protein, integral membrane protein 2B, transmembrane protein 47, C-src tyrosine-protein kinase, and the decorin precursor protein. All the genes identified have not been previously associated with germ cell mitosis, but are known to be involved with the cell plasma membrane and/or cell signaling pathways.

Overexpression of follistatin in trout stimulates increased muscling

Deletion or inhibition of myostatin in mammals has been demonstrated to markedly increase muscle mass by hyperplasia, hypertrophy, or a combination of both. Despite a remarkably high degree of conservation with the mammalian protein, the function of myostatin remains unknown in fish, many species of which continue muscle growth throughout the lifecycle by hyperplasia. Transgenic rainbow trout (Oncorhynchus mykiss) overexpressing follistatin, one of the more efficacious antagonists of myostatin, were produced to investigate the effect of this protein on muscle development and growth. P(1) transgenics overexpressing follistatin in muscle tissue exhibited increased epaxial and hypaxial muscling similar to that observed in double-muscled cattle and myostatin null mice. The hypaxial muscling generated a phenotype reminiscent of well-developed rectus abdominus and intercostal muscles in humans and was dubbed "six pack." Body conformation of the transgenic animals was markedly altered, as measured by condition factor, and total muscle surface area increased. The increased muscling was due almost exclusively to hyperplasia as evidenced by a higher number of fibers per unit area and increases in the percentage of smaller fibers and the number of total fibers. In several individuals, asymmetrical muscling was observed, but no changes in mobility or behavior of follistatin fish were observed. The findings indicate that overexpression of follistatin in trout, a species with indeterminate growth rate, enhances muscle growth. It remains to be determined whether the double muscling in trout is due to inhibition of myostatin, other growth factors, or both.

Identification of a molecular marker for type A spermatogonia by microarray analysis using gonadal cells from pvasa‐GFP transgenic rainbow trout (Oncorhynchus mykiss)

The spermatogonia of fish can be classified as being either undifferentiated type A spermatogonia or differentiated type B spermatogonia. Although type A spermatogonia, which contain spermatogonial stem cells, have been demonstrated to be a suitable material for germ cell transplantation, no molecular markers for distinguishing between type A and type B spermatogonia in fish have been developed to date. We therefore sought to develop a molecular marker for type A spermatogonia in rainbow trout. Using GFP-dependent flow cytometry (FCM), enriched fractions of type A and type B spermatogonia, testicular somatic cells, and primordial germ cells were prepared from rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa regulatory regions (pvasa-GFP rainbow trout). The gene-expression profiles of each cell fraction were then compared with a microarray containing cDNAs representing 16,006 genes from several salmonid species. Genes exhibiting high expression for type A spermatogonia relative to above-mentioned other types of gonadal cells were identified and subjected to RT-PCR and quatitative PCR analysis. Since only the rainbow trout notch1 homologue showed significantly high expression in the type A spermatogonia-enriched fraction, we propose that notch1 may be a useful molecular marker for type A spermatogonia. The combination of GFP-dependent FCM and microarray analysis of pvasa-GFP rainbow trout can therefore be applied to the identification of potentially useful molecular markers of germ cells in fish.

Isolation of highly pure and viable primordial germ cells from rainbow trout by GFP-dependent flow cytometry.

A highly pure and viable primordial germ cell (PGC) population appears to be an essential tool for establishing a cell line that can differentiate into a germ cell lineage and for studying the molecular biology and biochemistry of fish PGCs. Therefore, the aim of the present study was to establish a flow cytometric method for isolating highly pure and viable PGCs. As the material for PGC isolation, we used transgenic rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa-gene regulatory sequences (pvasa-GFP). Four independent transgenic strains were subjected to fluorescence microscopy and GFP-dependent flow cytometric analyses. We found that some of the pvasa-GFP transgenic strains exhibited ectopic background green fluorescence in the somatic cells aside from strong fluorescence in PGCs. Although flow cytometric analysis of genital ridge somatic cells in the four pvasa-GFP transgenic strains revealed a wide range of GFP intensities, we proved that somatic cell contamination of the GFP-positive cell population was markedly reduced if transgenic strains without the ectopic background green fluorescence were used. In addition, the forward light-scattering (FS) property, which is an indication of relative cell size, and the side light-scattering (SS) property, which is determined by cell shape and granularity, were employed to remove non-PGC contaminants from the GFP-positive cell population. By isolating GFP-positive cells with high FS/SS values, we were able to effectively remove cell blebs and the apoptotic fraction. Consequently, the purities and survival rates of isolated PGCs were greatly improved compared with those using GFP intensity as a single indicator. Thus, our flow cytometric method, in combination with the selection of suitable transgenic strains without the ectopic background green fluorescence, is capable of isolating highly pure and viable PGCs from rainbow trout. By using this method in combination with cell-cryopreservation and cell transplantation techniques, the isolated PGCs may also be used for preserving the genetic resources of endangered fish species and domesticated fish strains carrying commercially valuable traits. Mol. Reprod. Dev. 67: 91-100, 2004.

Production of biologically-active recombinant goldfish gonadotropins in transgenic rainbow trout

The feasibility of using rainbow trout Oncorhynchus mykiss embryos as an expression system for proteins was investigated. For model proteins, we selected two goldfish gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH). To produce single-chain goldfish FSH (scgfFSH) and LH (scgfLH), cDNAs encoding glycoprotein hormone (GP) α and FSHβ were fused in tandem, and cDNAs encoding GPα and LHβ were fused in tandem. The fused cDNAs were ligated with β-actin promoter, and microinjected into fertilized rainbow trout eggs. After 4-days incubation, the embryos were subjected to western blotting and in vitro bioassays. The recombinant proteins produced by the embryos were immunoreactive to antisera against goldfish GPα, N-glycosylated, and biologically active. We conclude that scgfFSH and scgfLH were successfully produced in transgenic rainbow trout.

Analysis of cell-specificity and variegation of transgene expression driven by salmon prolactin promoter in stable lines of transgenic rainbow trout.

In order to identify the specificity and functionality of salmon prolactin (sPRL) promoter, transgenic rainbow trout carrying a construct comprising the 2.4 kb fragment of the 5' flanking region of Atlantic Chinook sPRL gene fused either to the reporter genes cat (sPRL-cat) or lacZ (sPRL-lacZ) were produced. sPRL-cat in transgenic F0 fish expressed strongly CAT only in the pituitary gland. Transgenic in F1-F4 lines harbouring sPRL-lacZ expressed beta-galactosidase (beta-gal) only in the follicular PRL-producing cells of the adenohypophysis. We observed heterocellular, mosaic distribution of beta-gal within PRL cell population and enormous variation of lacZ expression level between the littermates in the same transgenic line. Regardless of the transgene copy number, age or sex of transgenic fish, beta-gal expression was lactotroph-specific but variegated in all the nine F2 hemizygous lines analysed. One line harbouring a multicopy integration was followed up to F4 generation: the transgene was transmitted without modifications. Analysis of genomic DNA from pituitaries showed that lacZ sequences were highly methylated. LacZ expression was low and its transcripts, analysed by in situ hybridisation, showed a mosaic distribution within the pituitary gland. These data suggest that variegated expression of lacZ can occur at the transcription level owing to the silencing effect of lacZ gene. After proving the tissue-specific expression of reporter genes driven by the sPRL promoter, we tried to obtain the genetic ablation of PRL-producing cells,by transferring the same construct comprising diphtheria toxin DT-A gene (tox). However, the high mortality rate of sPRL-tox transformed embryos has embedded this study and no transgenic fish expressing tox were produced. The appropriateness of using transgenic strategies to analyse gene function in Salmonids is discussed, especially the implications of the multicopy integration patterns and of the variegated transgene expression.

Mass isolation of primordial germ cells from transgenic rainbow trout carrying the green fluorescent protein gene driven by the vasa gene promoter.

Mass isolation of live primordial germ cells (PGCs) was demonstrated for the first time in ectothermal vertebrates. To establish a stem cell-mediated gene transfer system in fish, a stem cell line that retains the ability to develop into gametes is necessary. PGCs are well suited for use as the initial material for such a stem cell line. We established transgenic rainbow trout (Oncorhynchus mykiss) strains carrying the green fluorescent protein (GFP) gene driven by a rainbow trout vasa-like gene (RtVLG) promoter/enhancer. Because GFP expression was specific to the PGCs, PGCs were successfully visualized in all developmental stages examined. Isolated genital ridges containing GFP-labeled PGCs were enzymatically dissociated. To isolate PGCs from the complex pools of dissociated genital ridges, GFP-labeled cells were sorted by flow cytometry. The sorted GFP-positive cells were large and round with a large nucleus, typical characters of PGC morphology. The expression of RtVLG was detected only in the GFP-positive cell population, confirming that these cells were PGCs. This simple and efficient technique to purify a large number of viable PGCs opens the way for establishing a stem cell line, which can differentiate into the germline. The purified PGCs would also be a novel tool for cellular and molecular study of vertebrate germline stem cells.

Gene knock-down in rainbow trout embryos using antisense morpholino phosphorodiamidate oligonucleotides.

Gene knock-down technology using antisense molecules has many applications for studying gene function, disrupting undesirable genetic traits, as well as providing effective therapy for a number of viral diseases. Encouraged by these applications, we developed a gene knock-down technique to interfere with gene expression using transgenic rainbow trout expressing the green fluorescent protein (GFP) gene as a model. One of the antisense morpholino phosphorodiamidate oligonucleotides (AMOs) used in this study (AtGFP-1) was 25 nucleotides in length and localized against codons 2 to 8 of GFP messenger RNA. Microinjection of AtGFP-1 into the blastodisc of fertilized eggs decreased the level of GFP gene expression in a dose-dependent manner, A comparison of the effects of various doses of AtGFP-1 suggested that 10 ng of AtGFP-1 was the optimal concentration in that it interfered with specific gene expression without being strongly toxic to trout embryos. Conversely, morpholino phosphorodiamidate oligonucleotides with the inverted AtGFP-1 sequence, which cannot bind to the target mRNA, did not inhibit GFP gene expression. AtGFP-1 did not affect the expression of nontargeted genes such as the skeletal muscle actin and foreign lacZ genes. These results also indicate that AtGFP-1 interfered with the expression of only the targeted gene. Western blot and reverse transcriptase polymerase chain reaction analyses revealed that the amount of GFP protein drastically decreased whereas the mRNA level was not affected by AtGFP-1, suggesting that AtGFP-1 blocked specific gene function at the translational level. Further, this gene inhibition persisted until the hatching stage. Another AMO, which was localized against the junction region between the 5? untranslated region and the starting codon of GFP mRNA (AtGFP-2), also caused inhibition effects. Thus AMOs can have potent and specific gene knock-down effects in trout embryos. This technology may be useful for examining the roles of selected genes and disrupting their expression during embryonic development of salmonid fish.