Red Fluorescent Protein Variant with a Dual-Peak Emission of Fluorescence.

The marine environment is a rich reservoir of diverse biological entities, many of which possess unique properties that are of immense value to biotechnological applications. One such example is the red fluorescent protein derived from the coral Discosoma sp. This protein, encoded by the DsRed gene, has been the subject of extensive research due to its potential applications in various fields. In the study, a variant of the red fluorescent protein was generated through random mutagenesis using the DsRed2 gene as a template. The process employed error-prone PCR (epPCR) to introduce random mutations, leading to the isolation of twelve gene variants. Among these, one variant stood out due to its unique spectral properties, exhibiting dual fluorescence emission at both 480 nm (green) and 550 nm (red). This novel variant was expressed in both Escherichia coli and zebrafish (Danio rerio) muscle, confirming the dual fluorescence emission in both model systems. One of the immediate applications of this novel protein variant is in ornamental aquaculture. The dual fluorescence can serve as a unique marker or trait, enhancing the aesthetic appeal of aquatic species in ornamental settings.

Growth Hormone Overexpression Induces Hyperphagia and Intestinal Morphophysiological Adaptations to Improve Nutrient Uptake in Zebrafish.

The excess of circulating growth hormone (GH) in most transgenic animals implies mandatory growth resulting in higher metabolic demand. Considering that the intestine is the main organ responsible for the digestion, absorption, and direction of dietary nutrients to other tissues, this study aimed to investigate the mechanisms by which gh overexpression modulates the intestine to support higher growth. For this purpose, we designed an 8-weeks feeding trial to evaluate growth parameters, feed intake, and intestinal morphometric indices in the adult gh-transgenic zebrafish (Danio rerio) model. To access the sensitivity of the intestine to the excess of circulating GH, the messenger RNA (mRNA) expression of intestine GH receptors (GHRs) (ghra and ghrb) was analyzed. In addition, the expression of insulin-like growth factor 1a (igf1a) and genes encoding for di and tripeptide transporters (pept1a and pept1b) were assessed. Gh-transgenic zebrafish had better growth performance and higher feed intake compared to non-transgenic sibling controls. Chronic excess of GH upregulates the expression of its cognate receptor (ghrb) and the main growth factor related to trophic effects in the intestine (igf1a). Moreover, transgenic zebrafish showed an increased intestinal absorptive area and higher expression of crucial genes related to the absorption of products from meal protein degradation. These results reinforce the ability of GH to modulate intestinal morphology and the mechanisms of assimilation of nutrients to sustain the energy demand for the continuous growth induced by the excess of circulating GH.

GH Overexpression Alters Spermatic Cells MicroRNAome Profile in Transgenic Zebrafish.

Overexpression of growth hormone (GH) in gh-transgenic zebrafish of a highly studied lineage F0104 has earlier been reported to cause increased muscle growth. In addition to this, GH affects a broad range of cellular processes in transgenic fish, such as morphology, physiology, and behavior. Reports show changes such as decreased sperm quality and reduced reproductive performance in transgenic males. It is hypothesized that microRNAs are directly involved in the regulation of fertility potential during spermatogenesis. The primary aim of our study was to verify whether gh overexpression disturbs the sperm miRNA profile and influences the sperm quality in transgenic zebrafish. We report a significant increase in body weight of gh-transgenic males along with associated reduced sperm motility and other kinetic parameters in comparison to the non-transgenic group. MicroRNA transcriptome sequencing of gh-transgenic zebrafish sperms revealed expressions of 186 miRNAs, among which six miRNA were up-regulated (miR-146b, miR-200a-5p, miR-146a, miR-726, miR-184, and miR-738) and sixteen were down-regulated (miR-19d-3p, miR-126a-5p, miR-126b-5p, miR-22a-5p, miR-16c-5p, miR-20a-5p, miR-126b-3p, miR-107a-3p, miR-93, miR-2189, miR-202-5p, miR-221-3p, miR-125a, miR-125b-5p, miR-126a-3p, and miR-30c-5p) in comparison to non-transgenic zebrafish. Some of the dysregulated miRNAs were previously reported to be related to abnormalities in sperm quality and reduced reproduction ability in other species. In this study, an average of 134 differentially expressed miRNAs-targeted genes were predicted using the in silico approach. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis demonstrated that the genes of affected pathways were primarily related to spermatogenesis, sperm motility, and cell apoptosis. Our results suggested that excess GH caused a detrimental effect on sperm microRNAome, consequently reducing the sperm quality and reproductive potential of zebrafish males.

Expression of GFP in transgenic tilapia under the control of the medaka B-actin promoter: establishment of a model system for germ cell transplantation

The Nile tilapia (Oreochromis niloticus) stands out as one of the most important fresh water edible fish, possessing remarkable characteristics that make it desirable for both commercial culture and as a laboratory model. For the utilization of tilapia in germ cell transplantation experiments, appropriate cell markers are required to evaluate the colonization behavior of donor-derived germ cells in recipient gonads. Here we report the production of a medaka β-actin/EGFP transgenic tilapia strain expressing the green fluorescent protein (GFP) reporter gene in several tissues including germ cells in testis and ovary. Fluorescent observations in F2 generation transgenic individuals showed GFP positive cells along the body axis in pre-hatched embryos, while in hatching embryos the GFP gene was strongly expressed in the area surrounding the gills, operculum and in the cephalic region. In early larvae, fluorescent cells were scattered throughout the body, forming aggregations around the dorsal-cephalic and mouth areas. At 38 days post-fertilization, juvenile fish expressed the GFP homogeneously in the whole body. GFP fluorescence was also observed in caudal fins, muscle, and in several internal organs (gills, heart, testes, and ovaries) in 140 and 240 day F2 and F3 individuals. Immunohistochemistry using a monoclonal anti-GFP antibody in juvenile and adult gonads showed that both mitotic and meiotic germ cells were labeled with GFP. The utilization of this transgenic line in a germ cell transplantation system could offer a fast and reliable screening of donor-derived transgenic offspring, as well as accurate tracing of donor-derived cell colonization in the recipient gonad by means of immunohistochemistry using GFP antibodies. In the future, germ cell transplantation using Nile tilapia also could help to preserve the genetic resources of threatened cichlids, through cryopreservation and interspecies transplantation of germ cells from endangered cichlids into O. niloticus recipients.(AU)

Expression of GFP in transgenic tilapia under the control of the medaka B-actin promoter: establishment of a model system for germ cell transplantation

The Nile tilapia (Oreochromis niloticus) stands out as one of the most important fresh water edible fish, possessing remarkable characteristics that make it desirable for both commercial culture and as a laboratory model. For the utilization of tilapia in germ cell transplantation experiments, appropriate cell markers are required to evaluate the colonization behavior of donor-derived germ cells in recipient gonads. Here we report the production of a medaka β-actin/EGFP transgenic tilapia strain expressing the green fluorescent protein (GFP) reporter gene in several tissues including germ cells in testis and ovary. Fluorescent observations in F2 generation transgenic individuals showed GFP positive cells along the body axis in pre-hatched embryos, while in hatching embryos the GFP gene was strongly expressed in the area surrounding the gills, operculum and in the cephalic region. In early larvae, fluorescent cells were scattered throughout the body, forming aggregations around the dorsal-cephalic and mouth areas. At 38 days post-fertilization, juvenile fish expressed the GFP homogeneously in the whole body. GFP fluorescence was also observed in caudal fins, muscle, and in several internal organs (gills, heart, testes, and ovaries) in 140 and 240 day F2 and F3 individuals. Immunohistochemistry using a monoclonal anti-GFP antibody in juvenile and adult gonads showed that both mitotic and meiotic germ cells were labeled with GFP. The utilization of this transgenic line in a germ cell transplantation system could offer a fast and reliable screening of donor-derived transgenic offspring, as well as accurate tracing of donor-derived cell colonization in the recipient gonad by means of immunohistochemistry using GFP antibodies. In the future, germ cell transplantation using Nile tilapia also could help to preserve the genetic resources of threatened cichlids, through cryopreservation and interspecies transplantation of germ cells from endangered cichlids into O. niloticus recipients.