Red Fluorescent Protein Expression in Transgenic Founder of Angelfish (Pterophyllum sp) Driven by Zebrafish Myosin Light Chain 2 Promoter

Angelfish (Pterophyllum sp.) are attractive fish popular with aquarists. The introduction of fluorescent protein genes into angelfish has been reported, but specific techniques have not been revealed. This study aimed to develop a strategy to produce red fluorescent protein (RFP) transgenic angelfish driven by the myosin light chain 2 (mylz2) promoter from zebrafish. A 1999 bp Mylz2 promoter fragment was isolated from zebrafish muscle genomic DNA. This promoter fragment was then cloned into the plasmid pDsred2-1 open-loop at restriction enzyme SacI and AgeI sites to create the final transgene construct pMylz2-RFP. Angelfish embryos at one cell stage were microinjected with approximately 100 pg of the plasmid pMylz2-RFP. From 524 microinjected embryos, 16 successfully hatched, while 12 showed red fluorescence signals. Two larvae survived to 2 months of age. They showed significant red fluorescence expression in the muscles, suggesting that the angelfish could be used as potential transgenic founders to evaluate the next generation of stable red fluorescence expression transgenic fish.

MLL/AF9 Expression Causes Leukemia in Zebrafish

Background Acute Leukaemia (AL) is a genetically heterogeneous disorder caused by somatic mutations and acquired chromosomal translocations. Translocations can lead to the formation of fusion genes such as the MLL/AF9 fusion, which results from the t(9;11)(p22;q23). A better understanding the molecular pathophysiology of AML, of the mechanisms of treatment resistance, disease relapse can be achieved by developing animal models. The MLL/AF9 fusion is frequently used to model AML in mice. However, to date, no MLL/AF9 leukemia models in zebrafish have been reported. Aim Our aim was to establish a transgenic zebrafish leukemia model using the human MLL/AF9 fusion gene. Methods To generate transgenic fish, two constructs (pTol2-Runx1+23: MLL-AF9-IRES-EGFP-cmlc-GFP and pTol2-Runx1+23: MLL-AF9-IRES-mCherry-cmlc-GFP) were injected together with Tol2 transposase mRNA into one-cell stage zebrafish embryos. We used the murine Runx1+23 enhancer to direct MLL/AF9 expression to hematopoietic stem cells and EGFP or mCherry as fluorescent markers. We selected transgenic embryos 24 hours post-fertilization based on the heart marker expression (cmlc). Results 29% (100 of 340 embryos) of the transgenics reached adulthood (6 weeks). After 6 to 24 months, 77% (77) of them developed signs of sickness. They became less active with protruding eyes and hump formation on the nose. Some started bleeding from the gills and/or showed tumor formation around the abdomen and head. Sick fish were euthanized and dissected. The autopsies showed pale and dysmorphic kidneys, pale and enlarged spleens, and in some cases white granular spots on the spleen. Histological sections revealed increased kidney, spleen, and liver cellularity with massive cellular infiltration of cells in these organs. In flow cytometry, kidney marrow cells from the transgenic fish showed a different forward scatter (FSC) and side scatter (SSC) profile compared to that of the normal zebrafish kidney marrow cells. The transgenic F 0 zebrafish showed increased numbers of lymphoid cells (12 fish), precursor cells (9 fish), or myeloid cells (6 fish). Peripheral blood smears showed many intermediate-sized mononuclear cells with an increased nuclear-cytoplasmic ratio, with nuclei containing dipsersed chromatin and inconspicuous nucleoli resembling blasts. There were occasional myelocytes and no mature granulocytes. These data are consistent with the development of acute leukemia in our MLL/AF9 transgenic fish. Whole-mount in situ hybridization (WISH) was performed on F 1 embryos. RNA probes for early hematopoietic markers (gata1, scl, runx1, ikaros, cmyb, mpx, and lyz) were hybridized to 24 and 48 hpf F1 transgenic embryos. There were expression changes of these markers compared to age-matched wild-type larvae, including low expression of gata1, scl, cmyb and high expression of lyz, mpx and ikaros in the caudal hematopoietic organ. We also performed transplantation experiments with the kidney marrow cells from diseased fish to test whether the disease was transplantable. The disease was serially transplantable into secondary and tertiary recipient fish. Transplanted fish had a significantly shorter latency to disease development of only 2 to 6 weeks. The morphological evidence and the serial transplantability of the disease proves that we have in fact succeeded in establishing an MLL/AF9-driven acute leukemia model in zebrafish. The long latency and incomplete penetrance observed in our F 0 MA9 zebrafish, along with a shorter latency in the transplanted fish, suggests that additional somatic mutations are required for leukemogenesis in this model. We performed whole exome sequencing to find cooperating somatic mutations and RNASeq to identify differentially expressed genes in MA9 leukemia fish. Whole exome sequencing on six samples identified putative somatic mutations in genes such as Stat5, Cyp2j20, Ms4a17a.3, Tapbp.1 and Herc5.3, which have been reported to be mutated in human cancer. RNA-seq analysis on seven samples showed 67 differentially expressed genes with a q value < 0.05 (e.g., cxcl32b.1, myof1, ctdsp2, egr3, il2rb) and nine enriched pathways with a P-value of < 0.054 (e.g.: KRAS, TP53, MEK) in our transgenic leukemic MLL/AF9 fish. Conclusion Our MLL/AF9 zebrafish acute leukemia model will be a helpful tool to understand leukemia biology and enable testing of new therapeutic strategies. Disclosures Browett: AbbVie: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees.

Differential expression of transposable elements in the medaka melanoma model

Malignant melanoma incidence is rising worldwide. Its treatment in an advanced state is difficult, and the prognosis of this severe disease is still very poor. One major source of these difficulties is the high rate of metastasis and increased genomic instability leading to a high mutation rate and the development of resistance against therapeutic approaches. Here we investigate as one source of genomic instability the contribution of activation of transposable elements (TEs) within the tumor. We used the well-established medaka melanoma model and RNA-sequencing to investigate the differential expression of TEs in wildtype and transgenic fish carrying melanoma. We constructed a medaka-specific TE sequence library and identified TE sequences that were specifically upregulated in tumors. Validation by qRT- PCR confirmed a specific upregulation of a LINE and an LTR element in malignant melanomas of transgenic fish.

Assessing wild genetic background and parental effects on size of growth hormone transgenic coho salmon

Experiments examining potential impacts of growth hormone (GH) transgenesis in fish typically use a single source strain, and do not address potential differential impacts in strains of different genetic backgrounds. Here, we examine the effects of differing genetic backgrounds when reared in culture on the growth of transgenic and non-transgenic coho salmon (Oncorhynchus kisutch) produced by mating sires from different rivers with transgenic dams from a single origin. We found a significant difference in size between offspring of sires originating from various river systems in British Columbia. This difference was independent of differences between transgenotypes (i.e., transgenic vs. non-transgenic offspring). However, the effects of strain or sire were relatively small compared to the effects of the transgene, which were consistent regardless of sire origin. Thus, results derived from studies of GH transgenic fish from a single source population can provide useful information for assessments of GH transgenic salmon from other systems. This has important implications for examining potential risks from introgression of a transgene into different populations.

Gene Transmission, Growth, and Exogeneous Growth Hormone Expression of G2 Transgenic Betta Fish (Betta imbellis)

Highlight ResearchThe F2 of GH-transgenic B. imbellis was successfully producedThe transgene inheritance by the F2 fish was more than 90%The growth and body size of transgenic fish was significantly higher than controlF2 fish reached a larger body size in a shorter period compared to the F1 AbstractIn our previous research, we had successfully produced G0 and G1 Pangasianodon hypophthalmus growth hormone (PhGH) transgenic B. imbellis, native ornamental betta from Indonesia, which its giant-sized variant has valuable price for the breeders. The G0 and G1 transgenic (TG) fish showed higher growth rate and body size compared to the non-transgenic (NT) fish. The study was aimed to produce and evaluate the consistencies of transgene transmission and expression in G2 generation. The growth rate and body size between TG and NT fish was also compared. The G2 generation was produced using crosses between TG and NT G1 fish: ♂TG × ♀TG, ♂TG × ♀NT, ♂NT × ♀TG, and ♂NT ×♀ NT. Fish were reared for 12 weeks, and transgene detection was performed using the polymerase chain reaction method (PCR) on isolated DNA from the caudal fin clips. The endogenous and exogenous GH expression analysis was conducted using the quantitative real-time PCR (qPCR) method. The results showed that the inheritance of the GH transgene by the G2 fish was more than 90% in all transgenic crosses. Endogenous GH was expressed at the same levels in the brain of TG and NT fish, but the exogenous GH expression was highly detected only in the TG fish. The G2 transgenic fish had a higher specific growth rate, up to 31%, compared to the control. The body length of TG crosses were 23−35% higher and had 111−135% higher body weight compared to NT fish. These results showed a promising approached in mass-producing stable lines of giant-sized betta using the GH-transgenic technology.

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.

Optogenetic Manipulation of Olfactory Responses in Transgenic Zebrafish: A Neurobiological and Behavioral Study

Olfaction is an important neural system for survival and fundamental behaviors such as predator avoidance, food finding, memory formation, reproduction, and social communication. However, the neural circuits and pathways associated with the olfactory system in various behaviors are not fully understood. Recent advances in optogenetics, high-resolution in vivo imaging, and reconstructions of neuronal circuits have created new opportunities to understand such neural circuits. Here, we generated a transgenic zebrafish to manipulate olfactory signal optically, expressing the Channelrhodopsin (ChR2) under the control of the olfactory specific promoter, omp. We observed light-induced neuronal activity of olfactory system in the transgenic fish by examining c-fos expression, and a calcium indicator suggesting that blue light stimulation caused activation of olfactory neurons in a non-invasive manner. To examine whether the photo-activation of olfactory sensory neurons affect behavior of zebrafish larvae, we devised a behavioral choice paradigm and tested how zebrafish larvae choose between two conflicting sensory cues, an aversive odor or the naturally preferred phototaxis. We found that when the conflicting cues (the preferred light and aversive odor) were presented together simultaneously, zebrafish larvae swam away from the aversive odor. However, the transgenic fish with photo-activation were insensitive to the aversive odor and exhibited olfactory desensitization upon optical stimulation of ChR2. These results show that an aversive olfactory stimulus can override phototaxis, and that olfaction is important in decision making in zebrafish. This new transgenic model will be useful for the analysis of olfaction related behaviors and for the dissection of underlying neural circuits.

The Maturity of Female Gonads of G2 Transgenic Mutiara Catfish (Clarias sp.)

The G2 trangsenic Mutiara catfish (Clarias sp.) (MTG) is a Mutiara catfish that is inserted with the CgGH gene (Clarias gariepinus Growth Hormone) through the transgenesis. The effect of transgenesis stimulates gonad growth of G2 transgenic Mutiara catfish (Clarias sp.) faster than non-transgenic fish. Study aimed to analyze the maturity of the gonads and the spawning ability of female G2 transgenic Mutiara catfish (Clarias sp.) to obtain superior broodstock candidates. Experimental method with completely randomized design (pair of parents used as treatment and repeated four times) for spawning was used for this study. Three pairs of parent G2 were crossed semi-artificially as treatment A (female 1 MTG G2 crossed with male 1 MTG G2), B (female 2 MTG G2 crossed with male 2 MTG G2) and C (female 3 MTG G2 crossed with male 3 MTG G2). The results showed that the performance of female G2 transgenic Mutiara catfish (Clarias sp.) (treatment A, B and C) was higher given non-transgenic with an average relative fecundity of 82,438 eggs / kg of broodstock, an average egg diameter of 1.76 mm and an average egg weight 1.75 mg. These indications suggest that GH transgenesis increases gonadal maturity. The gonad maturity profile reached the stage of complete maturity (full ripe) compared to Sangkuriang catfish (Clarias gariepinus) (immature gonads). Therefore it is necessary to compare the reproductive performance of G2 transgenic Mutiara catfish (Clarias sp.) with non-transgenic fish (Sangkuriang) as candidates for superior broodstock of catfish.

Knockdown of Novel lncRNA TCONS_00028652 in Zebrafish Affects Embryonic Vasculature Development

Long non-coding RNAs (lncRNA) are increasingly being regard as potential key regulators of biological process, however, little is known about the function of most of them. The involvement of a novel lncRNA (ENSDART000000150571), previously named TCONS_00028652, in intersegmental vessel development was investigated in this study. TCONS_00028652, having a single exon (568 bp), is located in chromosome 16 at position from 15786804 to 15786237, which was previously identified as an embryonic and adult heart-enrichedlncRNA. Bioinformatics analysis and annotation of TCONS_00028652 was performed using online databases. Its protein-coding potential was assessed using online softwares: the Coding Potential Assessing Tool (CPAT) and the Coding Potential Calculator (CPC). To verify its real existence, a cloned fragment was proliferated by designing primers against 5’ and 3’ exon-flanking sequence. Subsequently, spatiotemporal expression during zebrafish embryonic development was determined using real-time quantitative PCR (qPCR) and whole mount in situ hybridization. We found that lncRNA TCONS_00028652 was generally expressed throughout early stages of zebrafish embryonic development and predominantly in embryonic brain, tail, and heart. Knockdown of TCONS_00028652 using morpholino oligonucleotides (MO) resulted in intersegmental vessel defects, suggesting that TCONS_00028652 is indispensable for embryonic vascular development in zebrafish. Using Tg (flil:EGFP) transgenic fish expressing a cardiovascular marker gene, loss of function experiments confirmed that TCONS_00028652 was involved in embryonic intersegmental vessel development. Our results may lead to valuable understanding of lncRNAs functions in zebrafish embryonic development and molecular mechanisms of embryonic cardiovascular development. © 2021 Friends Science Publishers

A robust and flexible CRISPR/Cas9 based system for neutrophil-specific gene inactivation in zebrafish

CRISPR/Cas9-based tissue-specific knockout techniques are essential in probing the functions of genes in embryonic development and disease using zebrafish. However, the lack of capacity to perform gene-specific rescue or live-imaging in the tissue-specific knockout background has limited the utility of this approach. Here, we report a robust and flexible gateway system for tissue-specific gene inactivation in neutrophils. Using a transgenic fish line with neutrophil-restricted expression of Cas9 and ubiquitous expression of sgRNAs targeting rac2, specific disruption of the rac2 gene in neutrophils is achieved. Transient expression of sgRNAs targeting rac2 or cdk2 in the neutrophil-restricted Cas9 line also results in significantly decreased cell motility. Re-expressing sgRNA-resistant rac2 or cdk2 gene restored neutrophil motility in the corresponding knockout background. Moreover, active Rac and force bearing F-actins localize to both the cell front and the contracting tail during neutrophil interstitial migration in an oscillating fashion that is disrupted when rac2 is knocked out. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identifying and characterizing gene functions in a tissue-specific manner.