Transgenic Fish

2009 ◽  
pp. 1-39
Author(s):  
Ioannis Arvanitoyannis ◽  
Persephoni Tserkezou
Keyword(s):  
Transgenic Fish

scholarly journals A CRISPR/Cas9 vector system for neutrophil-specific gene disruption in zebrafish

2020 ◽  
Author(s):  
Yueyang Wang ◽  
Alan Y. Hsu ◽  
Eric M. Walton ◽  
Ramizah Syahirah ◽  
Tianqi Wang ◽  
...  
Keyword(s):  
Cell Motility ◽  
Neutrophil Migration ◽  
Transgenic Fish ◽  
Subcellular Location ◽  
Vector System ◽  
Specific Gene ◽  
Tissue Specific ◽  
Biological Studies

AbstractTissue-specific knockout techniques are widely applied in biological studies to probe the tissue-specific roles of specific genes in physiology, development, and disease. CRISPR/Cas9 is a widely used technology to perform fast and efficient genome editing in vitro and in vivo. Here, we report a robust CRISPR-based gateway system for tissue-specific gene inactivation in zebrafish. A transgenic fish line expressing Cas9 under the control of a neutrophil-restricted promoter was constructed. As proof of principle, we transiently disrupted rac2 or cdk2 in neutrophils using plasmids driving the expression of sgRNAs from U6 promoters. Loss of the rac2 or cdk2 gene in neutrophils resulted in significantly decreased cell motility, which could be restored by re-expressing Rac2 or Cdk2 in neutrophils in the corresponding knockout background. The subcellular location of Rac activation and actin structure and stress in the context of neutrophil migration was determined in both the wild-type and rac2 knockout neutrophils in vivo. In addition, we evaluated an alternative approach where the Cas9 protein is ubiquitously expressed while the sgRNA is processed by ribozymes and expressed in a neutrophil-restricted manner. Cell motility was also reduced upon rac2 sgRNA expression. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identification and characterization of gene functions in neutrophils.

scholarly journals OPTIMAL ELECTROPORATION CONDITION FOR SPERM MEDIATED GENE TRANSFER IN STRIPPED CATFISH (Pangasionodon hypophthalmus)

2010 ◽  
Vol 5 (1) ◽  
pp. 1
Author(s):  
Raden Roro Sri Pudji Sinarni Dewi ◽  
Alimuddin Alimuddin ◽  
Agus Oman Sudrajat ◽  
Komar Sumantadinata ◽  
Sularto Sularto
Keyword(s):  
Sperm Motility ◽  
Fluorescent Protein ◽  
Pulse Length ◽  
Transgenic Fish ◽  
Pulse Number ◽  
Pulse Interval ◽  
Hatching Rate ◽  
Exogenous Dna ◽  
Motility Of Sperm ◽  
Green Fluorescent

The success of transgenic fish production has been achieved through eggs fertilization using electroporated sperms carrying exogenous DNA. This study was conducted in order to obtain the optimal electroporation condition for stripped catfish sperm. A plasmid containing green fluorescent protein (GFP) gene driven by carp β-actin promoter was transferred into sperm using electrophoresis method towards transgenic stripped catfish (Pangasionodon hypophthalmus) production. Electroporation was carried out using square wave shock with pulse length of 30 ms and pulse interval of 0.1 sec. Treatments are combination between voltage (50 V, 75 V, and 100 V) and pulse number (1 and 3). Exogenous DNA concentration used was 10 μg/mL of Tris-EDTA. Results showed that increasing the voltage from 50 to 100 decreased sperm motility, while pulse number did not affect sperm motility. Voltage of 50 gave the best motility of sperm, although sperm viability relatively similar between treatments and control except at 100 V with 3 pulses number. Further, electroporation-treated sperms were able to fertilize eggs. Higher hatching rate of eggs was obtained in electroporation treatment at 50 V with pulse number of 1 and 3. The persistence of transferred GFP was detected in electroporated and incubated sperms (control). However, GFP was only detected in larvae from eggs that were fertilized by electroporated sperm. Thus, electroporation could be applied to produce transgenic stripped catfish. 

Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos

Development ◽  
1988 ◽  
Vol 103 (2) ◽  
pp. 403-412 ◽  
Author(s):  
G.W. Stuart ◽  
J.V. McMurray ◽  
M. Westerfield
Keyword(s):  
Germ Line ◽  
Transmission Rate ◽  
Transgenic Fish ◽  
Early Embryo ◽  
Zebrafish Embryos ◽  
Fish Genome ◽  
Bacterial Plasmid ◽  
Foreign Dna ◽  
High Concentrations ◽  
F2 Progeny

To generate stable lines of transgenic fish, early zebrafish embryos were injected with high concentrations of a linear bacterial plasmid. After injection, the foreign DNA was converted into a high molecular weight form and then amplified approximately tenfold during the initial rapid cleavages characteristic of the early embryo prior to gastrulation. While most of this DNA was subsequently degraded during gastrulation, some of the foreign sequences survived the gastrula stage and could be found in most of the injected fish at 3 weeks of age. Only about 5% of fish analysed 4 months after the injection retained foreign DNA in their fins, usually at less than one copy per cell. One of these fish was also found to contain about 100 copies per cell of foreign DNA in a fraction of its germ cells. Approximately 20% of the F1 offspring from this germ-line-positive parent inherited the foreign DNA, whereas 50% of F2 progeny obtained from an identified F1 individual inherited these sequences. The 50% transmission rate in F2 progeny was as expected for a single, heterozygous genomic insert. These observations indicate that injected DNA can be integrated into the fish genome, that the resulting transgenic fish are mosaic and that some of these mosaic individuals give rise to stable lines of transgenic fish.

Hematopoietic regulatory domain of gata1 gene is positively regulated by GATA1 protein in zebrafish embryos

Development ◽  
2001 ◽  
Vol 128 (12) ◽  
pp. 2341-2350
Author(s):  
Makoto Kobayashi ◽  
Keizo Nishikawa ◽  
Masayuki Yamamoto
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Regulatory Region ◽  
Ectopic Expression ◽  
Transgenic Fish ◽  
Functional Domain ◽  
Cis Acting ◽  
Gfp Reporter ◽  
Gata Motif

Expression of gata1 is regulated through multiple cis-acting GATA motifs. To elucidate regulatory mechanisms of the gata1 gene, we have used zebrafish. To this end, we isolated and analyzed zebrafish gata1 genomic DNA, which resulted in the discovery of a novel intron that was unknown in previous analyses. This intron corresponds to the first intron of other vertebrate Gata1 genes. GFP reporter analyses revealed that this intron and a distal double GATA motif in the regulatory region are important for the regulation of zebrafish gata1 gene expression. To examine whether GATA1 regulates its own gene expression, we microinjected into embryos a GFP reporter gene linked successively to the gata1 gene regulatory region and to GATA1 mRNA. Surprisingly, ectopic expression of the reporter gene was induced at the site of GATA1 overexpression and was dependent on the distal double GATA motif. Functional domain analyses using transgenic fish lines that harbor the gata1-GFP reporter construct revealed that both the N- and C-terminal zinc-finger domains of GATA1, hence intact GATA1 function, are required for the ectopic GFP expression. These results provide the first in vivo evidence that gata1 gene expression undergoes positive autoregulation.

GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene

Development ◽  
1997 ◽  
Vol 124 (20) ◽  
pp. 4105-4111 ◽  
Author(s):  
Q. Long ◽  
A. Meng ◽  
H. Wang ◽  
J.R. Jessen ◽  
M.J. Farrell ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Progenitor Cells ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Transgenic Fish ◽  
Green Fluorescent Protein Reporter ◽  
Erythroid Progenitor ◽  
Green Fluorescent ◽  
Fluorescent Protein Reporter

In this study, DNA constructs containing the putative zebrafish promoter sequences of GATA-1, an erythroid-specific transcription factor, and the green fluorescent protein reporter gene, were microinjected into single-cell zebrafish embryos. Erythroid-specific activity of the GATA-1 promoter was observed in living embryos during early development. Fluorescent circulating blood cells were detected in microinjected embryos 24 hours after fertilization and were still present in 2-month-old fish. Germline transgenic fish obtained from the injected founders continued to express green fluorescent protein in erythroid cells in the F1 and F2 generations. The green fluorescent protein expression patterns in transgenic fish were consistent with the pattern of GATA-1 mRNA expression detected by RNA in situ hybridization. These transgenic fish have allowed us to isolate, by fluorescence-activated cell sorting, the earliest erythroid progenitor cells from developing embryos for in vitro studies. By generating transgenic fish using constructs containing other zebrafish promoters and green fluorescent protein reporter gene, it should be possible to visualize the origin and migration of any lineage-specific progenitor cells in a living embryo.

scholarly journals Efficient and Stable Delivery of Multiple Genes to Fish Cells by a Modified Recombinant Baculovirus System

2018 ◽  
Vol 19 (12) ◽  
pp. 3767 ◽  
Author(s):  
Qian Wang ◽  
Jian Fang ◽  
Qihua Pan ◽  
Yizhou Wang ◽  
Ting Xue ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Recombinant Baculovirus ◽  
Transgenic Fish ◽  
Early Gene ◽  
Drug Selection ◽  
Baculovirus System ◽  
Fish Cells

The recombinant baculovirus has been widely used as an efficient tool to mediate gene delivery into mammalian cells but has barely been used in fish cells. In the present study, we constructed a recombinant baculovirus containing the dual-promoter cytomegalovirus (CMV) and white spot syndrome virus (WSSV) immediate-early gene 1 (ie1) (WSSV ie1), followed by a puromycin–green fluorescent protein (Puro-GFP, pf) or puromycin–red fluorescent protein (Puro-RFP, pr) cassette, which simultaneously allowed for easy observation, rapid titer determination, drug selection, and exogenous gene expression. This recombinant baculovirus was successfully transduced into fish cells, including Mylopharyngodon piceus bladder (MPB), fin (MPF), and kidney (MPK); Oryzias latipes spermatogonia (SG3); and Danio rerio embryonic fibroblast (ZF4) cells. Stable transgenic cell lines were generated after drug selection, which was further verified by Western blot. A cell monoclonal formation assay proved the stable heredity of transgenic MPB cells. In addition, a recombinant baculovirus containing a pr cassette and four transcription factors for induced pluripotent stem cells (iPSC) was constructed and transduced into ZF4 cells, and these exogenous genes were simultaneously delivered and transcribed efficiently in drug-selected ZF4 cells, proving the practicability of this modified recombinant baculovirus system. We also proved that the WSSV ie1 promoter had robust activity in fish cells in vitro and in vivo. Taken together, this modified recombinant baculovirus can be a favorable transgenic tool to obtain transient or stable transgenic fish cells.

scholarly journals A Novel AURKA Mutant-Induced Early-Onset Severe Hepatocarcinogenesis Greater than Wild-Type via Activating Different Pathways in Zebrafish

Cancers ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 927 ◽  
Author(s):  
Zhong-Liang Su ◽  
Chien-Wei Su ◽  
Yi-Luen Huang ◽  
Wan-Yu Yang ◽  
Bonifasius Putera Sampurna ◽  
...  
Keyword(s):  
Early Onset ◽  
Transgenic Fish ◽  
Aurora A Kinase ◽  
Mitotic Progression ◽  
Wild Type ◽  
Proliferation Markers ◽  
Long Latency ◽  
Important Regulator ◽  
Low Incidence ◽  
Screening Platform

Aurora A kinase (AURKA) is an important regulator in mitotic progression and is overexpressed frequently in human cancers, including hepatocellular carcinoma (HCC). Many AURKA mutations were identified in cancer patients. Overexpressing wild-type Aurka developed a low incidence of hepatic tumors after long latency in mice. However, none of the AURKA mutant animal models have ever been described. The mechanism of mutant AURKA-mediated hepatocarcinogenesis is still unclear. A novel AURKA mutation with a.a.352 Valine to Isoleucine (V352I) was identified from clinical specimens. By using liver-specific transgenic fish overexpressing both the mutant and wild-type AURKA, the AURKA(V352I)-induced hepatocarcinogenesis was earlier and much more severe than wild-type AURKA. Although an increase of the expression of lipogenic enzyme and lipogenic factor was observed in both AURKA(V352I) and AURKA(WT) transgenic fish, AURKA(V352I) has a greater probability to promote fibrosis at 3 months compared to AURKA(WT). Furthermore, the expression levels of cell cycle/proliferation markers were higher in the AURKA(V352I) mutant than AURKA(WT) in transgenic fish, implying that the AURKA(V352I) mutant may accelerate HCC progression. Moreover, we found that the AURKA(V352I) mutant activates AKT signaling and increases nuclear β-catenin, but AURKA(WT) only activates membrane form β-catenin, which may account for the differences. In this study, we provide a new insight, that the AURKA(V352I) mutation contributes to early onset hepatocarcinogenesis, possibly through activation of different pathways than AURKA(WT). This transgenic fish may serve as a drug-screening platform for potential precision medicine therapeutics.

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