Establishment of a transgenic zebrafish EF1α:Kaede for monitoring cell proliferation during regeneration

Moon, Hyun-Yi; Kim, Oc-Hee; Kim, Hyun-Taek; Choi, Jung-Hwa; Yeo, Sang-Yeob; Kim, Nam-Soon; Park, Doo-Sang; Oh, Hyun‐Woo; You, Kwan‐Hee; Zoysa, Mahanama De; Kim, Cheol-Hee

doi:10.1016/j.fsi.2013.02.021

Cited by 8 publications

(5 citation statements)

References 21 publications

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“…In these experiments, we also show that multiple constructs can be prepared and delivered in parallel, with minimal set-up overhead. We transfected three different Tol2 plasmids with the eef1a1/1 (formerly known as ef1a ) promoter (Negrutskii and El’skaya, 1998, Moon et al, 2013) driving expression of various nuclear fluorescent reporters, mNeon-Green, Cherry, and mCerulean (Fig. 4A).…”

Section: Resultsmentioning

confidence: 99%

Lipid-Based Transfection of Zebrafish Embryos: A Robust Protocol for Nucleic Acid Delivery in Zebrafish

Terzi,

Lao,

Jacobo

2024

Preprint

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Zebrafish, a widely used model organism in developmental and biomedical research, offers several advantages such as external fertilization, embryonic transparency, and genetic similarity to humans. However, traditional methods for introducing exogenous genetic material into zebrafish embryos, particularly microinjection, pose significant technical challenges and limit throughput. To address this, we developed a novel approach utilizing Lipofectamine ® LTX for the efficient delivery of nucleic acids into zebrafish embryos by lipid-based transfection. Our protocol bypasses the need for microinjection, offering a cost-effective, high-throughput, and user-friendly alternative. This protocol outlines new strategies for gene delivery in zebrafish to enhance the efficiency and scope of genetic studies in this model system.

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Section: Resultsmentioning

confidence: 99%

Lipid-Based Transfection of Zebrafish Embryos: A Robust Protocol for Nucleic Acid Delivery in Zebrafish

Terzi,

Lao,

Jacobo

2024

Preprint

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“…Instead of using flow cytometry, we therefore measured the red intensity using microscope images, and then calculated the relative values and analyzed the changes in the mean relative red intensity. The Kaede protein has been used to analyze the proliferation in zebrafish fin regeneration [35], although the proliferation was not quantified. Pulse labeling with histone 2B-GFP has been used to identify low or non-proliferating cells [36].…”

Section: Discussionmentioning

confidence: 99%

An optical labeling-based proliferation assay system reveals the paracrine effect of interleukin-6 in breast cancer

Itou¹,

Tanaka²,

Sato³

et al. 2015

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Proliferation analysis is one of the basic approaches to characterize various cell types. In conventional cell proliferation assays, the same sample cannot be observed over time, nor can a specific group within a heterogeneous population of cells, for example, cancerous cells, be analyzed separately. To overcome these limitations, we established an optical labeling-based proliferation assay system with the Kaede protein, whose fluorescence can be irreversibly photoconverted from green to red by irradiation. After a single non-toxic photoconversion event, the intensity of red fluorescence in each cell is reduced by cell division. From this, we developed a simple method to quantify cell proliferation by monitoring reduction of red fluorescence over time. This study shows that the optical labeling-based proliferation assay is a viable novel method to analyze cell proliferation, and could enhance our understanding of mechanisms regulating cell proliferation machinery. We used this newly established system to analyze the functions of secreted interleukin-6 (IL-6) in cancer cell proliferation, which had not been fully characterized. Reduction in proliferation was observed following IL-6 knockdown. However, after co-culturing with IL-6-expressing cells, the proliferation of Kaede-labeled IL-6-knockdown cells was restored. These data indicate that in basal-like breast cancer cells, IL-6 exhibits a paracrine effect to positively regulate cell proliferation. Our results thus demonstrate that cancer cells can secrete signaling molecules, such as IL-6, to support the proliferation of other cancer cells.

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“…The zebrafish Eef1g (eukaryotic translation elongation factor 1 gamma) (Burket et al 2008 ) and Ef1α (elongation factor 1) (Moon et al 2013 ) promoter sequences were PCR-amplified using the primers specific to the promoters (Supplemental Table 1) and genomic DNA samples isolated from zebrafish. To link the promoters to the eGFP expression plasmid, the promoter sequences were further PCR-amplified using primers containing MluI (New England Biolabs (NEB), USA, Catalog no.…”

Section: Methodsmentioning

confidence: 99%

Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model

et al. 2018

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ObjectivesTo investigate the effect of endogenous Cas9 on genome editing efficiency in transgenic zebrafish.ResultsHere we have constructed a transgenic zebrafish strain that can be screened by pigment deficiency. Compared with the traditional CRISPR injection method, the transgenic zebrafish can improve the efficiency of genome editing significantly. At the same time, we first observed that the phenotype of vertebral malformation in early embryonic development of zebrafish after ZFERV knockout.ConclusionsThe transgenic zebrafish with expressed Cas9, is more efficient in genome editing. And the results of ZFERV knockout indicated that ERV may affect the vertebral development by Notch1/Delta D signal pathway.Electronic supplementary materialThe online version of this article (10.1007/s10529-018-2605-5) contains supplementary material, which is available to authorized users.

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Establishment of a transgenic zebrafish EF1α:Kaede for monitoring cell proliferation during regeneration

Cited by 8 publications

References 21 publications

Lipid-Based Transfection of Zebrafish Embryos: A Robust Protocol for Nucleic Acid Delivery in Zebrafish

Lipid-Based Transfection of Zebrafish Embryos: A Robust Protocol for Nucleic Acid Delivery in Zebrafish

An optical labeling-based proliferation assay system reveals the paracrine effect of interleukin-6 in breast cancer

Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model

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