Ligand-Controlled Site-Specific Recombination in Zebrafish

Chekuru, Avinash; Kuscha, Veronika; Hans, Stefan; Brand, Michael

doi:10.1007/978-1-4939-7169-5_6

Cited by 4 publications

(2 citation statements)

References 19 publications

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“…Importantly, the zebrafish genome contains orthologues of about 82% of human diseaserelated genes, (18) including those affecting the skeleton. Both tissuespecific overexpression via site-specific recombinases (Cre) (32) and gene-specific knockout via clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (30) are available in zebrafish, along with antisense oligonucleotide gene knockdown approaches (33) and ideal conditions to carry out forward and reverse genetic screens. (28,34,35) Furthermore, single-nucleotide genome editing can be performed by CRISPR-Cas9-mediated knock-ins.…”

Section: Introductionmentioning

confidence: 99%

Skeletal Biology and Disease Modeling in Zebrafish

Dietrich

Fiedler

Kurzyukova

et al. 2020

Journal of Bone and Mineral Research

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Zebrafish are teleosts (bony fish) that share with mammals a common ancestor belonging to the phylum Osteichthyes, from which their endoskeletal systems have been inherited. Indeed, teleosts and mammals have numerous genetically conserved features in terms of skeletal elements, ossification mechanisms, and bone matrix components in common. Yet differences related to bone morphology and function need to be considered when investigating zebrafish in skeletal research. In this review, we focus on zebrafish skeletal architecture with emphasis on the morphology of the vertebral column and associated anatomical structures. We provide an overview of the different ossification types and osseous cells in zebrafish and describe bone matrix composition at the microscopic tissue level with a focus on assessing mineralization. Processes of bone formation also strongly depend on loading in zebrafish, as we elaborate here. Furthermore, we illustrate the high regenerative capacity of zebrafish bones and present some of the technological advantages of using zebrafish as a model. We highlight zebrafish axial and fin skeleton patterning mechanisms, metabolic bone disease such as after immunosuppressive glucocorticoid treatment, as well as osteogenesis imperfecta (OI) and osteopetrosis research in zebrafish. We conclude with a view of why larval zebrafish xenografts are a powerful tool to study bone metastasis. © 2021 American Society for Bone and Mineral Research (ASBMR).

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Section: Introductionmentioning

confidence: 99%

Skeletal Biology and Disease Modeling in Zebrafish

Dietrich

Fiedler

Kurzyukova

et al. 2020

Journal of Bone and Mineral Research

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“…All incubations were conducted in the dark. Cre mRNA were conducted as previously described (Chekuru et al, 2017). Cre mRNA injections into 3C tyr-positive progeny at the 1cell stage was conducted five times with at least 30 transgenic animals analyzed.…”

Section: -Hydroxytamoxifen Cre Mrna and Heat Treatmentsmentioning

confidence: 99%

Cre-Controlled CRISPR (3C) Mutagenesis: Fast and Easy Conditional Gene Inactivation in Zebrafish

Hans¹,

Zöller²,

Hammer³

et al. 2020

Preprint

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Conditional gene inactivation is a powerful tool to determine gene function when constitutive mutations result in detrimental effects. The most commonly used technique to achieve conditional gene inactivation employs the Cre/loxP system and its ability to delete DNA sequences flanked by two loxP sites. However, targeting critical exons or an entire gene with two loxP sites is time and labor consuming. To circumvent these issues, we developed Cre-Controlled CRISPR (3C) mutagenesis. 3C mutagenesis is simple, fast and allows gene inactivation in a Cre-dependent manner. In contrast to loxP-flanked alleles, the recombined cells become fluorescently visible enabling the isolation of these cells and their subjection to various omics techniques. Moreover, 3C will be scalable and will enable the conditional inactivation of multiple genes simultaneously. Hence, 3C mutagenesis provides a valuable alternative to the production of loxP-flanked alleles and should be applicable to all model organisms amenable to single integration transgenesis.

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Targeted knock-in of CreER T2 in zebrafish using CRISPR/Cas9

et al. 2018

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New genome-editing approaches, such as the CRISPR/Cas system, have opened up great opportunities to insert or delete genes at targeted loci and have revolutionized genetics in model organisms like the zebrafish. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity. Using a CRISPR/Cas9-mediated knock-in strategy, we inserted a zebrafish codon-optimized CreER transgene at the otx2 gene locus to generate a conditional Cre-driver line. We chose otx2 as it is a patterning gene of the anterior neural plate that is expressed during early development. By knocking in CreER upstream of the endogenous ATG of otx2, we utilized this gene's native promoter and enhancer elements to perfectly match CreER and endogenous otx2 expression patterns. Next, by combining this novel driver line with a Cre-dependent reporter line, we show that only in the presence of tamoxifen can efficient Cre-loxp-mediated recombination be achieved in the anterior neural plate-derived tissues like the telencephalon, the eye and the optic tectum. Our results imply that the otx2:CreER transgenic fish will be a valuable tool for lineage tracing and conditional mutant studies in larval and adult zebrafish.

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Ligand-Controlled Site-Specific Recombination in Zebrafish

Cited by 4 publications

References 19 publications

Skeletal Biology and Disease Modeling in Zebrafish

Skeletal Biology and Disease Modeling in Zebrafish

Cre-Controlled CRISPR (3C) Mutagenesis: Fast and Easy Conditional Gene Inactivation in Zebrafish

Targeted knock-in of CreER T2 in zebrafish using CRISPR/Cas9

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