RecA-Mediated, Targeted Mutagenesis in Zebrafish

Cui, Zongbin; Yang, Ying; Kaufman, Christopher D.; Agalliu, Dritan; Hackett, Perry B.

doi:10.1007/s10126-002-0059-0

Cited by 20 publications

(14 citation statements)

References 59 publications

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“…A stable potential knockout/knockin line could be generated and bred to homozygosity until the F 3 generation. The rate of homologous recombination with the characteristic of germ line transmission was 1.7Â10 _ 6 in this study, which was close to the results previously described (Cui et al, 2003). Even so, the method established here can still be regarded as efficient and practical for the manipulation of genes in zebrafish without embryonic stem (ES) cells.…”

Section: Discussionsupporting

confidence: 75%

“…Injection of such oligonucleotides into newly fertilized eggs has highly specific effects on a wide variety of genes. Large-scale mutagenesis screening methods are available for zebrafish, allowing the identification of important genes in development and diseases (Lin et al, 1994;Gaiano et al, 1996;Cui et al, 2003). However, in comparison with the mouse, for which the gene knockout method was developed a long time ago (Bradley et al, 1984), for zebrafish the method to produce a line of targeted gene modification through homologous recombination between the introduced DNA and the corresponding chromosomal segments is lacking.…”

Section: Introductionmentioning

confidence: 99%

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Integration of Double-Fluorescence Expression Vectors into Zebrafish Genome for the Selection of Site-Directed Knockout/Knockin

Zhang

Xiong

et al. 2006

Mar Biotechnol

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Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/ knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7Â10 _ 6 . In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F 1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P 0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.

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Section: Discussionsupporting

confidence: 75%

Section: Introductionmentioning

confidence: 99%

Integration of Double-Fluorescence Expression Vectors into Zebrafish Genome for the Selection of Site-Directed Knockout/Knockin

Zhang

Xiong

et al. 2006

Mar Biotechnol

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“…However, there are likely more sensitive indicators of UVB damage, as 0.005 J/cm 2 was sufficient to stimulate some aspects of DNA repair in zebrafish, although the specific mechanisms have not been identified [33]. While to our knowledge, a systematic characterization of UVA dosing regimes in zebrafish embryos has not been presented, data have been gathered for responses of zebrafish to UVB as part of environmental studies.…”

Section: Uvb and Uva Sensitivities Of Zebrafishmentioning

confidence: 99%

Photobiological effects of UVA and UVB light in zebrafish embryos: Evidence for a competent photorepair system

Dong

Svoboda

Tiersch

et al. 2007

Journal of Photochemistry and Photobiology B: Biology

110

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The consequences of UVB and UVA irradiation on hatch rate, mortality, and malformation were studied in embryonic zebrafish (Danio rerio). The use of zebrafish embryos has expanded from traditional developmental models to diverse studies, including many techniques utilizing light exposure. To characterize useful indicators of photodamage, the responses and threshold limits of UV radiation as a function of embryonic stage and fish source were evaluated. Significant differences in UVB susceptibility were observed in embryos at 3, 6-7, 12, and 24 h postfertilization (hpf), with the 1000-cell stage (3 hpf) having greatest tolerance to UVB. Embryos derived from zebrafish raised in outdoor ponds were more tolerant to UVB than were embryos from laboratory-raised fish. Combinations of UVB and UVA exposure were used to confirm the presence of a competent photorepair system in zebrafish that could return otherwise malformed embryos to a normal phenotype. Overall, embryonic zebrafish had large tolerances (LD 50 of 850 J/cm 2 ) to UVA, confirming their suitability for photoactivation and photorepair studies.

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“…Cui et al (2003) have reported RecA-mediated, targeted mutagenesis in zebrafish. Previously we have shown the possibility of drug selection for gene transfer into established piscine non-ES cell lines (Chen et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Retention of the Developmental Pluripotency in Medaka Embryonic Stem Cells after Gene Transfer and Long-term Drug Selection for Gene Targeting in Fish

et al. 2004

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Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

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RecA-Mediated, Targeted Mutagenesis in Zebrafish

Cited by 20 publications

References 59 publications

Integration of Double-Fluorescence Expression Vectors into Zebrafish Genome for the Selection of Site-Directed Knockout/Knockin

Integration of Double-Fluorescence Expression Vectors into Zebrafish Genome for the Selection of Site-Directed Knockout/Knockin

Photobiological effects of UVA and UVB light in zebrafish embryos: Evidence for a competent photorepair system

Retention of the Developmental Pluripotency in Medaka Embryonic Stem Cells after Gene Transfer and Long-term Drug Selection for Gene Targeting in Fish

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