Transgene manipulation in rainbow trout using Cre recombinase

Kume, Sachi; Katayama, Naoto; Ichida, Kensuke; Hattori-Ihara, Shoko; Nagasawa, Kazue; Yoshizaki, Goro

doi:10.1007/s12562-014-0742-x

Cited by 2 publications

(4 citation statements)

References 24 publications

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“…It is noteworthy that Cre recombinase (originally isolated from P1 bacteriophage) produced by the transgene successfully excised the loxP-flanked sequence in rainbow trout raised at 108C, which is much cooler than optimum temperature for the host bacteria of the P1 bacteriophage. In a previous study, we demonstrated that Cre recombinase can function in rainbow trout; however, we delivered large amounts of cre mRNA into each embryo in order to induce in vivo DNA excision effectively in that study [41]. Thus, this cell type-specific and transgene-mediated DNA excision by Cre recombinase may open the possibility to switch on and off the expression of desired genes in target cells and allow us to develop cell-lineage tracing, cell-specific ablation, and cell type-specific conditional gene manipulation in rainbow trout.…”

Section: Discussionmentioning

confidence: 99%

“…Gamete collection and insemination were performed as previously described [40]. The pvasa-cre plasmid (2 nl of 500 ng/ ll) was microinjected into the blastodisk of individual 1-cell-stage embryos via a method described previously [41]. The microinjected embryos were incubated at 108C and reared until they reached sexual maturity.…”

Section: Gamete Collection Microinjection and Maintenance Of Injectmentioning

confidence: 99%

“…Production of a Reporter Transgenic Fish Tg(hsc:LRLG) Line P1 fish carrying the heat-shock-cognate71 promoter: loxP-DsRed-loxPEgfp (hsc:LRLG) gene were developed in our previous study [41]. A P1 male was mated with a wild-type female to obtain F1 progeny.…”

Section: Production Of Cre Transgenic Tg(vasa-cre) Fish Linesmentioning

confidence: 99%

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Germ Cell-Specific Excision of loxP-Flanked Transgenes in Rainbow Trout Oncorhynchus mykiss1

Katayama

Kume

Hattori-Ihara

et al. 2016

Biology of Reproduction

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Cre/loxP-mediated DNA excision in germ cell lineages could contribute substantially to the study of germ cell biology in salmonids, which are emerging as a model species in this field. However, a cell type-specific Cre/loxPsystem has not been successfully developed for any salmonid species. Therefore, we examined the feasibility of Cre/loxP-mediated, germ cell-specific gene excision and transgene activation in rainbow trout. Double-transgenic (wTg) progeny were obtained by mating a transgenic male carryingcrewith a transgenic female carrying thehsc-LRLGgene;crewas driven by rainbow troutvasaregulatory regions and thehsc-LRLGgene was made up of the rainbow troutheat-shock-cognate71promoter, theDsRedgene flanked by twoloxPsites, and theEgfpgene. PCR analysis, fluorescence imaging, and histological analysis revealed that excision of theloxP-flanked sequence and activation ofEgfpoccurred only in germ cells of wTg fish. However, progeny tests revealed that the excision efficiency ofloxP-flanked sequence in germ cells was low (≤3.27%). In contrast, the other wTg fish derived from two differentcre-transgenic males frequently excised theloxP-flanked sequence in germ cells (≤89.25%). Thus, we showed for the first time successful germ cell-specific transgene manipulation via the Cre/loxPsystem in rainbow trout. We anticipate that this technology will be suitable for studies of cell function through cell targeting, cell-linage tracing, and generating cell type-specific conditional gene knockouts and separately for developing sterile rainbow trout in aquaculture.

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Section: Discussionmentioning

confidence: 99%

Section: Gamete Collection Microinjection and Maintenance Of Injectmentioning

confidence: 99%

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Germ Cell-Specific Excision of loxP-Flanked Transgenes in Rainbow Trout Oncorhynchus mykiss1

Katayama

Kume

Hattori-Ihara

et al. 2016

Biology of Reproduction

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“…Subsequently, the Cre/ lox P recombination system was used to remove the marker genes from genetically modified organisms. This has been performed successfully in yeast and other organisms [9,10,11,12,13]. In this paper, the Cre/ lox P site-specific recombination system was, for the first time, applied to genetic transformation of A. limacinum , where it has provided the experimental basis for directional transformation of A. limacinum without leaving behind an antibiotic marker.…”

Section: Introductionmentioning

confidence: 99%

Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum

et al. 2015

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Abstract:The Cre/loxP site-specific recombination system was applied to Aurantiochytrium limacinum to obtain a transformant without the antibiotic resistance marker gene. First, the enhanced green fluorescent protein gene (egfp) and chloramphenicol resistance gene (Cm r ), along with the two loxP loci, were integrated into the genome of A. limacinum OUC88 using 18S rDNA sequences as the homologous recombination sites. Then plasmid pSH65, containing a zeocin resistance gene (Ble r ) was transferred into A. limacinum OUC_CG. After induction with galactose, repeated passage in culture and PCR-based assessment, the pSH65 plasmid was lost and A. limacinum OUC_EG host was shown to no longer have resistance to 100 mg chloramphenicol/L or 5 mg zeocin/L. Through southern blotting and fluorescence detection, egfp was found to be integrated into the genome of A. limacinum OUC_EG, and EGFP was successfully expressed in the cells. The successful application of the Cre/loxP system demonstrates an experimental basis for genetic modification of A. limacinum so as to obtain transformed strains with no antibiotic resistance marker genes.

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Transgene manipulation in rainbow trout using Cre recombinase

Cited by 2 publications

References 24 publications

Germ Cell-Specific Excision of loxP-Flanked Transgenes in Rainbow Trout Oncorhynchus mykiss1

Germ Cell-Specific Excision of loxP-Flanked Transgenes in Rainbow Trout Oncorhynchus mykiss1

Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum

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