Simple and efficient transgenesis with <i>I‐SceI</i> meganuclease in the newt, <i>Cynops pyrrhogaster</i>

Casco-Robles, Martin Miguel; Yamada, Shouta; Miura, Tomoya; Chiba, Chikafumi

doi:10.1002/dvdy.22463

Cited by 22 publications

(19 citation statements)

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“…Reports in amphibians such as Xenopus [22,32] and newts [24], and also in fish [21,33] described the high efficiency and simplicity of I-SceI meganuclease-mediated gene transfer. However, to date this technique has apparently not been tested in mammals.…”

Section: Discussionmentioning

confidence: 99%

“…One of these meganucleases, I-SceI [19] was first described approximately 30 years ago, as a sequence-specific endonuclease that recognized large (>12 base pair) sequence sites [20]. Transgenesis using the I-SceI meganuclease was efficient for production of transgenic killifish Medaka [21], Xenopus [22,23], and newts [24]. This method simply consists of digestion of a transgene construct carrying I-SceI recognition sites flanking the transgene, and cytoplasmic injection of a digestion mixture into fertilized eggs.…”

Section: Introductionmentioning

confidence: 99%

“…Transgene expression was documented; furthermore, it was transmitted through the germ line and also expressed in offspring [22]. Ogino et al [22] and Casco-Robles et al [24] reported that a high proportion of the Xenopus and newt embryos had a nonmosaic expression pattern, and also demonstrated that the number of transgene integration sites was usually one or two, and that copy numbers ranged from one to four. Unfortunately, the mechanisms underlying the great success of the I-SceI transgenesis technique remain unclear.…”

Section: Introductionmentioning

confidence: 99%

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Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos

et al. 2013

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a b s t r a c tAlthough transgenic methods in mammals are inefficient, an easy and highly efficient transgenesis system using I-SceI meganuclease (intron-encoded endonuclease from S. cerevisiae) was recently described in Xenopus. The method consisted of injection into fertilized eggs of an I-SceI reaction mixture with a plasmid DNA carrying the transgene, flanked by the meganuclease recognition sites (pIS). In the present study, the effects of I-SceI on gene transfer were tested apparently for the first time in mammals, in particular, in cattle. Various conditions were evaluated, including three concentrations of the plasmid pIS Pax6egfp, carrying I-SceI recognition sites flanking egfp under Pax6 promoter and two injection times (before IVM and after IVF) of pIS CAGegfp, carrying I-SceI sites fanking egfp under CAG promoter. In addition, the quantity of transgene was measured using quantitative polymerase chain reaction, and presence of transgene signals was evaluated using fluorescence in situ hybridization analysis. Transgene expression rates were higher (P < 0.05) for groups treated after IVF (79.1%, 91/115 and 63.0%, 75/ 119) than before IVM (32.6%, 31/95 and 34.7%, 33/95), with and without I-SceI, respectively. Interestingly, injection with pIS plus I-SceI after IVF increased frequency (P < 0.05) of nonmosaic transgene-expressing embryos (58.3%, 42/72 vs. 29.7%, 25/84) for pIS plus I-SceI and pIS alone. Based on fluorescence in situ hybridization analysis, injection with I-SceI increased (P < 0.05) the proportion of embryos with transgene signals in all blastomeres compared with pIS alone (44.0%,11/25 vs. 6.9%, 2/29) for pIS plus I-SceI and pIS alone. In addition, transgene copy number was numerically higher for the group treated with pIS plus I-SceI compared with pIS alone. In conclusion, I-SceI gene transfer increased transgene signals in bovine embryos.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos

et al. 2013

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“…Can such systems be developed for amphibians, or can alternative methods be designed? The recent demonstration of a simpler and more efficient method of creating transgenic newts (Casco-Robles et al, 2010) suggests that this is possible.…”

Section: Looking Distally: Perspective For the 21 St Centurymentioning

confidence: 99%

Looking proximally and distally: 100 years of limb regeneration and beyond

Stocum

Cameron

2011

Developmental Dynamics

106

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The experimental study of amphibian limb regeneration spans most of the 20 th century and the first decade of the 21 st century. We first review the major questions investigated over this time span: (1) the origin of regeneration blastema cells, the mechanism of tissue breakdown that liberates cells from their tissue organization to participate in blastema formation, (3) the mechanism of dedifferentiation of these cells, (4) how the blastema grows, (5) how the blastema is patterned to restore the missing limb structures, and (6) why adult anurans, birds and mammals do not have the regenerative powers of urodele salamanders. We then look forward in a perspective to discuss the many unanswered questions raised by investigations of the past century, what new approaches can be taken to answer them, and what the prospects are for translation of basic research on limb regeneration into clinical means to regenerate human appendages. Developmental Dynamics 240:943-968,

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“…In addition, the transgenic manipulation of aquaculture fish species using gene(s) related to quantitative traits has received attention due to its potential as a powerful tool in the molecular genetic breeding of farmed fishes for productivity enhancement (Nam et al, 2001(Nam et al, , 2007. The microinjection of DNA into fertilized eggs has long been utilized to produce transgenic fish strains, although novel methodologies to induce homologous and/or targeted transgenesis have been challenged recently in some fish models (Rembold et al, 2006;Grabher and Wittbrodt, 2007;Casco-Robles et al, 2010). In microinjection-mediated gene delivery, it is widely accepted that the exogenously introduced DNA integrates into vertebrate genomes in an approximately…”

Section: Introductionmentioning

confidence: 99%

Concatemer-Associated Transgene Expression Patterns in Transgenic Marine Medaka Oryzias dancena Strains

Cho¹,

Kim²,

Nam

2015

Fisheries and aquatic sciences

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To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four stable transgenic marine medaka Oryzias dancena germlines harboring β-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be significantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.

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Simple and efficient transgenesis with I‐SceI meganuclease in the newt, Cynops pyrrhogaster

Cited by 22 publications

References 61 publications

Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos

Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos

Looking proximally and distally: 100 years of limb regeneration and beyond

Concatemer-Associated Transgene Expression Patterns in Transgenic Marine Medaka Oryzias dancena Strains

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