High-throughput transgenesis in Xenopus using I-SceI meganuclease

Ogino, Hitoshi; McConnell, William B.; Grainger, Robert M.

doi:10.1038/nprot.2006.208

Cited by 120 publications

(104 citation statements)

References 35 publications

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“…Transgenesis was performed using the I-SceI method following the protocol already described 43 . Briefl y, aft er in vitro fertilization of the oocytes, one-cell-stage Xenopus embryos were injected with 5 nl of the injection mix, composed of the construct DNA (fi nal concentration 5 ng μ l − 1 ), 5 U of I-SceI enzyme ( New England Biolabs ) and 1X I-SceI buff er.…”

Section: Methodsmentioning

confidence: 99%

An evolutionarily conserved three-dimensional structure in the vertebrate Irx clusters facilitates enhancer sharing and coregulation

et al. 2011

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Developmental gene clusters are paradigms for the study of gene regulation; however, the mechanisms that mediate phenomena such as coregulation and enhancer sharing remain largely elusive. Here we address this issue by analysing the vertebrate Irx clusters. We fi rst present a deep enhancer screen of a 2-Mbp span covering the IrxA cluster. Using chromosome conformation capture, we show that enhancer sharing is widespread within the cluster, explaining its evolutionarily conserved organization. We also identify a three-dimensional architecture, probably formed through interactions with CCCTC-binding factor, which is present within both Irx clusters of mouse, Xenopus and zebrafi sh. This architecture brings the promoters of the fi rst two genes together in the same chromatin landscape. We propose that this unique and evolutionarily conserved genomic architecture of the vertebrate Irx clusters is essential for the coregulation of the fi rst two genes and simultaneously maintains the third gene in a partially independent regulatory landscape.

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Section: Methodsmentioning

confidence: 99%

An evolutionarily conserved three-dimensional structure in the vertebrate Irx clusters facilitates enhancer sharing and coregulation

et al. 2011

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“…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%

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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“…Recently, efficient transgenesis methods using I-SceI meganuclease have been reported in fish (Thermes et al, 2002;Grabher et al, 2004) and applied to amphibia (Ogino et al, 2006;Pan et al, 2006) and ascidians (Deschet et al, 2003). I-SceI meganuclease is an intron homing enzyme isolated from Saccharomyces cerevisiae mitochondria and is suggested to facilitate the early integration of transgenes into the host genome (Grabher and Wittbrodt, 2007).…”

Section: Introductionmentioning

confidence: 99%

The Ars insulator facilitates I‐SceI meganuclease‐mediated transgenesis in the sea urchin embryo

Ochiai

Sakamoto

Suzuki

et al. 2008

Developmental Dynamics

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For the efficient generation of transgenic sea urchins, we have adopted an I-SceI meganuclease-mediated transgenesis method. Several types of promoter-GFP gene constructs flanked by two I-SceI recognition sequences were co-injected with I-SceI into sea urchin fertilized eggs. Using cell-lineage-specific promoter constructs, the frequency of transgene expression was elevated, and their level of mozaicism was reduced. The addition of the Ars insulator sequence, which is known to block the enhancer activity and protect transgenes from position effects, led to a reduction in ectopic transgene expression and an elevation of transgene expression frequency in this I-SceI-mediated system. However, the magnitude of the effects of the Ars insulator was dependent upon the promoter constructs. QPCR analysis also showed that the Ars insulator increases the transgene copy number. These results suggest that the I-SceI-mediated method using the Ars insulator is advantageous for transgenesis in the sea urchin embryo. Developmental Dynamics 237:2475-2482, 2008.

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High-throughput transgenesis in Xenopus using I-SceI meganuclease

Cited by 120 publications

References 35 publications

An evolutionarily conserved three-dimensional structure in the vertebrate Irx clusters facilitates enhancer sharing and coregulation

An evolutionarily conserved three-dimensional structure in the vertebrate Irx clusters facilitates enhancer sharing and coregulation

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

The Ars insulator facilitates I‐SceI meganuclease‐mediated transgenesis in the sea urchin embryo

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