Replication of injected DNA templates in Xenopus embryos

Etkin, Laurence D.; Pearman, Bradley; Ansah-Yiadom, Richmond

doi:10.1016/0014-4827(87)90207-2

Cited by 20 publications

(9 citation statements)

References 14 publications

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“…Total numbers of embryos showing transgene signal in at least one of their blastomeres did not differ between transgene alone and transgene plus meganuclease groups; however, I-SceI-mediated gene transfer increased the proportion of blastomeres with transgene signals per embryo. Perhaps mosaic expression of green fluorescent protein reflected persistence of injected plasmids as extrachromosomal episomes which are inherited only by a subset of cells [47,48] and also to late integration in the genome. This phenomenon was frequently observed after pronuclear microinjection and intracytoplasmic sperm injection-mediated gene transfer in mammals [26,27], and it continues to be one of the main limitations of most of the current transgenesis techniques in mammals.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…In a control experiment, the expression of E5 was driven by the promoter of another homeobox gene, Xanf-1. The expression of Xanf-1 also occurs in neuroectoderm, but, unlike Otx-2, does not have distinct spatiotemporal domains and ceases before the tadpole stage (21,22). Correspondingly, the signal from the Xanf-IIES construct appeared uniformly orange in the tadpole brain (Fig.…”

Section: A' And?'mentioning

confidence: 97%

“…The representative mosaic fluorescent image composed from clones of cells, which acquired the plasmid during blastomere cleavage (21), reflects the in situ hybridization data accurately (Fig. 4, A to C).…”

Section: A' And?'mentioning

confidence: 99%

"Fluorescent Timer": Protein That Changes Color with Time

Terskikh

Fradkov

Ermakova

et al. 2000

Science

363

283

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We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression. We used in vivo labeling with E5 to measure expression from the heat shock-dependent promoter in Caenorhabditis elegans and from the Otx-2 promoter in developing Xenopus embryos. Thus, E5 is a "fluorescent timer" that can be used to monitor both activation and down-regulation of target promoters on the whole-organism scale.

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“…Although injection of mRNAs into fertilized eggs has been valuable for assessing the function of genes involved in early amphibian embryogenesis (25), the mRNAs and their protein products are too short-lived to be useful for the study of metamorphosis. The injection of plasmid DNAs into fertilized eggs only results in transient and mosaic gene expression (26,27).…”

Section: Discussionmentioning

confidence: 99%

Metamorphosis is inhibited in transgenic Xenopus laevis tadpoles that overexpress type III deiodinase

Huang

Marsh-Armstrong

Brown

1999

Proc. Natl. Acad. Sci. U.S.A.

162

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One of the genes that is up-regulated by thyroid hormone (TH) during Xenopus laevis metamorphosis encodes a type III deiodinase (D3) that inactivates TH. Transgenic X. laevis tadpoles overexpressing a GFP-D3 fusion protein were produced. These transgenic tadpoles had high levels of deiodinase activity and were resistant to exogenous TH added 1 week after fertilization. They developed normally throughout embryogenesis and premetamorphic stages but became retarded in their development late in prometamorphosis when endogenous TH reaches its highest level. Gill and tail resorption were delayed and most of the animals arrested and died. One tadpole completed its metamorphosis without resorbing its tail. These results demonstrate that D3 can modulate the action of TH in vivo, and document the value of the new transgenic method for functional analysis of genes involved in metamorphosis.One of the direct response genes that is up-regulated by thyroid hormone (TH) during Xenopus laevis metamorphosis is a type III deiodinase (D3) (originally called gene 15) (1). This enzyme inactivates TH by cleaving an iodine from the inner ring of the hormone (2). The basal expression, the developmental profile, the induction by TH (1), and the cell and tissue localization (3, 4) of D3 gene expression suggest that this enzyme plays a role in modulating the tadpole's response to TH.Two organs that have different expression patterns of D3 and that respond differently to TH during metamorphosis are the limb and the tail. Limb buds, which are one of the first organs to undergo a metamorphic change, have no detectable basal expression of D3 and high levels of thyroid hormone receptor ␣ (TR␣) (1). They undergo TH-induced changes early during prometamorphosis, when the endogenous TH level is low (5). Limb growth can be induced by low levels of exogenous TH. In contrast, the tail, which is the last organ to change, has a basal expression of D3 and low levels of TR␣ (1), and undergoes TH-induced changes late during metamorphic climax, when the endogenous TH level is maximal and TR␤ has been induced (6). Tail resorption can be induced only by high levels of exogenous TH. As the endogenous level of TH rises during prometamorphosis, so does the level of D3 in the tail. When endogenous TH is at its highest level, D3 expression decreases. A class of delayed TH-response genes that includes several proteases is then induced, and the tail resorbs rapidly (7). Both the basal and up-regulated expression of D3 occur within cells adjacent to collagen lamellae that underlie the epidermis and surround the notochord (4). Coincident with the down-regulation of D3, these same cells up-regulate the delayed-response genes (4).The correlation of D3 gene expression with other TH response genes extends to a variety of tissues during metamorphosis (3). Basically, decrease of D3 expression correlates with increase of expression of TH response genes. This expression pattern suggests that D3 can protect tissues from the action of TH.In this paper, we report the p...

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Replication of injected DNA templates in Xenopus embryos

Cited by 20 publications

References 14 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

"Fluorescent Timer": Protein That Changes Color with Time

Metamorphosis is inhibited in transgenic Xenopus laevis tadpoles that overexpress type III deiodinase

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