Replication, integration and expression of exogenous DNA injected into fertilized eggs of Xenopus laevis

Etkin, Laurence D.; Pearman, Bradley; Roberts, Mary P.; Bektesh, Susan L.

doi:10.1111/j.1432-0436.1984.tb01395.x

Cited by 94 publications

(44 citation statements)

References 39 publications

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“…1 consist mainly of head-to-tail plasmid multimers, supporting a homologaus recombination mechanism for their formation also. Over the ·period analyzed, the conformational changes result in a relative enrichment for hmw forms, which are generally considered to be a prerequisite for successful integration of donor DNA into the host genome (Brinster et al 1985;Etkin and Pearman 1987;Etkin et al 1984). The signal comigrating with the hmw DNA of the recipient is already visible 6 h after injection at the early blastula stage (see also Chong and Vielkind 1989), in contrast to findings in Xenopus, where such signals become detectable no earlier than late blastula stage (Etkin et al 1984).…”

Section: Discussionmentioning

confidence: 87%

“…Only linear plasmid molecules were found to be multimerized in random head-to-head and head-to-tail orientations due to ligation of linear termini. In Xenopus and mammalian cells, on the other band, the fonnation of linear, exclusively head-to-tail multimers after injection of circular DNA has been previously shown (Rusconi and Schaffner 1981;Folger et al 1982;Etkin et al 1984). A homologaus recombination mechanism was postulated that, in the case of Xenopus, is also thought tobe responsible for the formation ofhead-to-tail concatemers after injection of linearized plasmid DNA (Marini et al 1988).…”

Section: Discussionmentioning

confidence: 99%

“…Generally, for such analyses, stable transgenic animals have been produced in Caenorhabditis elegans (Fire 1986), sea urchins , Drosophila (Rubin and Spradling 1982), Xenopus (Etkin et al 1984), mouse (Palmiter and Brinster 1986;Jaenisch 1988), and recently in fish (for a review see Maclean et al 1987;Stuart et al 1988;Guyomard et al 1989). However, stable transgenic systems have several limitations: (1) production of stable transgenic lines is time con,suming and therefore only a limited nurober of regulatory sequences can be tested; (2) integration site and integrated copy nurober can obscure the true characteristics of such sequences; and (3) genomic imprinting, as reported in the mouse (Surani et al 1988), may additionally alter the expression of the transgene.…”

Section: Introductionmentioning

confidence: 99%

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Transient expression of foreign DNA during embryonic and larval development of the medaka fish (Oryzias latipes)

Winkler

Vielkind²,

Schartl

1991

Mol Gen Genet

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Summary. Species of small fish are becoming useful tools for studies on vertebrate development. Wehave investigated the developing embryo of the Japanese medaka for its application as a transient expression system for the in vivo analysis of gene regulation and function. The temporaland spatial expression patterns ofbacterial chloramphenicol acetyltransferase and galactosidase reporter genes injected in supercoiled plasmid form into the cytoplasm of one cell of the two-cell stage embryo was promoter-specific. The transient expression was found to be mosaic within the tissue and organs reflecting the unequal distribution of extrachromosomal foreign DNA and the intensive cell mixing movements that occur in fish embryogenesis. The expression data are consistent with data on DNA fate. Foreign DNA persisted during embryogenesis and was still detectable in some 3-and 9-month-old adult fish; it was found in high molecular weight form as weil as in circular plasmid conformations. The DNA was replicated during early and late embryogenesis. Our data indicate that the developing medaka embryo is a powerful in vivo assay system for studies of gene regulation and function.

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Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Transient expression of foreign DNA during embryonic and larval development of the medaka fish (Oryzias latipes)

Winkler

Vielkind²,

Schartl

1991

Mol Gen Genet

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“…First attempts to create transgenic frogs had been reported in pioneering studies by Etkin et al (1984) more than 2 decades ago. Linearized plasmid DNA was injected into fertilized Xenopus eggs and was found to be maintained in the form of long extrachromosomal concatemers during early stages of development, eventual integration into the genome occurring at later stages.…”

Section: Introductionmentioning

confidence: 99%

I‐SceI meganuclease‐mediated transgenesis in Xenopus

Pan

Chen

Loeber

et al. 2005

Developmental Dynamics

112

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Several experimental approaches have been described to generate transgenic frogs. Here, we report on the application of a novel method in Xenopus, making use of I-SceI meganuclease. The characteristic feature of this endonuclease is that it has an extended recognition site of 18 bp, which is expected to exist only once in 7 ؋ 10 10 bp of random DNA sequences. Various reporter constructs flanked by two I-SceI recognition sites were injected together with the I-SceI meganuclease into one-cell stage Xenopus embryos. We observed an overall transgenesis frequency of 10% or more under optimized condition. The injected genes were integrated into the genome and transmitted to F1 offspring. Southern blot analysis showed that between one and eight copies of the transgene were integrated. Meganuclease-aided transgenesis, thus, provides a simple and highly efficient tool for transgenesis in Xenopus. Developmental Dynamics 235:247-252, 2006.

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“…Cleavage-stage embryos were obtained by in vitro fertilization of ovulated eggs (9,17). After antibody injection, embryos were fixed, sectioned, and stained with Feulgen-Fast green as previously described (20).…”

Section: Methodsmentioning

confidence: 99%

Mitosis-specific monoclonal antibodies block cleavage in amphibian embryos.

Davis¹,

Wright

Penkala

et al. 1989

Cell Struct. Funct.

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ABSTRACT. By microinjecting monoclonal antibodies that bind specifically to mitotic and meiotic cells of a variety of species, we studied the biological activity of antigens recognized by these antibodies. The antibodies recognize a family of phosphoprotein antigens that are found throughout the cytoplasm of mitotic cells and particularly at microtubule organizing centers, including centrosomes and kinetochores. Their binding is dependent on phosphorylation of the polypeptides. Immunoglobulins were introduced into Xenopus laevis and Rana pipiens oocytes or cleaving embryos using glass micropipettes. The ability of the antibody-injected oocytes to undergo mitosis or meiosis was compared with those injected with control mouse immunoglobulins. The antibodies failed to block chromosome condensation and germinal vesicle breakdown in progesterone-treated oocytes. However, functional mitotic spindles were not assembled in cleavage stage frog embryos injected with antibodies. In vitro, the binding of the antibodies to the antigens inhibited the dephosphorylation of the antigens by alkaline phosphatase. The antibody binding to the activated microtubule organizing centers (MTOC) seems to block not only the nucleation of microtubules and the organization of the mitotic spindle, but also the dephosphorylation of proteins associated with the MTOC that normally occurs at the mitosis-G1 transition.

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Replication, integration and expression of exogenous DNA injected into fertilized eggs of Xenopus laevis

Cited by 94 publications

References 39 publications

Transient expression of foreign DNA during embryonic and larval development of the medaka fish (Oryzias latipes)

Transient expression of foreign DNA during embryonic and larval development of the medaka fish (Oryzias latipes)

I‐SceI meganuclease‐mediated transgenesis in Xenopus

Mitosis-specific monoclonal antibodies block cleavage in amphibian embryos.

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