Transient Transgenesis in Xenopus laevis Facilitated by AAV-ITRs

Fu, Yuchang; Kan, Donghui; Evans, Sylvia M.

doi:10.1385/1-59259-678-9:167

Cited by 6 publications

(5 citation statements)

References 10 publications

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“…Given the rapid pace of development in this animal model, precise minute‐to‐hour control of gene delivery can facilitate the dissection of biological events. At present, conventional transgenic methods allow some spatiotemporal control of gene expression [13,19], however precise cellular restriction usually offers little leeway in the timing of gene delivery, and inducible systems offer little leeway in the restricting sites of expression [20]. Tetracycline‐regulated gene shutoff may provide one alternative to conventional expression systems [21], however toxicity and the incompleteness of gene shut‐off complicate this approach.…”

Section: Discussionmentioning

confidence: 99%

“…AAV DNA vector injections were performed as described by [13]. The nβgalITR expression vector was generously provided by Sylvia Evans (Scripps Institute).…”

Section: Methodsmentioning

confidence: 99%

“…For lacz development, embryos were fixed in MEMFA for 1 h, washed in 0.1 M phosphate buffer (PB) for 5 min, then transferred embryos into βgal solutions consisting of 32.93 mg potassium ferricyanide, 42.24 mg potassium ferrocyanide and 0.1 ml 15% Xgal (15 mg in 100 μl DMF) in 10 ml 0.1 M PB [13].…”

Section: Methodsmentioning

confidence: 99%

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Electroporation‐mediated gene transfer in free‐swimming embryonic Xenopus laevis

2000

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Xenopus laevis are a rich resource for vertebrate embryology and cell biology. Transplantation and transgenesis have provided much information about the developmental mechanisms of embryogenesis and molecule function, however existing methods have faced limitations regarding either the precise localization of gene expression or flexibility in the timing of gene transfer. Here we have found that electroporation of tailbud (stage 29/30) embryos is a rapid and efficient method of combining cell-specific expression with variation in temporal delivery. At the low voltages required for electroporation, embryos resumed normal swimming behavior and development. We conclude that electroporation has wide experimental application to Xenopus developmental and cell biology. ß

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Section: Discussionmentioning

confidence: 99%

“…AAV DNA vector injections were performed as described by [13]. The nβgalITR expression vector was generously provided by Sylvia Evans (Scripps Institute).…”

Section: Methodsmentioning

confidence: 99%

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Electroporation‐mediated gene transfer in free‐swimming embryonic Xenopus laevis

2000

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“…Competent E. coli strain XL2-Blue cells were transformed by ligase mixture using electroparation under the conditions described above. Recombinant E. coli clones were collected in LB medium with Zeocin (25 mg/mL) [10]. Preparation of Recombinant X-33 Strain P. pastoris Yeast Clones.…”

Section: Construction Of the Yeast Vector With The Marker Gene Clonimentioning

confidence: 99%

Induction of the PAOX1-Promoter and Synthesis of GFP Protein in Wild X-33 Strain Recombinant Pichia pastoris Yeast Cells

2005

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Recombinant clones of X-33 strain Pichia pastoris containing the marker gene yEGFP were prepared. The optimal methanol concentration in the medium for induction of heterologous expression was determined in the recombinant clones.The biotechnology of yeast is a very valuable branch of modern industry. Thus, several recombinant proteins have been produced in yeast for pharmacological applications [1]. Most recombinant proteins are synthesized in systems based on type I yeast of Saccharomyces cerevisiae and Pichia pastoris [2].The promoter AOX1-gene, which is induced by methanol, is used to prepare recombinant proteins of P. pastoris. Various recombinant proteins that exhibit functional activity are produced in P. pastoris cells [3]. The yield of active protein from yeast is known to depend on several factors, the most important of which are:1) The nature of the heterologous gene and the corresponding properties of the recombinant protein;2) The genotype of the strain, which sets the base level for expression of the heterologous gene in the cell (genetically determined mechanisms for regulating transcription, translation, splicing, etc.);3) The morphological features of the strain (structural features of the cell wall and certain organelles) and metabolic characteristics of the yeast cells belonging to various strains; 4) Differences in the genetic stability of recombinant clones of the yeast strains. The most well studied strain GS115 is auxotrophic for histidine synthesis and was created through mutagenesis [4]. Our investigations are directed mainly at the study of wild yeast strains, in particular, the X-33 strain.We prepared the genetic construction pPICZB/yEGFP containing the marker gene yEGFP in order to monitor expression of heterologous genes in X-33 strain P. pastoris cells [5]. In order to prepare the recombinant yeast vector, we cloned the marker gene yEGFP into yeast vector pPICZB. For this the marker gene was cut from the pUG-36 plasmid at restriction sites for the enzyme XbaI. Vector pPICZB was cleaved from the XbaI site. This produced the linear pDNA vector pPICZB with 3.3 kb and yEGFP with 0.721 kb. These DNA fragments were analyzed in agarose gel with subsequent extraction of the aforementioned fragments from the gel for ligation. Ligation produced recombinant yeast vector pPICZB/yEGFP (Fig. 1). It can be seen that this vector contains the marker gene for the pAOX1-promoter and the gene conferring resistance for the antibiotic Zeocin. After ligation, competent E. coli cells were transformed with ligase mixture. Rccombinant clones were selected in medium with added Zeocin. According to genetic maps, plasmid recombinant vector PICZB/yEGFP should have a molecular weight (MW) of 4.048 kb and contain the yEGFP gene in the correct orientation, i.e., the start of the yEGFP gene should be located under the AOX1-promoter. pDNA was isolated from the two E. coli recombinant clones collected by us. Restrictive analysis was used to find clones with correct insertion of the yEGFP gene. Restriction analysis was p...

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“…advent of both transient transgenics and germ-line transgenics, the ability to identify tissue-speci®c transcription regulatory element genes and to express gene products in speci®c cell lineages has expanded greatly in both Xenopus and zebra®sh [Higashijima et al, 1997;Fu et al, 1999;Jessen et al, 1999;MarshArmstrong et al, 1999;Motoike et al, 2000;Osada et al, 2000;Sparrow et al, 2000]. Zebra®sh lines can be generated that carry a stable transgene that drives the expression of green¯uorescent protein (GFP) behind promoter elements of a speci®c gene.…”

Section: Emerging Technologiesmentioning

confidence: 99%

Vertebrate model systems in the study of early heart development:Xenopus and zebrafish

Lohr¹,

Yost²

2000

Am. J. Med. Genet.

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Xenopus and zebrafish serve as outstanding models in which to study vertebrate heart development. The embryos are transparent, allowing observation during organogenesis; they can be obtained in large numbers; and they are readily accessible to embryologic manipulation and microinjection of RNA, DNA, or protein. These embryos can live by diffusion for several days, allowing analysis of mutants or experimental treatments that perturb normal heart development. Xenopus embryos have been used to understand the induction of the cardiac field, the role of Nkx genes in cardiac development, and the role transforming growth factor beta molecules in the establishment and signaling of left-right axis information. Large-scale mutant screens in zebrafish and the development of transgenics in both Xenopus and zebrafish have accelerated the molecular identification of genes that regulate conserved steps in cardiovascular development.

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Transient Transgenesis in Xenopus laevis Facilitated by AAV-ITRs

Cited by 6 publications

References 10 publications

Electroporation‐mediated gene transfer in free‐swimming embryonic Xenopus laevis

Electroporation‐mediated gene transfer in free‐swimming embryonic Xenopus laevis

Induction of the PAOX1-Promoter and Synthesis of GFP Protein in Wild X-33 Strain Recombinant Pichia pastoris Yeast Cells

Vertebrate model systems in the study of early heart development:Xenopus and zebrafish

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