Triploid and gynogenetic diploid <i>Xenopus laevis</i>

Tompkins, Robert

doi:10.1002/jez.1402030207

Cited by 63 publications

(17 citation statements)

References 10 publications

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“…Gynogenesis utilizing high pressure has been used to great success in X. laevis (Müller et al, 1978;Tompkins, 1978;Reinschmidt et al, 1979;Columbelli et al, 1984;Krotoski et al, 1985;Tompkins and Reinschmidt, 1991). Given that X. tropicalis embryos develop at a slightly Fig.…”

Section: Optimization Of the Gynogenetic Methodsmentioning

confidence: 99%

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A gynogenetic screen to isolate naturally occurring recessive mutations in Xenopus tropicalis

Noramly

Zimmerman²,

Cox

et al. 2005

Mechanisms of Development

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In the rapidly developing, diploid amphibian Xenopus tropicalis, genetics can be married to the already powerful tools of the amphibian system to overcome a disability that has hampered Xenopus laevis as a model organism: the difficulties inherent in conducting genetic analyses in a tetraploid organism with a longer generation time. We describe here a gynogenetic screen to uncover naturally occurring recessive mutations in wild X. tropicalis populations, a procedure that is both faster and easier than conventional genetic screens traditionally employed in model organisms to dissect early developmental pathways. During the first round of our screen, gynogenetic diploids from over 160 females comprising four different wild-caught populations were examined. Forty-two potential mutant phenotypes were isolated during this round of gynogenesis. From this group, we describe 10 lines that have genetically heritable recessive mutations. A wide range of developmental defects were obtained in this screen, encompassing effects limited to individual organs as well phenotypes characterized by more global changes in tadpole body morphology. The frequency of recessive mutations detected in our screen appears lower than that seen in other vertebrate genetic screens, but given constraints on the screening procedure used here, is likely to be consistent with rates seen in other animals, and clearly illustrates how wild-caught animals can be a productive source of developmental mutations for experimental study. The development of genetic strategies for the Xenopus system, together with new genomic resources, existing technologies for transgenesis, and other means for manipulating gene expression, as well as the power of performing embryonic manipulations, will provide an impressive set of tools for resolving complex cell and developmental phenomena in the future.

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Section: Optimization Of the Gynogenetic Methodsmentioning

confidence: 99%

“…While three generations are required to isolate homozygous mutants in the traditional manner, the approach we used, based on the methods developed for making mutants in wild-caught animals in X. laevis (Tompkins, 1978) reduces this requirement to two generations (Fig. 1A).…”

Section: Introductionmentioning

confidence: 99%

A gynogenetic screen to isolate naturally occurring recessive mutations in Xenopus tropicalis

Noramly

Zimmerman²,

Cox

et al. 2005

Mechanisms of Development

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“…Hazen and Co., Alburg, VT) and Xenopus laeuis (South African Snake Farm, Capetown, South Africa), were used. In addition, a few non-chimeric 3N adult Xenopus laeuis (9,24) were also used.…”

Section: Methodsmentioning

confidence: 99%

Fixation‐Associated quantitative variations of DNA fluorescence observed in flow cytometric analysis of hemopoietic cells from adult diploid frogs

Holtfreter

Cohen

1990

Cytometry

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We have examined, by flow cytometry, the apparent DNA content of frog blood cells that had been fixed with either 50% ethanol, 70% ethanol, or 66% methanol, before being stained with either mithramycin, propidium iodide, or Hoechst 33258. After 50% ethanol fixation, regardless of the dye used, the DNA content of the hemopoietic cells appeared unimodal, but after either 70% ethanol or 66% methanol fixation, it appeared bimodal. Cell sorting revealed that the lower and upper modes are represented by erythrocytes (RBCs) and leukocytes (WBCs), respectively. In amphibians, the chromatin of metabolically inactive RBCs is highly condensed relative to the chromatin of metabolically active WBCs. The bimodal distribution of DNA contents seen with 66% methanol and 70% ethanol, but not 50% ethanol, seems to reflect this disparity in the degree of chromatin condensation existing between the RBCs and WBCs. This, in turn, implies that the accessibility of fluorescent DNA dyes to the chromatin of fixed frog hemopoietic cells, especially of RBCs, can be affected by the concentration of alcohol used for their fixation.Key terms: DNA fluorescent dye accessibility, fixation artifacts, frog hemopoietic cells, erythrocyte chromatin compaction, ploidy We and others have been using diploidhiploid chimeras to investigate the developmental origin of hemopoietic cell in the frogs, Rana pipiens and Xenopus laeuis. Chimerism was established prior to larval life by fusing defined regions of diploid (2N) and triploid (3N) tailbud stage embryos (3,11,22). In principle, the ploidy of blood cells in chimeric tadpoles or adults should indicate the embryonic site(s) of that cell type. During the course of some control studies that involved different fixation protocols, we noted that the amount of DNA in RBCs and WBCs of normal (i.e., non-chimeric) 2N or 3N perimetamorphic tadpoles, froglets, and mature frogs appeared to differ considerably as if the RBCs and WBCs from a single animal were of a different ploidy. These observations compelled us to investigate the possibility of fixation artifacts, and to establish a reproducible fixation protocol for flow cytometry that allows fluorescent DNA dyes to bind stoichiometrically to the chromatin of all hemopoietic cells (i.e., independent of their developmental stage or degree of chromatin compaction) such that the dye bound to the chromatin accurately reflects the cells' ploidy. MATERIALS AND METHODSMaterials Non-chimeric diploid adult frogs, Rana pipiens (J.M. Hazen and Co., Alburg, VT) and Xenopus laeuis (South African Snake Farm, Capetown, South Africa), were used. In addition, a few non-chimeric 3N adult Xenopus laeuis (9,24) were also used. MethodsCell suspension medium (CSM). Hemopoietic cells were suspended in Leibovitz-15 medium (L-15) (Gibco, Grand Island, NY) that was diluted to the osmolarity of adult frog serum (220 mOsm) and supplemented with 1.25 x lop5 M HEPES buffer, 100 U/ml penicillin, 100 kg/ml streptomycin, and 2% fetal bovine 'Address reprint requests to Dr.

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“…Haploid embryos develop abnormally, potentially obscuring detection of recessive phenotypes. However, if haploid embryos are subjected to several thousand pounds of hydrostatic pressure within a few minutes of fertilization, the extrusion of the second meiotic polar body is suppressed, thereby generating diploid embryos with only a maternal gene complement (Tompkins, 1978). One can then score these embryos for developmental phenotypes.…”

Section: Uncovering Mutations In X Tropicalis: Gynogenesis and Inbrementioning

confidence: 99%

Xenopus, the next generation: X. Tropicalis genetics and genomics

Hirsch

Zimmerman

Grainger

2002

Developmental Dynamics

143

102

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A small, fast-breeding, diploid relative of the frog Xenopus laevis, Xenopus tropicalis, has recently been adopted for research in developmental genetics and functional genomics. X. tropicalis shares advantages of X. laevis as a classic embryologic system, but its simpler genome and shorter generation time make it more convenient for multigenerational genetic, genomic, and transgenic approaches. Its embryos closely resemble those of X. laevis, except for their smaller size, and assays and molecular probes developed in X. laevis can be readily adapted for use in X. tropicalis. Genomic manipulation techniques such as gynogenesis facilitate genetic screens, because they permit the identification of recessive phenotypes after only one generation. Stable transgenic lines can be used both as in vivo reporters to streamline a variety of embryologic and molecular assays, or to experimentally manipulate gene expression through the use of binary constructs such as the GAL4/ UAS system. Several mutations have been identified in wild-caught animals and during the course of generating inbred lines. A variety of strategies are discussed for conducting and managing genetic screens, obtaining mutations in specific sequences, achieving homologous recombination, and in developing and taking advantage of the genomic resources for Xenopus tropicalis.

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Triploid and gynogenetic diploid Xenopus laevis

Cited by 63 publications

References 10 publications

A gynogenetic screen to isolate naturally occurring recessive mutations in Xenopus tropicalis

A gynogenetic screen to isolate naturally occurring recessive mutations in Xenopus tropicalis

Fixation‐Associated quantitative variations of DNA fluorescence observed in flow cytometric analysis of hemopoietic cells from adult diploid frogs

Xenopus, the next generation: X. Tropicalis genetics and genomics

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