J. Vielkind

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

SummarySpecies of small fish are becoming useful tools for studies on vertebrate development. We have 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 temporal and spatial expression patterns of bacterial 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 well 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.

The induction of a specific pigment cell type by total genomic DNA injected into the neural crest region of fish embryos of the genus Xiphophorus

SummaryWe report genetic transformation in an intact higher organism, i.e., in xiphophorine fish. The gene to be transferred (Tu) is responsible for the formation of T-melanophores in the platyfish and is involved in the formation of melanomas in platyfish-swordtail hybrids. After injection of Tu-donor DNA into the neural crest region of embryos from Tu-free fish, some of the recipients developed T-melanophores. In a few cases, one or two single T-melanophores were formed during late embryogenesis. In most cases, many T-melanophores developed in young fish and were arranged in several colonies or in a pattern. DNase-degraded Tu-donor DNA, Tu-free fish DNA, as well as DNA from E. coli and adenovirus-2, did not induce T-melanophores. When using DNA from different strains of Tu-donor fish which differed in a regulating gene linked to Tu, the percentages of fish showing T-melanophores paralleled the degree of phenotypic expression of the Tu gene in the DNA donor. The results suggest that the Tu gene has been successfully transferred together with the linked regulating gene.

An approach to genetic transformation in the Xiphophorine fish

SummaryThe particular suitability of the Xiphophorine fish system for achieving genetic transformation is presented, and it was analyzed whether information carrying donor DNA might be available to the propigment cells of embryos ofXiphophorus helleri, which are the target cells for the transformation. Heterologous2H3H-labelled donor DNA fromE. coli, which was taken for technical reasons instead of homologous fish DNA, undergoes degradation both after injection into the neural crest region and after injection into the yolk sac (molecular weight at 0 h: 50×106; at 2 h: 1×106; at 5 h: <3×105; at 10 h: <1×104). It is concluded therefore, that informative donor DNA is present for about 2 to 3 hours after injection. The DNA of the recipient embryo is labelled radioactively during that time at which informative DNA is present only, if the donor DNA is injected into the neural crest region. The probability that a foreign gene might become available to the propigment cells and might induce transformation is discussed.

Uptake of bacterial H3-DNA into fish embryos

H3-DNA vonSalmonella typhimurium wurde in den Dotter vonPlatypoecilus maculatus-Embryonen injiziert und ihr Verbleib untersucht. Nach 45 Stunden findet man 45% der injizierten H3-DNA-Radioaktivität in der säurelöslichen Fraktion des Dotters. Von diesem Zeitpunkt an nimmt die Radioaktivität in der säureunlöslichen Fraktion der Embryonen ständig zu, bis nach 140 Stunden ein Wert von 50% erreicht ist. Der hohe Anteil an radioaktivem säurenunlöslichem Material im Dotter, der 140 Stunden nach der Injektion noch ca. 35% beträgt, lässt vermuten, dass hier noch hochmolekulare Bakterien-DNA vorhanden ist. Somit erscheint die Möglichkeit einer Aufnahme dieser hochmolekularen DNA in die Embryozellen nicht ausgeschlossen.

DNA-mediated transformation in the platyfish-swordtail melanoma system.

A genetic marker, the tumor geneTu, which causes the formation of abnormal melanophores, the T-melanophores, in the skin of Xiphophorine fish has been transferred by donor DNA from aTu genotype to recipient embryos lackingTu. Abnormal melanophores which are identical to the T-melanophores of the donor genotype occurred only in recipients treated withTu-DNA and not in those treated withTu-free control DNA.

Electron microscopic studies on melanotic and amelanotic melanomas in xiphophorin fish

Electron microscopic studies on heritable melanomas in platyfish-swordtail hybrids reveal that the slowly growing melanotic melanomas of both pigmented and albino hybrids consist of well-differentiated cells, whereas the very rapidly growing melanotic melanomas of pigmented hybrids and amelanotic melanomas of albino hybrids consist of incompletely differentiated cells. There seems to be a correlation between the degree of cytological differentiation of the melanoma cell and the rate of tumor growth. Es wurden elektronenmikroskopische Untersuchungen an genetisch bedingten Melanomen von Bastarden vonPlatypoecilus maculatus undXiphophorus helleri (Poeciliidae) durchgeführt. Langsam wachsende melanotische Melanome von pigmentierten sowie von albinotischen Bastarden bestehen aus gut differenzierten Melanocyten und Macromelanophoren, während schnell wachsende Melanome von pigmentierten Bastarden sowie amelanotische Melanome von albinotischen Bastarden aus unvollständig differenzierten Melanocyten bestehen. Der Grad der cytoplasmatischen Differenzierung der Melanomzellen ist daher mit der Wachstumsrate der Melanome korreliert.

DNA synthesis in genetically determined melanomas of platyfish-swordtail hybrids.

Explants from genetically determined melanotic and amelanotic melanomas of platyfish-swordtail hybrids were cultivated in H3-thymidine-containing medium with or without additional amounts of amino acids. The incorporation of label into the DNA extracted from the explants was measured, and the DNA content per unit protein was determined. The results revealed a significant difference between the two melanoma types; the rate of DNA synthesis as well as the DNA to protein ratio of amelanotic melanoma explants was about twice that of melanotic ones. This is consistent with the results of earlier investigations which had shown that the amelanotic melanomas grow more rapidly and contain more incompletely differentiated cells than the melanotic ones. The data, furthermore, demonstrated that both melanoma types exhibit extreme individual variations in DNA synthesis and in the DNA to protein ratio. The present study failed to confirm the results of earlier autoradiographic studies which suggested that additional amounts of amino acids in the culture medium stimulate the incorporation of labeled thymidine into the melanoma DNA. The results of the present experiments on melanomas in vitro do not rule out the possibility that amino acids stimulate melanoma growth in vivo by initiating DNA synthesis and cell division of melanoma cells. Explantate von genetisch bedingten melanotischen und amelanotischen Melanomen von Zahnkarpfenbastarden wurden in H3-thymidinhaltigem Medium mit oder ohne zusätzliche Aminosäuren kultiviert. Der Einbau des H3-Thymidins in die DNA der Melanome wurde durch Messung der Radioaktivität in DNA-Extrakten verfolgt und der DNA-Gehalt pro Einheit Protein bestimmt. Es zeigte sich, daß die beiden Melanomtypen sich merklich voneinander unterscheiden; sowohl die DNA-Syntheserate als auch das DNA-ProteinVerhältnis war bei den amelanotischen Melanomen etwa doppelt so hoch wie bei den melanotischen. Dies stimmt mit dem Befund früherer Untersuchungen überein, daß die amelanotischen Melanome schneller wachsen und einen höheren Anteil an unvollständig differenzierten Zellen aufweisen als die melanotischen Melanome. Die Ergebnisse zeigten außerdem, daß innerhalb eines Melanomtyps starke individuelle Unterschiede in der DNA-Syntheserate und im DNA-Protein-Verhältnis vorhanden sind. Sie konnten jedoch den Befund früherer autoradiographischer Untersuchungen, daß ein Zusatz von Aminosäuren zum Kulturmedium den Einbau von radioaktiv markiertem Thymidin in die DNA der Melanome stimuliert, nicht stützen. Die vorliegenden Ergebnisse an Melanomen in vitro schließen allerdings nicht aus, daß Aminosäuren in vivo das Melanomwachstum anregen, indem sie DNA-Synthese und Zellteilung der Melanomzellen begünstigen.

Culture of embryos from viviparous fishes of the genusXiphophorus

A procedure is described for the in vitro culture ofXiphophorus embryos. The method permits the raising of embryos of viviparous fish species from an early stage of embryo-genesis (first somite formation) beyond the stage of birth and into fertile adults.

A possibility to achieve genetic transformation in the platyfish-swordtail system (Platypoecilus maculatus - Xiphophorus helleri)

In order to determine how informative homologous donor DNA might be made available to propigment cells of the recipientXiphophorus helleri for transformation, labelled heterologous DNA fromE. coli was injected into the neural crest region or the yolk sac of embryos of the recipient. On the basis of the degradation rate of the donor DNA and the incorporation rate of radioactivity into the recipient DNA, it is concluded that injection into the neural crest region may be a suitable method to make available informative homologous donor DNA for transformation.