Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-02-24
1999-12-28
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 8, 435 29, 435441, 435455, C12Q 168, C07H 2104
Patent
active
060079932
DESCRIPTION:
BRIEF SUMMARY
The invention relates to an in vitro test procedure for the detection of chemically-induced embryotoxic (for example also teratogenic) effects based on differentiated pluripotent embryonic stem (ES) cells from the mouse and rat and using embryonic germ (EG) cells obtained established from primordial germ cells.
The detection of teratogenic properties of chemical agents occurs at this time by determination of the reproduction toxicity of test substances following single or multi-administrations to pregnant laboratory mammals and by tests of the embryotoxicity in the early stages of pregnancy (Holz and Siegemund, "Der Einsatz von Tieren in der Forschung und Entwicklung im Verbraucher- und Umweltschutz, Abschlussbericht zur Basiserhebung des Batelle-Instituts 1988 {The Use of Animals in the Research and Development in the Protection of the Consumer and the Environment, Final Report to the Base Determination of the Batelle Institute 1988}). Furthermore, in vitro tests are performed with mammal embryos (Neubert and Merker, Cell culture techniques--applicability for studies on prenatal differentiation and toxicity, de Gruyter, Berlin--New York (1981)) and with embryonic organs for teratogenicity tests. These tests procedures have however the disadvantage that they require the use of a large number of live mammals, in particular rats and mice. In vitro test procedures, in which primary cell cultures of limb buds (for example, "Limb Buds", Kochhar, Teratology 11, 273-287 (1975)), or brain parts of embryonic rats (Flint and Orton, Toxicol. Appl. Pharmacol. 76, 383-395 (1984)) or permanent cell lines of embryonic or adult mammal tissue, such as tumor cells of the ovary or embryonic palate cells are employed, do not fulfill, strictly speaking, the requirements which are imposed on the teratogenicity tests during the embryogenesis, namely giving indications of possible disgeneses or developmental disturbances.
Efforts have been made for a couple of years to employ permanent cultures of totipotent/pluripotent embryonic stem cells (ES cells) for the detection of embryotoxic and mutagenic substances (Laschinski et al., Reproductive Toxicol. 5, 57-65 (1991), Newall and Beedles, Toxicol. in Vitro 8, 697-701 (1994), Sehlmeyer and Wobus, Mutation Res. 324, 69-76 (1994)). Embryonic stem cells are the totipotent cells of the early embryo from which there develops the complete mammal organism. Disturbances during the germ-layer and prenatal development can lead to the necrosis of the embryos (preimplantative death), to developmental disturbances, maldevelopment or, respectively, malformations.
There are known as in vitro systems of totipotent or, respectively, pluripotent cells cells (Evans and Kaufman, Nature 282, 507-509 (1981)), and obtained from primordial germ cells (PGC) of about 9-day old embryos and are cultivated as permanent cell lines (Stewart et al., Dev. Biol. 161, 626-628 (1994)).
Both ES cells as well as EG cells are totipotent and participate in vivo in the normal embryogenesis after retransfer into the blastocyst and are in the position to colonize the germination path (Bradley et al., Nature Bd. 3 309, 255-256 (1984), Matsui et al., Cell 70, 841-847 (1992)).
ES cells can differentiate in vitro after differentiation in embryo-like aggregates, so-called embryoid bodies, derivatives of all three germ layers, i.e. in cardiogenic cells (Doetschmann et al., J. Embryol. Exp. Morphol. 87, 27-45 (1985)), in myogenic cells (Rohwedel et al., Dev. Biol. 164, 87-101 (1994)), neuronic and haematopoietic cells (Wiles and Keller, Development 111, 259-267 (1991)).
It has been demonstrated in previous experiments that the teratogenic retinoic acid (RA) led to grave changes of the expression of tissue-specific genes if it took effect at specific times of the embryoid body differentiation, and to activation, repression, or modulation of the expression of the myocardial-specific genes or somatic-specific genes (Wobus et al., Roux's Arch. Dev. Biol. 204, 1994 (36-45). This activation, repressing, or modulation of the gene expressio
REFERENCES:
patent: 5346812 (1994-09-01), Voellmy et al.
Lai, FASEB J. 9(4), A942 (Apr. 1995).
Franz Wolfgang-Michael
Wobus Anna Magdalene
Horlick Kenneth R.
Insitut fur Pflanzengenetik und Kulturpflanzenforschung
Kasper Horst M.
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