Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-09-14
2003-12-02
Chen, Shin-Lin (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S455000, C435S006120, C536S023100, C536S023500, C530S350000
Reexamination Certificate
active
06656735
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a methodefor identification of transcription factor responsive elements, target genes and/or co-factors of transcription factors.
2. Description of the Prior Art
Among the numerous genes that control metazoan development, homeogenes play a key role in cell lineage decisions and in establishing the morphological identity of multicellular domains. They are expressed in all tissue layers of the organism and at different stages during development. They code for homeoproteins, a large family of transcription factors characterized by a highly conserved 60 amino-acids DNA-binding domain, the homeodomain.
The mammalian Engrailed homeoproteins, Engrailed-1 and Engrailed-2 (En1 and En2) are expressed first in the anterior neuroepithelium at the 1 somite—(in the case of Enl) and 5 somite—(in the case of En2) stages in a domain, which will later develop into midbrain-hindbrain territory. Then, En1 is expressed in the spinal chord, in the dermomyotome of the somites and the ventral ectoderm of the limb bud. Gene inactivation experiments showed, that En1 is necessary for the development of mid/hindbrain, the sternum and the ventral limb bud, whereas En2 mutants only display a slight mid/hindbrain patterning phenotype.
However, gene replacement experiments of En1 by En2 suggest, that both proteins have identical biochemical properties and that the different phenotypes in brain development observed in En1 and En2 mutants are due to temporal differences in the expression of both genes during early neurogenesis. Therefore, in this study En1 and En2 are often summarized and referred to as Engrailed.
As illustrated in the case of Engrailed, analysis of expression patterns and of the morphological modifications which were produced in murine embryos mutated for distinct homeogenes, have been invaluable to understand the function of these homeogenes during development. However, the understanding of their precise mode of action requires the identification of the genetic networks executing their functions. Considering the Engrailed mutant phenotypes potential targets might be involved in tissue patterning (secreted molecules), in cell type specification (other transcription factors), in cell migration (cell and substrate adhesion molecules) and in cell morphogenesis (structural proteins.
In Drosophila, genetic approaches unraveling functional interactions in mutants and the search of DNA-binding sites in polytene chromosomes have been available to screen for target genes of any given transcription factor. The application of these techniques led to the identification of a number of putative engrailed target genes, such as hedgehog, decapentaplegic, cubitus-interrupus (ci), polyhometic and &bgr;3-tubulin. Only ci and &bgr;3-tubulin were shown to be direct target genes of engrailed. The genetic methods used in the fruit fly and which in principle might give access to such genetic pathways are not easily applicable to vertebrates due to the very high number of offsprings that would have to be screened, and to the inavailability of polytene chromosomes. In vertebrates, most putative homeoprotein target genes have either been discovered fortuitously by promoter analysis or by homology with genes already identified in the invertebrates, primarily in Drosophila. In most cases, the evidence that the identified genes are true target genes has remained rather circumstantial. It is particularly noteworthy that, with some exceptions illustrated by a recent study on NCAM, almost none of these target genes have been shown to be regulated by homeoproteins in vivo.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for the identification of transcription factor responsive elements, target genes and/or co-factors of transcription factors, said method enabling a systematic determination of these elements, target genes and co-factors and avoiding the disadvantages known from the prior art.
This object is achieved according to the present invention by means of a method for identification of transcription factor responsive elements, target genes and/or co-factors of transcription factors with the following steps of:
(a) introduction of a gene trap vector into a eukaryotic cell in culture, wherein said gene trap vector exhibits at least the following elements in functional arrangement:
a reporter gene
a polyadenylation site
a selectable marker gene
(b) selection of cells containing the vector;
(c) contacting the selected cells with a transcription factor, the corresponding transcription factor responsive element, target gene and/or corresponding co-factor of which is to be analyzed;
(d) identifying and cultivating such cells, which show an alteration of the reporter gene activity;
(e) identifying the target genes and/or co-factors of the transcription factors and/or the transcription factor responsive elements.
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Prochiantz Alain
Wurst Wolfgang
Chen Shin-Lin
GSF Forschungszentrum Fuer Umwelt und Gesundheit GmbH
Townsend & Townsend and Crew LLP
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