Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
2000-07-17
2003-05-20
Nguyen, Dave T. (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S018000, C800S025000, C536S023100, C536S023200, C435S004000, C435S320100, C435S455000
Reexamination Certificate
active
06566581
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a transgenic non-human mammal, to a method for producing the latter, to the utilization thereof, to a cell tissue therefrom, to a method for producing such cell tissue, to the utilization thereof, to a recombinant DNA expression vector and to the utilization of such vector.—The term non-human mammal refers to taxonomically higher units than animal species. Transgenic animals are organisms carrying an additional gene not originating from their species, that is a foreign gene in their genome. For the purpose of the invention in particular such transgenic animals are meant that have the foreign gene also in the germ cells, that is which hand on the foreign gene vertically, i.e. from generation to generation. If a special transgenic animal has been created, further corresponding transgenic animals may be obtained by breeding. The transgenic animals are known in the art in various embodiments, and various methods for producing transgenic animals are also known. As an example only, reference is made to document R. Jaenisch, Science, Vol. 240, 10, 1988, page 1468 ff., and the documents cited therein. The term cell tissue comprises complete organs or parts of organs of an animal, however also specific cell lines that can be isolated and cultivated therefrom, i.e. Increased in number.
A recombinant DNA expression vector is an instrument for producing a transgenic animal carrying, among other features, the foreign DNA to be integrated in the cells of the animal.
BACKGROUND OF THE INVENTION
The general technological background of the special transgenic animal provided by the invention is the following. Cancer, in particular lung cancer, is one of the most widespread diseases of mankind, and has up to now therapy predictions offering little success only. In the framework of the development of better therapies for cancer diseases it is, among other conditions, required and also legally laid down, for ethical reasons, to perform pre-clinical examinations in animals models with possible active substances obtained from basic research or by screening tests. In the case of examinations of prospective active substances for cancer therapies, it is therefore required to provide animals or (animal) cell tissues having the respective cancer diseases to be investigated, in order that the physiological effects, possibly also side effects, of the active substances can be tested in a qualitative and quantitative manner.
Cancer diseases are caused in many cases by the effects of so-called oncoproteins. These are proteins that have different structures compared to corresponding proteins in a healthy organism. These oncoproteins are capable, through not yet fully understood processes, to transform normal cells into uncontrollably proliferating cells, i.e. cancer cells. The formation of oncoproteins in an organism is in turn caused by so-called oncogenes, i.e. genes coding for the oncoprotein. Oncogenes may be introduced into a cell by viruses, may however also be formed by way of mutation of (certain) “healthy” genes, the proto-oncogenes. Such mutations can for instance take place by translocation (displacement) of a gene responsible for the production of a protein within the genome, by point mutations (replacement of a base and/or individual bases in the DNA of a gene responsible for the production of a protein by different base, with the consequence of the formation of a protein of modified amino acid sequence, the oncogene), by deletion (removal of one or more bases) or also by mutations in the region of a so-called promoter applicable for the respective gene. As a promoter is designated a DNA region of a gene by means of which the transcription (of the DNA code into a corresponding RNA) and thus finally also the expression (formation) of the protein correlated with the gene can be controlled. In a natural manner, a specific promoter is usually assigned to each gene, this promoter being arranged ahead of the latter in the genome. Ahead means that the promoter in the DNA sequence has a certain distance to the starting point of a transcription. For initiating a transcription, it is then also required that so-called transcription factors (often specific for the cell type) are taken up by the promoter.
In particular in connection with lung cancer, the so-called Raf proto-oncogenes play a special role. These genes are highly conservative with regard to evolution, and code kinases specific for serine/threonine of the cytoplasm playing in turn a role in the mitogenic signal transduction. Known in the art are for instance the genes A, B and c-Raf-1. For a survey, reference is made to documents U. R. Rapp et al., The Oncogene Handbook, Elsevier Science Publishers, Netherlands, page 115-154, 1988, and U. R. Rapp, Oncogene, 6, 495, 1991. To the family of the Raf genes belongs, among others, the c-Raf-1 gene expressing the c-Raf-1 kinase ubiquitarily in an organism. The c-Raf-1 gene comprises three conserved regions, i.e. these regions are in accordance with corresponding regions of other Raf genes of the family. The region CR1 is a regulatory domain around a cys finder consensus sequence, the region CR2 is a region having a high content of serine or threonine, and CR3 is the kinase domain. With regard to further detailed information, reference is made to document U.S. Pat. No. 5,618,670. From this document are also known (partial) sequences of the natural form of the CR3 region of the c-Raf-1 gene of mice and (partial) sequences of various point mutations thereof. From document U.S. Pat. No. 5,156,841 are known plasmids and eucariotic expression vectors containing A-Raf and v-Raf oncogenes, however in different connections, namely the genic production of Raf oncoproteins for immunological investigations.
DETAILED DESCRIPTION OF THE INVENTION
The invention is based on the technical problem to provide non-human mammals in sufficiently high number and with a pathology being uniform and reproducible with regard to tumor formation, for the purpose of pre-clinic examinations of prospective anti-cancer substances or therapies.
For achieving this object, the invention teaches a transgenic non-human mammal whose cells express a constitutively active oncogenic mutant of the kinase-domain of the c-Raf-1 gene or a protein coded by a corresponding normal allele or a derivative of the A, B or c-Raf-1 gene. —The term constitutively active means, in the context of the invention, that the protein per se is always active, i.e. the physiological effect of the protein is always obtained even without the condition of further reaction cascades in a cell or an organism. In contrast thereto, the activation of the not constitutively active Raf-1 protein requires for instance the bonding of the Ras protein with the Raf-1 protein. The term constitutively active therefore refers, for the purpose of the invention, only to the protein or the corresponding gene code and not to the gene itself or the gene activation. The reference to the Raf-1 gene means Raf-1 genes or variants thereof existing in any organism, at least however such Raf-1 genes existing in mammals. By the fact that the mammal is a transgenic animal having the mentioned features, identical animals, with regard to the pathology of the tumors induced by the expression of the mutant, can be obtained in any number by way of the natural reproduction from a transgenic base animal. Thereby pre-clinic examinations of active substances or therapies can be performed with the required reproducibility and the required statistical significance, and that also with defined control groups.
For performing pre-clinic examinations of active substances and therapies against lung cancer it is recommended that the expression of the protein coded by the constitutively active oncogenic mutant of the kinase-domain of the c-Raf-1 gene or by a corresponding allele or a derivative of the A, B or c-Raf-1 gene takes place in lung cells, since then the animals develop reproducible lung tumors.
In structural regard, a mammal as described above is characterized by
McGlew and Tuttle , P.C.
Nguyen Dave T.
Nguyen Quang
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