Chemistry: molecular biology and microbiology – Spore forming or isolating process
Patent
1992-08-13
1998-04-14
Guzo, David
Chemistry: molecular biology and microbiology
Spore forming or isolating process
4353201, 536 2372, 536 241, C12N 510, C12N 1586, C12N 1540
Patent
active
057390266
DESCRIPTION:
BRIEF SUMMARY
The present invention is related to DNA expression systems based on alphaviruses, which systems can be used to transform animal cells for use in the production of desired products, such as proteins and vaccines, in high yields.
The rapid development of biotechnology is to a large extent due to the introduction of recombinant DNA technique, which has revolutionized cellbiological and medical research by opening new approaches to elucidate the molecular mechanisms of the cell. With the aid of the techniques of cDNA cloning, large numbers of interesting protein molecules are characterized each year. Therefore, a lot of research activity is today directed to elucidate the relationship between structure and function of these molecules. Eventually this knowledge will increase our possibilities to preserve healthiness and combat diseases in both humans and animals. Indeed, there is today a growing list of new "cloned" protein products that are already used as pharmaceuticals or diagnostics.
In the recombinant DNA approaches to study biological questions, DNA expression systems are crucial elements. Thus, efficient DNA expression systems, which are simple and safe to use, give high yields of the desired product and can be used in a variety of host cells, especially also in mammalian cells, are in great demand.
Many attempts have been made to develop DNA expression systems, which fulfill these requirements. Often, viruses have been used as a source of such systems. However, up to date none of the existing vital expression systems fulfill all these requirements in a satisfying way. For instance, the Baculovirus expression system for cDNA is extremely efficient but can be used only in insect cells (see Reference 1 of the list of cited references; for the sake of convenience, in the following the cited references are only identified by the number they have on said list). As many important molecules will have to be produced and processed in cells of mammalian origin in order for them to become active, this system cannot be used in such cases. Furthermore, the Baculovirus cDNA expression system is not practically convenient for analysis of the relationship between structure and function of a protein because this involves in general the analysis of whole series of mutant variants. Today it takes about 6-8 weeks to construct a single Baculo recombinant virus for phenotype analyses. This latter problem is also true for the rather efficient Vaccinia recombinant virus and other contemporary recombinant virus cDNA expression systems (2,3). The procedure to establish stably transformed cell lines is also a very laborious procedure, and in addition, often combined with very low levels of protein expression.
Hitherto, most attempts to develop viral DNA expression systems have been based on viruses having DNA genomes or retroviruses, the replicative intermediate of the latter being double stranded DNA.
Recently, however, also viruses comprising RNA genomes have been used to develop DNA expression systems.
In EP 0 194 809 RNA transformation vectors derived from (+) strand RNA viruses are disclosed which comprise capped viral RNA that has been modified by insertion of exogenous RNA into a region non-essential for replication of said virus RNA genome. These vectors are used for expression of the function of said exogenous RNA in cells transformed therewith. The RNA can be used in solution or packaged into capsids. Furthermore, this RNA can be used to generate new cells having new functions, i.e. protein expression. The invention of said reference is generally claimed as regards host cells, (+) strand RNA viruses and the like. Nevertheless, it is obvious from the experimental support provided therein that only plant cells have been transformed and in addition only Bromo Mosaic virus, a plant virus, has been used as transformation vector.
Although it is stated in said reference that it is readily apparent to those skilled in the art to convert any RNA virus-cell system to a useful expression system for exogenous DNA using principals d
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Garoff Henrik
Liljestrom Peter
Bioption AB
Guzo David
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