Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1992-05-28
1993-12-21
Wax, Robert A.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
4351721, 435 691, 4353201, 4352523, 435 6, 435 33, 435 34, 435 42, 536 231, 536 243, C12N 1500, C12N 500, C12P 2106
Patent
active
052720713
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
The present invention relates to a process for the modification of the expression characteristics of a gene which is naturally present within the genome of a stable cell line or cloned microorganism. In the preferred embodiment, the present invention relates to a process for the activation and expression of a gene that is present within a stable cell line and normally transcriptionally silent or inert. As a result, the protein product of that gene is expressed. This phenomenon occurs without transfecting the cell with the DNA that encodes the product. Rather, the resident gene coding for the desired product is identified within a cell and activated by inserting an appropriate regulatory segment through a technique called homologous recombination. Positive and/or negative selectable markers can also be inserted to aid in selection of the cells in which proper homologous recombination events have occurred. As an additional embodiment, a specified gene can be amplified for enhanced expression rates, whether that gene is normally transcriptionally silent and has been activated by means of the present invention, or endogenously expresses product.
BACKGROUND OF THE INVENTION
It is well known that each cell within an organism contains the genetic information that encodes all of the proteins found within that organism. However, only a very small percentage of the genes present within a given cell type is actually transcribed. The intracellular mechanisms that regulate the array of genes to be transcribed are now understood. Cell specific proteins present within the nucleus interact with DNA regulatory Segments that are linked with particular genes. This interaction of nuclear proteins with DNA regulatory sequences is required for gene transcription. This results in mRNA biosynthesis and ultimate expression of the encoded protein (Mitchell and Tjian, Science, 245:371, 1989).
These DNA regulatory segments or elements for each gene lie upstream from and, in some cases, within or even downstream of the coding regions. Through an interaction with cell specific nuclear proteins, DNA regulatory segments affect the ability of RNA polymerase, the rate limiting enzyme in protein expression, to gain access to the body of the gene and synthesize a mRNA transcript. Thus, these DNA segments and the resident nuclear proteins play a critical role in the regulation of expression of specific genes (Johnson and McKnight, Ann. Rev. Biochem., 58:799, 1989).
The DNA regulatory segments are binding sites for the nuclear proteins. These nuclear proteins attach to the DNA helix and apparently alter its structure to make the desired gene available for RNA polymerase recognition, which facilitates gene transcription. The expression of these cell specific regulatory proteins determines which genes will be transcribed within a cell and the rate at which this expression will occur. As an example of the specificity of this system, pituitary cells but not liver cells express pituitary proteins, even though the genes for the pituitary proteins are present within all liver cells. Nuclei of the liver cells do not contain the specific DNA binding proteins which interact with the elements of pituitary genes resident within the liver cells.
CURRENT METHODS EMPLOYED TO EXPRESS PROTEINS USING RECOMBINANT DNA
TECHNOLOGY
With the knowledge that specific DNA regulatory sequences are required to activate gene transcription within a particular cell type, scientists have expressed foreign genes within a particular cell type through genetic engineering. In general, DNA regulatory segments that are recognized by the cell's nuclear proteins are placed upstream from the coding region of a foreign gene to be expressed. In this way, after insertion into the cell, foreign DNA may be expressed since the cell's nuclear regulatory proteins now recognize these DNA regulatory sequences. This technology has been employed to produce proteins that have been difficult to obtain or purify from natural sources by traditional purification strategies.
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REFERENCES:
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Zhu, J. et al., "Construction of stable laboratory and industrial yeast expressing a foreign gene by integrative transformation using a dominant selection system", Gene, vol. 50, Nos. 1-3, 1986, pp. 225-237.
Le Mouellic, et al., "Targeted replacement of the homeobox gene Hox-3.1 by the Escherichia coli lacZ in mouse chimeric embryos," Proc. Natl. Acad. Sci. USA 87:4712-4716 (1990).
Mansour et al., "Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes", Nature 336(24):348-52 (1988).
Larsen et al., "Reporession mediates cell-type-specific expression of the rat growth hormone gene", Proc. Natl. Acad. Sci. USA 83:8283-8287 (1986).
Brent et al., "Functional Characterization of the Rat Growth Hormone Promoter Elements Required for Induction by Thyroid Hormone with and without a Co-transfected .beta. Type Thyroid Hormone Receptor," J. Biol. Chem. 264(1):178-182 (1989).
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Applied Research Systems ARS Holding N.V.
Escallon Miguel
Wax Robert A.
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