Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1997-10-31
2003-09-30
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Vector, per se
C536S024100
Reexamination Certificate
active
06627436
ABSTRACT:
FIELD OF THE INVENTION
The invention concerns an expression vector that permits high level expression of genes (especially heterologous genes) and fragments thereof in both prokaryotic and eukaryotic systems. The invention also pertains to derivatives of such a vector that contain a prokaryotic or eukaryotic (especially mammalian) gene. The invention further pertains to prokaryotic or mammalian cells containing such an expression vector or derivative.
BACKGROUND OF THE INVENTION
The capacity to clone polynucleotides into autonomously replicating vectors has had a profound impact on medicine, biotechnology, and biological research. Typically, the fundamental differences between prokaryotic and eukaryotic cells has required the use of separate vectors whenever expression is desired in both classes of cells, or whenever a gene cloned in one class of cell is to be expressed in the other.
Because of fundamental differences between mammalian and prokaryotic cells, previously described vectors could not be used interchangeably to direct expression in both prokaryotic and mammalian cells. As a consequence, researchers seeking to express a gene of interest in both prokaryotic and mammalian cells needed to re-isolate and re-clone that gene into separate vectors, one suitable for prokaryotic expression, and one suitable for expression in mammalian cells. Each step involves isolation and characterization of clones containing the gene of interest, requiring a significant investment of time and biological reagents.
Thus, a large number of specialized prokaryotic vectors have been described (e.g., plasmids such as those capable of replication in
E. coli
such as, for example, pBR322, ColE1, pSC101, pACYC 184, &pgr;VX). Such plasmids are, for example, disclosed by Sambrook, J. et al. (In:
Molecular Cloning, A Laboratory Manual,
2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). Sambrook, J. et al., herein incorporated by reference, provide a review of the characteristics of mammalian vectors (In:
Molecular Cloning, A Laboratory Manual,
2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).
For expression in mammalian cells, eukaryotic genes are typically cloned first into a bacterial vector and then subcloned into a vector suitable for eukaryotic expression. Thus, although many vectors have been described that are capable of replicating in both prokaryotic and eukaryotic cells, such vectors are designed to express a particular inserted polynucleotide in only one class of cell.
Such vectors are illustrated by U.S. Pat. No. 4,970,155 (Ikasinski, G. F.), which describes a prokaryotic plasmid that contains eukaryotic transcription and replication elements, such that an inserted polynucleotide can be expressed only in a eukaryotic cell. Similarly, U.S. Pat. No. 5,266,490 (Davis, S. et al.) describes an expression vector that contains an SV40 origin of replication, a eukaryotic transcription unit of the early immediate human cytomegalovirus (CMV) promoter region; and a generic polylinker and an SV40 splice/polyadenylation site. The vector also contains a pBR322 origin of replication and an antibiotic resistance gene under control of a prokaryotic promoter.
It is, however, desirable to have vectors that are capable of expressing an inserted gene in both prokaryotic and eukaryotic cells without any requirement to modify the vector or reclone the inserted gene. Recently, He, B. et al. (Gene 164: 75-79 (1995) described expression vectors that direct the synthesis of proteins from a common set of signals in both prokaryotic and eukaryotic cells. To allow transcription from a common promoter the vectors rely upon a phage RNA polymerase. To direct initiation of translation to the same start codon the vectors utilize an internal ribosome entry site from encephalomyocarditis virus that has been modified to include a prokaryotic ribosome-binding site at an appropriate distance upstream from the desired start codon. Mole, S. E. et al. (
Nucl. Acids Res.
15: 9090 (1987) describe pSEMCatR1, a prokaryotic-eukaryotic shuttle vector compatible with pUR and lambda gt11 expression systems. Kaehler, R. et al. (DD 206791) discuss a hybrid expression vector which contains prokaryotic and eukaryotic control units directly connected to the sequence being expressed. Alting-Mees, M. A. (Strategies 5:58-61 (1992) describes the Stratagene® vector pBK-CMV, which is intended to be suitable for gene expression in both prokaryotes and eukaryotes. However, genes cloned into pBK-CMV are expressed only inefficiently in eukaryotic cells.
In sum, for prior vectors, the isolation and characterization of clones containing the gene of interest requires a significant investment of time and biological reagents. The present invention eliminates the need to subclone from one vector system to another by combining the features of both vector systems into a single vector.
REFERENCES:
patent: 5266490 (1993-11-01), Davis et al.
Zheng et al. A new expression vector for high level protein production, one step purification and direct isotopic labeling of calmodulin-binding peptide fusion proteins. Gene vol. 186 pp. 55-60, 1977.*
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Aruffo, Transient expression of proteins using COS cells. In Current Protocols in Molecular Biology (Ausubel et al. Eds.) pp. 16.13.1-16-13-7 John Wiley & Sons, New York, 1998.*
He et al. Phage RNA polymerase vectors that allow efficient gene expression in both prokaryotic and eukaryotic cells. Gene vol. 164 pp. 75-79, 1995.*
Stratagene Catalog Stratagene Cloning Systems p. 27, 1994.*
Padgett et al. Creating seamless junctions independent of restriction sites in PCR cloning. Gene vol. 168 pp. 31-35, 1996.*
He, Biao et al., 1995,Gene164:75-79.
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Padgett, K. A. et.,Strategies9:14-16 (1996).
Padgett Kerstien A.
Sorge Joseph A.
Brusca John S.
Palmer & Dodge LLP
Stratagene
Williams Kathleen M.
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