Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-22
2002-09-17
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S069100, C435S320100, C435S325000, C435S455000, C536S023100, C536S024100
Reexamination Certificate
active
06451539
ABSTRACT:
FIELD OF THE INVENTION
The invention provides novel expression vectors that allow stable, high-level expression of a polypeptide of interest in a host cell, particularly mammalian cells. The invention also includes a transfection system for mammalian cells using the constructs described herein. The invention described herein provides an efficient mechanism by which any desired polypeptide can be expressed at high levels using novel cell lines generated as described herein.
BACKGROUND OF THE IVVENTION
Vectors based on lytic viruses such as polyoma have been used for short-term expression, but tend to be unstable, and replicate many times per cell cycle (Lebkowski, J. S., et al., MOL. CELL. BIOL. 4:1951-1960, 1984). Vectors based on bovine papilloma virus have also been developed but do not consistently replicate once per cell cycle (Gilbert, D. M., et al., CELL 50:59-68, 1987; Ravnan, J.-B., et al., J. VIROL. 66:6946-6952, 1992). Further bovine papilloma virus-based vectors show a high frequency of rearrangements (Ashman, C. R., et al., SOMATIC CELL MOL. GENET. 11:499-504, 1985; DuBridge, R. B., et al., MOL. CELL. BIOL. 7:379-387, 1987).
In human and primate cells, vectors based on Epstein-Barr virus (EBV) have been developed (Yates, J., et al., PROC. NATL. ACAD. Sci. USA. 81:3806-3810, 1984; Reisman, D., et al., MOL. CELL. BIOL. 5:1822-1832, 1985; Lupton, S., et al., MOL. CELL. BIOL. 5:2533-2542, 1985). These vectors typically replicate once per cell cycle (Adams, A., J. VIROL. 61:1743-1746, 1987; Yates, J. L., et al., J. VIROLOGY 65:483-488, 1991; Haase, S. B., et al., NUC. ACIDS RES. 19:5053-5058, 1991) and are stably maintained over the long-term with a low mutation frequency (DuBridge, R. B., et al., MOL. CELL. BIOL. 7:379-387, 1987; DuBridge, R. B., et al., MUTAGENESIS 3:1-9, 1988; Drinkwater, N. R., et al., PROC. NATL. ACAD. SCI. USA 83: 3402-3406, 1986). These vectors have been used for cloning and expression studies in human and simian cells (Margolskee, R. F., et al., MOL. CELL. BIOL. 8:2837-2847, 1988; Young, J. M., et al., GENE 62:171-185, 1988; Belt, P.B.G.M., et al., GENE 84:407-417, 1989; Peterson, C., et al., GENE 107:279-284, 1991). Stable transformation frequencies are high because integration into the genome is not required, and recovery of cloned sequences is achieved by plasmid extraction. However, rodent cells are not permissive for EBV replication, and no rodent counterpart of EBV has been described (Yates, J. L., et al., NATURE (LONDON) 313:812-815, 1985).
U.S. Pat. No. 4,959,317 (Sauer, et al.) discloses the use of Cre-Lox site-specific recombination to achieve gene transfer in eukaryotic cells. However, the system described does not provide efficient or stable integration of transferred DNA into the host genome (see e.g., (Sauer, et al., (1993) Methods in Enzymology 225: page 898). This is largely due to the fact that excision of transferred DNA out of the genome, by way of intramolecular exchange, predominates over integration of DNA into the genome, by way of intermolecular site-specific recombination.
U.S. Pat. No. 5,928,914 (Leboulch, et al.) describes methods and compositions for transforming cells, resulting in efficient and stable site-specific integration of transgenes. Transformation is achieved by introducing into a cell an acceptor vector, preferably a retroviral vector, which integrates into the genome of the cell. The acceptor vector comprises two incompatible lox sequences, L1 and L2. A donor vector is then introduced into the cell comprising a transgene flanked by the same L1 and L2 sequences. Stable gene transfer is initiated by contacting the lox L1 and L2 sequences with Cre recombinase.
SUMMARY OF THE INVENTION
In one aspect, the invention provides an expression vector comprising (a) a first polynucleotide encoding a first, crippled, selectable marker (b) a second polynucleotide encoding a heterologous polypeptide of interest; and (c) a third polynucleotide encoding a second, amplifiable selectable marker. Suitable first selectable markers include sequences coding antibiotic (e.g., neomycin) resistance containing one or more crippling mutations. In one embodiment, the amplifiable selectable marker is dihydrofolate reducatase (dhfr).
The invention also includes the following constructs: a plasmid designated pESN1dhfr; a plasmid designated pESN2dhfr; plasmid designated pESN3dhfr; a plasmid designated pneo*dhfr5′del (e.g., pneo1dhfr5′del, pneo2dhfr5′del, pneo3dhfr5′del); and a plasmid designated pdhfr3′del.
In another aspect the invention includes a method for producing a polypeptide of interest in a host cell, comprising
(a) introducing an expression vector or construct described herein into a host cell,
(b) selecting host cells which express the first and second selectable markers under conditions that select for stably integrated expression vectors,
(c) growing the stably-transfected host cells under conditions which favor expression of the polypeptide of interest, and
(d) isolating the polypeptide of interest.
In certain embodiments, the heterologous polypeptide is a viral protein (e.g., an HIV protein) or is CAB2 or CAB4 and the host cell is a mammalian or insect cell. Host cell lines that produces a polypeptide of interest using this method are also included in the present invention.
In another aspect, the invention includes a transfection system comprising
(a) a first construct comprising, in a suitable backbone, a sequence encoding a first selectable marker and a sequence encoding a second selectable marker, wherein the second selectable marker contains at least one disabling mutation in its coding sequence; and
(b) a second construct comprising, in a suitable backbone, a polynucleotide sequence of interest and a sequence encoding a third selectable marker, wherein the third selectable marker is the same selectable marker as the second selectable marker except that the third selectable marker contains at least one disabling mutation that is in a different region of the coding sequence than the disabling mutation in said second selectable marker. In certain embodiments, the first selectable marker encodes for antibiotic resistance, for example, by encoding wild-type or functionally impaired neomycin phosphotransferase II, the second selectable marker encodes dhfr which disabled by at least one mutation in the 5′ coding region and the third selectable marker encodes dhfr which is disabled by at least one different mutation in the 3′ coding region. The disabling mutations may be, by way of example, point mutations or deletions. Where the constructs are plasmids, the transfection system may further comprise
(c) first, second, and third promoters operably linked to said first and second selectable markers and said transgene, respectively;
(d) sequences encoding polyadenylation sites operably linked to said first and second selectable markers and said transgene; and
(e) sequence encoding origins of replication operably linked to said first, second selectable markers and said transgene. Promoters such as CMV promoter, an RSV promoter or an SV-40 early promoter may be used and each sequence may be operably linked to a different promoter.
In another aspect, the invention includes a method for producing a mammalian cell line for expression of a selected polynucleotide sequence, comprising
(a) introducing into a selected mammalian cell, having a genome, a first construct comprising a sequence encoding a first selectable marker and a sequence encoding a second selectable marker, wherein the second selectable marker contains at least one disabling mutation in its coding sequence,
(b) selecting for a mammalian cell expressing the first selectable marker, wherein said first construct stably integrates into the genome;
(c) introducing into the mammalian cell a second construct comprising a polynucleotide sequence of interest and a sequence encoding a third selectable marker, wherein the third selectable marker is the same selectable marker as the second selectable marker except that the third selectable
Innis Michael
Scott Elizabeth M.
Blackburn Robert P.
Chiron Corporation
Davis Katharine F
Dollard Anne S.
Guzo David
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