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text: Pict Bow Bow - Main Hand 22.1 DPS (33-56) +4 Dexterity "{{{quality}}}" cannot be used as a page name in this wiki. Retrieved from "http://aoc.wikia.com/wiki/Pict_Bow?oldid=19088" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Missing Icon Bow Weapons Add category Cancel Save

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The Townhouses is the solo project of Melbourne-based muso Leigh Hannah. Freshly released sophomore LP Diaspora features collaborations with Italian maestro Giorgio Tuma, Sydney's Guerre and Rainbow Chan, Melbourne's Wintercoats as well as Felix Weatherbourne.
Leigh describes The Townhouses as a "kaleidoscopic adventure of worldly sounds, viewed through a contemporary lens." Stream Diaspora below and tell us if you agree.
Click here for a pay-what-you-want download of The Townhouses' Diaspora.


text: Hey guys, and happy April! To kick off the arrival of spring I'm sharing a different take on the classic granny square in a few beautiful and cheery spring shades! You may recognize these colors from my post a couple of weeks ago in the Granny Ripple Tutorial. If you missed it, you can check it out here. [Read more…]


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On the other hand, the herd mentality has pushed EPHE to all-time highs and nearly 16% above a critical moving average. It's hard to imagine a scenario where EPHE does not experience a near-term 10% pullback. In my estimation, there are better values in funds like iShares MSCI Singapore ( EWS) and iShares MSCI Malaysia ( EWM). Both have more attractive price-to-earnings ratios, significantly better annual yields (3.6%) and vibrant trade relations throughout the Asia Pacific region. Additionally, neither EWM nor EWS are technically "overbought." There's a feeling by some of the investment community that the European Monetary Union may finally be getting itself out of its three-year nightmare. This has benefited some of the more financially responsible members of the alliance such as Germany and Austria. German stocks have recently hit four-year highs, while iShares Austria ( EWO) has amassed a staggering 33% since its late July bottom. It also rests in overbought territory -- more than 11% above its trend line. (See the chart below.) I might be confident that a number of European multinationals -- SAP ( SAP), Sanofi ( SNY), Louis Vuitton -- can ultimately thrive. On the other hand, the strong possibility of sharp declines in the euro lead me to a dollar-hedged alternative. Enter WisdomTree Europe Hedged Equity ( HEDJ). Not only does it hedge against potential declines in the euro, it provides diversification across leading corporations throughout Europe. Equally beneficial, HEDJ has its greatest exposure in consumer staples and consumer discretionary, where names like Bayer ( BAYRY), Unilever ( UN) and Anheuser-Busch Inbev ( BUD) have an impressive worldwide footprint.
You can listen to the ETF Expert Radio Show "LIVE", via podcast or on your iPod. You can follow me on Twitter @ETFexpert. This article is commentary by an independent contributor, separate from TheStreet's regular news coverage.


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Vogue S-4035; ca. 1949; One-piece dress. Instep-length skirt straight in front, very full godet in back, joins the bodice at a slightly lower waist-line. Front shoulder yokes, very deep back yoke. Shaped bias folds below front yoke. Gathers below collarless neck-line release bust fullness. Short kimono sleeves, narrow cuffs.
Featured in Vogue Pattern Book, Spring 1950
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Birthday Bash Flowers and Balloon Bouquet
Say "Happy Birthday" with flowers and balloons! These flower balloon bouquet bursts with festive beauty in a presentation that will make the birthday girl or boy ecstatic! Bright orange lilies, lavender chrysanthemums, and lavender carnations are gracefully arranged with green button poms, purple monte casino asters, and lush greens in a whitewash willow handled basket for an incredible display. Colorful curled ribbons and a "Happy Birthday" balloon complete the centerpiece. Same day florist delivery available! Your birthday balloon delivery will bring a smile to your special recipients face and joy to their heart. Send birthday flowers and balloons to extend your best wishes. You're not only congratulating them on another year but wishing them a grand year to come as well!
Orange Asiatic Lilies
Purple Carnations
Green Button Poms
Happy Birthday Balloon
Basket with Curly Ribbon
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Delivery Information
Same Day Flowers are delivered in the USA by a local florist partner. Send Flowers online or balloons today by placing the online order prior to 3pm in the delivery zip code. Or schedule a flower delivery for a future date easily at check-out. Cut-off times for gift delivery today may vary during peak holidays.
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Substitutions may be necessary to ensure your arrangement or specialty gift is delivered in a timely manner and depending on availability.
Substitution Policy
Substitutions may be necessary to ensure your arrangement is delivered in a timely manner. The utmost care and attention is given to your order to ensure that it is as similar as possible to the requested item.
• For green and blooming plants, similar plants may be substituted of equal or greater value.
• For one-of-a-kind plants, such as orchids, we will make every attempt to match the plant type, but may substitute with another color.
• If the plant container shown online is not available, a similar container will be used.
• In arrangements of assorted flowers, the colors shown online will be used if at all possible, even if this means substituting other kinds of flowers of equal or greater value.
• For one-of-a-kind flower arrangements, such as all roses or all lilies, we will make every attempt to match the flower type, but may substitute with another color.
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Specialty Gifts
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text:
Popular Now
Popular Now
Popular Now
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01803 865913
+44 1803 865913
Series: Norrlinia Volume: 3
By: CE Sonck(Author)
24 pages, 8 b/w distribution maps
Finnish Museum of Natural History
Language: Swedish
The Taraxacum flora of the Northern part of Finnish Lapland was studied and specimens were collected in 1954, 1967, and later more regularly, almost every year from 1973 to 1990. A list is presented of species belonging to the sections Boreigena, Ceratophora, Erythrosperma, Borea, and Vulgaria (Ruderalia). In addition, 3 species from the section Spectabilia are included. The total number of species is 143. A list of the localities is given for each species, and 8 distribution maps are presented. Taxonomic problems especially concerning sectio Borea are discussed. Many species, belonging to sect. Vulgaria, were probably introduced in the 1940's with hay-transports of the German troops. The collected specimens are kept in the Botanical Museum, University of Helsinki.
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text:
|Publication number
|Publication type
|Application number
|Publication date
|Nov 13, 2001
|Filing date
|Dec 30, 1999
|Priority date
|Nov 1, 1999
|Fee status
|Also published as
|Publication number
|Michael Innis, Elizabeth M. Scott
|Original Assignee
|Chiron Corporation
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (10), Non-Patent Citations (13), Referenced by (30), Classifications (26), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is related to provisional patent application Ser. No. 60/162,930, filed Nov. 1, 1999, from which priority is claimed under 35 USC �119(e)(1) and which application is incorporated herein by reference in its entirety.
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.
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., M
In human and primate cells, vectors based on Epstein-Barr virus (EBV) have been developed (Yates, J., et al., P
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.
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 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; and
(d) selecting for a mammalian cell expressing a functional product encoded by the second selectable marker, wherein the functional product is encoded by a sequence produced by a recombination event between said second and third selectable markers, and the resulting mammalian cell is capable of expressing the selected polynucleotide sequence. In certain embodiments, the selected polynucleotide sequence encodes a polypeptide and expressing the selected polynucleotide sequence results in expression of the polypeptide. Mammalian cells produced by this method are also provided.
In another aspect, the invention includes a method for producing a polypeptide of interest in a host mammalian cell, said method comprising:
(a) introducing into said 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 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 encoding the polypeptide 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;
(d) selecting for a mammalian cell expressing a functional product encoded by the second selectable marker, wherein the functional product is encoded by a sequence produced by a recombination event between said second and third selectable markers, and the resulting mammalian cell is capable of expressing the polypeptide of interest; and
culturing the mammalian cell under conditions to produce the polypeptide of interest.
In one embodiment, the first selectable marker encodes for neomycin resistance and second and third selectable markers encode dhfr. In certain embodiments, the constructs can be introduced into said cells by electroporation or by calcium phosphate transfection. A mammalian cell line that produced according to these methods are also included.
These and other embodiments will be readily apparent to one skilled in the art in light of the teachings of this specification.
This invention will be more fully described with reference to the drawings in which:
FIG. 1 depicts the nucleotide sequence of CAB2 (SEQ ID NO:31). Lower case represents original sequence.
FIG. 2 is a schematic representation of expression vectors designated pESNdhfr, pESN1dhfr, pESN2dhfr and pESN3dhfr. The vectors differ in the location and/or number of mutations in the Neo gene. pESNdhfr contains a wild-type Neo sequence. pESN1dhfr contains a mutation which alters amino acid residue 182 of the Neo gene product. pESN2dhfr contains a mutation which alters amino acid residue 261 of the Neo gene product and pESN3dhfr contains mutations which alter both amino acid residues 182 and 261 of the Neo gene product.
FIG. 3 is a schematic depicting an expression vector designated pcDNA3. The vector is 5446 nucleotides and contains, in a pUC19 vector backbone (bases 4450-5310), the following sequences: a CMV promoter (bases 209-863); a T7 promoter (bases 864-882); a polylinker (bases 889-994); a Sp6 promoter (bases 999-1016); a BGH polyA sequence (bases 1018-1249); and SV40 promoter (bases 1790-2215); an SV40 origin of replication (bases 1984-2069); an open reading frame (ORF) encoding neomycin resistance (Ner) (bases 2151-2932); an SV40 polyA sequence (bases 3120-3250); and an ORF encoding ampicillin resistance (Ampr) (bases 4450-5310).
FIG. 4 is a schematic representation of a DNA construct designated pneo*dhfr*1, which comprises a mutant neomycin resistance gene (neo*) and a disabled DHFR marker gene, where XX represent mutations in the DHFR gene.
FIG. 5 depicts a vector designated pXdhfr*2, which also carries a transgene and a disabled DHFR gene mutated at a different site as indicated by XX.
FIG. 6 depicts homologous recombination between pXdhfr*2 and pneo*dhfr1 at the two copies of the disabled DHFR marker gene.
FIG. 7 shows the product of homologous recombination in which the disabled DHFR gene has been rescued and the transgene is positioned in the high expression locus of a mammalian cell genome by insertion of the transgene proximate to the mutant neo* gene.
FIG. 8 depicts the pdhfr*2 DNA construct; a transgene, such as a recombinant protein gene, can be inserted into the polylinker of pdhfr*2 to create pXdhfr*2.
FIG. 9 depicts a selection scheme, wherein the DNA construct identified as pdhfr3′del employs a deletion mutant of a DHFR gene and insertion of a transgene in a polylinker forms the construct designated pXdhfr3′del (e.g., pdhfr3′del).
FIG. 10 shows a DNA construct pneo*dhfr5′del (e.g., pneo1dhfr5′del, pneo2dhfr5′del, pneo3dhfr5′del) in which a DHFR gene is disabled by 5′ deletions and the disabled gene is covalently linked to a mutant neo* gene.
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 constructs comprise (i) a first sequence encoding a first selectable marker; (ii) a second sequence encoding a polypeptide product of interest (e.g., "transgene"); (iii) a third sequence encoding a second selectable marker. Typically, the three sequences are operably linked to at least one strong promoter, for example a CMV promoter to increase expression and promote correct splicing of the product of interest.
The invention also includes a transfection system for mammalian cells using the constructs described herein. The system uses first and second constructs as described above, although each construct has a disabling mutation in the second selectable marker. The disabling mutations are in different regions of the second selectable marker so that homologous recombination between the two produces a functional product. This system allows for high expression of a desired polypeptide. Mammalian cells modified by the transfection system described herein are also provided, as are methods for making these transfected cells.
Thus, the invention described herein eliminates the tedious process of identification of high expression loci in mammalian cells and provides an efficient mechanism by which any desired polypeptide can be expressed at high levels using the novel cell lines generated as described herein.
A "nucleic acid" molecule can include, but is not limited to, procaryotic sequences, eucaryotic mRNA, CDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA. A "coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3′ to the coding sequence.
Typical "control elements", include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3′ to the translation stop codon), sequences for optimization of initiation of translation (located 5′ to the coding sequence), and translation termination sequences. "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.
"Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomie, CDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. "Recombinant host cells," "host cells," "cells," "cell lines," "cell cultures," and other such terms denoting procaryotic microorganisms or eucaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.
Techniques for determining nucleic acid and amino acid "sequence identity" also are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. In general, "identity" refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). A preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art.
Two nucleic acid fragments are considered to "selectively hybridize" as described herein. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit a completely identical sequence from hybridizing to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern blot, Northern blot, solution hybridization, or the like, see Sambrook, et al., supra or Ausubel et al., supra). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence "selectively hybridize," or bind, to each other to form a hybrid molecule. A nucleic acid molecule that is capable of hybridizing selectively to a target sequence under "moderately stringent" typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe. Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe. Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
With respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions. The selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, see Sambrook, et al., supra or Ausubel et al., supra)
A first polynucleotide is "derived from" second polynucleotide if it has the same or substantially the same basepair sequence as a region of the second polynucleotide, its cDNA, complements thereof, or if it displays sequence identity as described above. A first polypeptide is "derived from" a second polypeptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide, or (ii) displays sequence identity to the second polypeptides as described above.
"Encoded by" refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed are polypeptide sequences which are immunologically identifiable with a polypeptide encoded by the sequence.
"Purified polynucleotide" refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about 90%, of the protein with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well-known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.
The term "transfection" is used to refer to the uptake of foreign DNA by a cell. A cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.
"Nucleic acid expression vector" or "Expression cassette" refers to an assembly which is capable of directing the expression of a sequence or gene of interest. The nucleic acid expression vector includes a promoter which is operably linked to the sequences or gene(s) of interest. Other control elements may be present as well. Expression cassettes described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include a bacterial origin of replication, one or more selectable markers, a signal which allows the plasmid construct to exist as single-stranded DNA (e.g. a M13 origin of replication), a multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication). Vector backbones are discussed in further detail below. A "vector" is capable of transferring gene sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.
A "selectable marker" or "reporter marker" refers to a nucleotide sequence included in a gene transfer vector that has no therapeutic activity, but rather is included to allow for simpler preparation, manufacturing, characterization or testing of the gene transfer vector. Suitable selectable markers are discussed further below.
Expression Vectors
In one embodiment, the expression vectors comprise polynucleotides encoding (i) a first crippled, selectable marker; (ii) a product of interest, also referred to as the "transgene"; and (iii) a second, amplifiable selectable marker. By "crippled" is meant a sequence encoding a selectable marker that includes one or more mutations that diminish, but do not destroy, the function of the marker. In one embodiment the first crippled selectable marker is a neomycin resistance (Neor) sequence in which amino acid residue 182 (Glu) is mutated to Asp. (see, FIG. 2, pESN1.dhfr and Yanofsky et al., infra). In another embodiment, the first crippled selectable marker is a Neor sequence in which amino acid residue 261 (Asp) is mutated to Asn (N). (FIG. 2, pESN2.dhfr). In yet another embodiment, the first crippled selectable marker is a Neo sequence containing both the above described mutations, e.g., Glu182 to Asp and Asp261 to Sn. (See, FIG. 2, pESN3,dhfr). Other suitable selectable markers and methods of making and testing crippling mutations are described below.
Also included in the expression vectors is a sequence encoding a product of interest (i.e., a "transgene"). Suitable transgenes include polynucleotides encoding for any polypeptide of interest. In one embodiment, the transgene encodes a chimeric protein such as "CAB". CAB combines features of membrane co-factor protein (MTP) and decay accelerating factor (SAF) to inhibit complement activation. See, e.g., Higgins et al. (1997) J. Immunology 158(6):2872-2881. In certain embodiments, the sequence encoding the transgene can also be designed, as described herein, such that aberrant mRNA splicing of the product is reduced or eliminated.
The expression vectors also include a second selectable, amplifiable marker. The second amplifiable marker facilitates selection of those cells which express the transgene at high levels, for example dihydrofolate reducatase (DHFR). Suitable markers are discussed in detail below.
The expression vectors of the present invention can be produced following the teachings of the present specification in view of techniques known in the art. Sequences, for example encoding transgenes, may be commercially available, for example, green fluorescent protein (G.P.) is available from Clontech, Palo Alto, Calif., and lucifers is available from Promega, Madison, Wis. Exemplified herein are expression vectors which direct high-level expression of the chimeric protein CAB and expression of HIV proteins. However, it is to be understood that the expression vectors, constructs and methods of the present invention are applicable to high-level expression of any polypeptide of interest.
Another standard source for the polynucleotides used in the invention is, of course, polynucleotides isolated from an organism (e.g, bacteria), a cell, or selected tissue. Nucleic acids from the selected source can be isolated by standard procedures, which typically include successive phenol and phenol/chloroform extractions followed by ethanol precipitation. After precipitation, the polynucleotides can be treated with a restriction endonuclease which cleaves the nucleic acid molecules into fragments. Fragments of the selected size can be separated by a number of techniques, including agarose or polyacrylamide gel electrophoresis or pulse field gel electrophoresis (Care et al. (1984) Nuc. Acid Res. 12:5647-5664; Chu et al. (1986) Science 234:1582; Smith et al. (1987) Methods in Enzymology 151:461), to provide an appropriate size starting material for cloning.
Another method of obtaining the nucleotide components of the expression vectors or constructs is PCR. General procedures for PCR are taught in MacPherson et al., PCR: A P
Another method for obtaining polynucleotides is by enzymatic digestion. For example, nucleotide sequences can be generated by digestion of appropriate vectors with suitable recognition restriction enzymes. Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using standard techniques.
Polynucleotides are inserted into suitable backbones, for example, plasmids, using methods well known in the art. For example, insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary or blunt ends on each molecule that can pair with each other and be joined with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of a polynucleotide. These synthetic linkers can contain nucleic acid sequences that correspond to a particular restriction site in the vector DNA. Other means are known and available in the art. A variety of sources can be used for the component polynucleotides.
Constructs for Transfection Systems
The invention also includes using first and second constructs in particular methods of producing and selecting stably transfected cell lines, particularly mammalian cell lines. In the transfection system described herein, the first construct comprises polynucleotides encoding first and second selectable markers. The first selectable marker is typically under the control of a strong promoter. As described above, the first selectable marker preferably contains at least one crippling mutation. In addition, in these embodiments, the second marker contains a disabling mutation, preferably in the 3′ or 5′ end of the coding region. By "disabling" is meant a mutation that reduces or, preferably eliminates, a functional gene product.
An exemplary first construct is shown in FIG. 4 and identified as pneo*dhfr*1. The first selectable marker encoded by this construct is an antibiotic resistance marker which imparts resistance to neomycin (neo*), preferably a crippled mutant of neo, designated neo* in the Figures. The neo* gene is operably linked to a strong promoter (e.g., CMV or RSV, shown 3′ to the neo* gene), and may be generally linked to other expression regulatory elements such as polyadenylation sequences (e.g., a bovine growth hormone (BGH) polyadenylation site). Further, an origin of replication useful for propagation in a microorganism may also be included (e.g., the f1 ori). The BGH polyA sequence and the f1 origin are typically 5′ to the neo* gene.
In this example, the second selectable marker of pneo*dhfr*1 construct encodes, for example, dihydrofolate reductase (DHFR). However, the function of the second selectable marker is disabled. Referring to FIG. 4, XX represent disabling mutations proximate the 5′ end of the DHFR gene. The DHFR gene is driven by the SV40 early promoter and, at its 3′ end, are sequences coding for SV40 polyA, ColEl, AmpR, and an appropriate restriction site, such as BglIII shown in FIG. 4.
The second vector construct of the transfection system comprises a polynucleotide encoding a heterologous polypeptide of interest (labeled "transgene" in the Figures) and a polynucleotide encoding the same second marker (i.e., the "second" selectable marker)that is used in the first construct. However, in the second construct, the second marker sequence contains a disabling mutation in a different position relative to the first construct, preferably on the opposite end of the coding region. The location of the disabling mutation in the second selectable marker of the first construct, and the location at the disabling mutation in the selectable marker of the second construct, are such that a single recombination event between the two copies of the coding sequences of the selectable marker can generate a functional selectable marker. Once the first construct has randomly integrated into the genome of the host cell homologous recombination events between the integrated first construct and second construct results in insertion of the transgene under the control of the strong promoter. Functional recombinants can be readily selected by assaying for the second marker. Thus, unlike other targeting systems, for example, as described in Detloff et al. (1994) Molecular and Cellular Biology 14:3936-6941, the transfection system described herein does not rely on homologous recombination events to alter the transgene of interest.
An exemplary second construct is shown in FIG. 5, and is designated pXdhfr*2. Here, the transgene is operably linked to the CMV promoter and includes a 3′ BGH polyA tail and f1 origin of replication. The second selectable marker, DHFR, is disabled by mutation(s) at the opposite end of the coding region to the disabled marker in the first construct. Thus, in FIG. 5, the mutations (designated XX) are in the 3′ end of the DHFR sequence. As in the pneo*dhfr*1 construct, the disabled DHFR marker is followed by nucleotide sequences encoding SV40 polyA, ColEl , AmpR, and a matching restriction site, in this case BglIII.
First and second constructs for use in transfection systems can be produced following the guidance of the present specification using methods known in the art, for example as described above.
General Methods
The expression vectors and methods described herein are useful in improving expression of a transgene of interest. Stable, high-level expression of genes in mammalian cells is critically dependent on both the site of integration and the copy number. The selection pressure imposed by the usual concentration of the neomycin analog G418, for example, is low and yield cell clones with widely different expression levels. Aberrant splice sites with the transgene may also hamper expression. The expression vectors described herein solve this and other problems by using a crippled first selectable marker operably linked a transgene of interest and a second, amplifiable selectable marker, which, in certain embodiments, contains a disabling mutation. The crippled marker and second marker allow for selection of high-expressing clones. In addition, expression may be increased by altering the transgene such that aberrant slicing is corrected. A strong promoter optionally containing an intron and/or enhancer may also be added to promote expression of the transgene.
The invention also provides a novel transfection system using first and second constructs. These highly efficient methods are capable of generating cells which produce high levels of the desired polypeptide. Cells are transformed with the first construct which integrates randomly into the target cellular genome. Exemplary target cells include, but are not limited to, mammalian cells and call lines (e.g., BHK, VERO, HT1080, 293, RD, COS-7, and CHO cells). The cells are grown in the presence of the appropriate substrate for the first selectable marker, for example, G418 if the first selectable marker encodes neomycin. Cells that survive selection in high concentration of the antibiotic have integrated the neomycin resistance gene at a high expression locus.
The cells having an integrated first construct are then used to make the cell lines of the invention by the following technique. The second construct is introduced into cells carrying an integrated copy of the first-construct. Homologous recombination between the two disabled DHFR genes results in a functional DHFR gene, as well as insertion of the desired gene encoding the functional polypeptide at the high expression locus (see, FIG. 7). Cell lines that survive selection with the appropriate toxin or antibiotic (e.g., methotrexate) have successfully undergone homologous recombination.
The second step can be repeated with any number of variations in the desired gene encoding the functional polypeptide (i.e. transgene) in order to obtain high level expression of the polypeptide of interest. This technique will now be described in greater detail with reference to the Figures.
The transfection system described herein is useful to express any polypeptide of interest. As used herein, the term "transgene" refers to exogenous DNA inserted in the genome of a mammalian cell whose expression at an elevated level in the cell is desired. The transgene employed in this invention encodes a functional polypeptide, which is an amino acid sequence that possesses a biological activity or an amino acid sequence that is a precursor of a protein having a biological activity. The transgene will generally encode a native or recombinant protein, although the expression of other polypeptides, such as epitopes or other immunologically active polypeptides, arc contemplated within the scope of this invention. Examples of proteins that can be expressed using the method of this invention are hormones; cytokines, such as growth factors; enzymes; receptors; oncogenes; polypeptide vaccines, viral proteins, and structural and secretory proteins. In one embodiment, the expression vectors contain sequences encoding CAB2, described above. These sequences have been further modified such that aberrant splicing is corrected, see Examples. In another embodiments, the expression vectors contain sequences encoding viral polypeptides, particular derived from HIV. These viral polypeptides include, but are not limited to, envelope (Env) polypeptides (e.g., gp120, gp160, gp140, gp41, and monomers or multimers thereof), Gag polypeptides (e.g., Gag, Gag-protease, Gag-polymerase), rev, tat, etc. In addition, the expression vectors can include transgenes encoding for synthetic HIV polypeptides, for example constructs described in the Examples.
The transgene employed in the constructs of the invention can be cloned sequences that retain intronic regions. If the exonic structure of the gene is known, the coding exons can be inserted in the constructs.
Expression of the polypeptide of interest can be directed by a promoter homologous to the polypeptide coding sequences (for example, human glucose-6-phosphate dehydrogenase under the control of its own transcription promoter sequences). Further, other homologous or heterologous expression control elements (e.g., affecting transcription, translation, or post-translational events) may be used.
It should be understood that expression of the transgene in the mammalian cells of the invention can be stable or transient. Even transient expression at a higher than normal level is useful for functional studies in the cells or for the production and recovery of proteins of interest.
Selectable Markers
Any suitable sequence encoding for a selectable marker can be used as the first or second markers in the compositions and methods described herein. Typically, the selectable marker genes employed in this invention can be obtained from readily available sources.
In one embodiment of the invention (depicted in the accompanying Figures), the first selectable marker encodes a gene which confers resistance to antibiotics. For example, in one aspect, the first selectable marker comprises a neomycin (neo) resistance gene. In preferred embodiments, mutant neo genes, designated neo*, can be used to establish a high expression locus in chromosomal DNA of the mammalian calls. (see, e.g., Yanofsky et al. 9(1990) PNAS USA 87:3435-39). The neomycin resistance gene of transposon Tn5 encodes for neomycin phosphotransferase II, which confers resistance to various antibiotics, including G418 and kanamycin. A single base substitution significantly impairs the function of the enzyme. Neo resistance genes used as selectable markers can be identified by restriction enzyme digestion, because base change results in the loss of an XhoII restriction site. See also Blazquez et al., (Molecular Microbiology, (2991) 5(6):1511-1518) for techniques for creating mutant neo genes for use in this invention. The optimum amount of substrate (e.g., G418) needed for selection can be individually determined for each cell line. Other similar selectable markers include, but are not limited to, those listed below.
Temperature-sensitive selectable markers can also be employed in the practice of the invention. For example, temperature-sensitive neo will be nearly wild type in function at non-stringent temperature and have low activity at stringent temperature. After electroporation and initial selection of pneo*ts-dhfr*1, insertion can be performed using G418 at non-stringent temperature, After colonies begin to grow, stringent temperature can be used to kill off colonies carrying low expression insertions.
As described above, the second selectable marker is included to enhance expression. In the expression vectors, the second selectable marker is typically an amplifiable marker, for example, DHFR. DHFR is necessary for purine biosynthesis. In the absence of erogenous purines, this enzyme is required for growth. Methotrexate is a potent competitive inhibitor of DHFR, so increasing methotrexate concentration selects for cells that express increased levels of DHFR. Extremely high levels of expression of the transfected normal DHFR gene are needed for selection in cell lines with high endogenous DHFR levels. To increase expression to even, higher levels, the transgene can be amplified by standard DHFR amplification methods.
For the constructs used in the transfection methods, the second selectable marker is found on both the first and second constructs of the claimed transfection system. However, these copies of the second selectable marker are disabled by mutations in different regions. These mutations are such that homologous recombination between the two mutated copies will rescue function of the gene. In one embodiment, the second (disabled) selectable marker encodes for DHFR.
The disabling mutations to the second selectable marker of the first and second constructs can be introduced by a variety of methods, for example, the DHFR gene or other marker gene can be disabled by the deletion of nucleotides in the native gene sequence or by substitution of nucleotides. Bullerjahn et al. (J. of Bio Chem. (1992) 267:864-870) provide an overview of how the activity of dihydrofolate reductase (DMM) enzyme is affected by residue deletions and/or substitutions. They describe deletions in critical areas (achieved by expressing an altered gene), which result in reduction of activity with complete loss of activity following deletion of 6 amino acids. In addition, a mutant DHFR gene is available that encodes an enzyme resistant to methotrexate (Simonsen et al., (1983) PNAS USA 80:2495-2499).
In some instances, it may also be desirable to amplify the gene encoding the second selectable marker (e.g., DHFR). This can be accomplished, for example, by increasing the concentration of substrate (e.g., methotrexate). Thus, cells harboring copies of the DHFR gene, multiplied many times, can be selected by sequential increases in the concentration of methotrexate to high levels. This technique also makes it possible to identify stable transformants. That is, whereas this phenotype is frequently unstable and is lost after several cell cycles in the absence of selective pressure, the unstable configuration of DHFR genes becomes stable under continuous selective pressure. Stably amplified cells contain the amplified DHFR genes within their chromosomes.
It will be understood that other selectable markers, which permit isolation of stable transfectants, can be employed in this invention as either first or second (disabled) markers. An example of another selectable marker is adenosine deaminase (ADA). A medium supplemented with thymidine, 9-β-D-xylofuranosyl adenine (Xyl-A), and 2′-deoxycoformycin (dCF) is employed. Xyl-A can be converted to Xyl-ATP and incorporated into nucleic acids, resulting in cell death. Xyl-A is detoxified to its inosine derivative by ADA. dCF is a transition state analogue inhibitor of ADA, and is needed to inactivate ADA endogenous to the parental cell type. As the level of endogenous ADA varies with cell type, the appropriate concentration of dCP for selection will vary as well. Kaufman et al., (1986) PNAS USA, 83:3136-3140. ADA-deficient CHO cells are also available.
Another suitable selectable marker for use in the invention is thymidine kinase (TK). In forward selection (TK− to TX+), complete medium is supplemented with hypoxanthine, aminopterin, thymidine, and glycine (HAT medium). In reverse selection (TK+ to TX−), complete medium is supplemented with 5-bromodeoxyuridine (BrdU). Under normal growth conditions, cells do not need thymidine kinase, because the usual means for synthesizing dTDP is through dCDP. Addition of BrdU to the medium will kill Tk+ cells, as BrdU is phosphorylated by TX and then incorporated into DNA. Selection of TK+ cells in HAT medium is primarily due to the presence of aminopterin, which blocks the formation of dTDP from dCDP. Cells, therefore, need to synthesize dTTP from thymidine, a pathway that requires TK. Thymidine kinase is widely used in mammalian cell culture because both forward and reverse selection conditions exist. Like ADA and DHFR, most mammalian cell lines express TK, removing the possibility of using the marker in those lines unless BrdU is used to select a TK− mutant. See Littlefield et al., (1964)Science 145:709-710.
An example of another suitable dominant selectable marker for use in the invention is xanthine-guanine phosphoribosyltransferase (XGPRT, gpt). Medium containing dialyzed fetal calf serum and xanthine, hypoxanthine, thymidine, aminopterin, mycophenolic acid, and L-glutamine can be employed. Aminopterin and mycophenolic acid both block the de novo pathway for synthesis of GMP. Expression of XGPRT allows cells to produce GMP from xanthine, allowing growth on medium that contains xanthine, but not guanine. XGPRT is a bacterial enzyme that does not have a mammalian homolog, allowing XGPRT to function as a dominant selectable marker in mammalian cells. The amount of mycophenolic acid necessary for selection varies with cell type and can be determined by titration in the absence and presence of guanine. See Mulligan at al., (1981) PNAS USA 78:2072-2076.
The selectable marker hygromycin-B-phosphotransferase (HPH) can also be employed. Complete medium in supplemented with hygromycin-B. Hygromycin-B is an aminocyclitol that inhibits protein synthesis by disrupting translocation and promoting mistranslation. The HPH gene has been used in mammalian systems, and vectors that efficiently express the gene are available. See Gritz et al., (1983)Gene 25:179-188; and Palmer et al., (1987) PNAS USA 84:1055-1059.
Another useful marker is chloramphenicol resistance, Resistance is mediated by chloramphenicol acetyltransferase (CAT), which inactivates chloramphenicol by converting it into mono- and bi-acetylated derivatives. These derivatives can be detected by thin layer chromatography. This enzyme is expressed in mammalian cells and is easily detected because it does not naturally occur in mammalian cells. The gene can be obtained from a derivative of PBR322 carrying transposon Tn9 by cleavage with suitable enzymes. It can be introduced into vector pSV2, yielding plasmid pSV2-cat (ATCC Accession No. 37155) a plasmid expresses the CAT gene from the early promoter of SV40.
Gossen et al., Science (1995) 268:1766-1769, describe fusion of a tetracycline resistance gene repressor to a viral transcription activation domain in order to induce rapid, greatly amplified gene expression in the presence of tetracycline. It is a modification of a preexisting system in which low levels of tetracycline prevented gene expression. The gene that codes for the tetracycline resistance gene repressor was mutagenized and a mutant fusion protein was created that depended on tetracycline for activation was identified. The construct can provide an on/off switch for high expression of a gene.
Another suitable marker is adeninephosphoribosyl transferase (APRT). The enzyme APRT, another enzyme of the purine salvage pathway, catalyzes the conversion of adenine to AMP. APRT positive cells can be selectable in a medium containing, for example, the glutamine analogue azaserine, which prevents de novo synthesis of purines. APRT-negative cells cannot be grown in a medium containing azaserine and adenine, and can be selected by treatment with 2,6-diaminopurine. This compound is toxic for normal cells, but APRT-negative cells survive because they do not incorporate it.
The expression of the selectable marker coding sequences can be placed under the control of, for example, promoter sequences derived from CMV, RSV, polyA′-BGH, SV40 or the like, and may include other expression control elements as well (e.g., sequences affecting transcription, translation or post-translation modifications).
Regulatory Sequences
In addition to selectable markers and transgenes, the constructs described herein may contain suitable regulatory elements. Regulatory elements (or control elements) are selected for use in the host cell of interest, for example, selectable markers may be included to allow propagation in microorganisms, (e.g., f1 origin of replication and ampicillin resistance encoding sequences). Such regulatory elements include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3′ to the translation stop codon), sequences for optimization of initiation of translation (located 5′ to the coding sequence), translation termination sequences, secretion signal sequences, and sequences that direct post-translational modification (e.g., glycosylation sites). Transcription promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters.
The selectable markers each be sandwiched between the promoter and a polyadenylation site, such as BGH polyA or SV40 polyA as shown in the Figures. A chimeric mRNA is transcribed from the promoters and stabilized by the polyadenylation signals located 3′ to the coding regions. During construction of the mammalian transcription unit, any bacterial promoters in the plasmids can be replaced with transcriptional regulatory sequences that are active in animal cells. Two such sequences are shown in the Figures, the CMV promoter and SV40 early promoter. Non-limiting examples of other promoters that can be employed are RSV and HSV-TK.
Ordinarily, gene expression will be constitutive, although regulatable promoters can be employed. Examples of suitable regulatable promoters are Tet, ecdysone and lac repressor sequences. Gene expression can also be controlled by transcription-regulation using heat, light, or metals, such as by the use of metallothionine genes or heat shock genes.
Exemplary "Backbone" Vectors
The above-described components of the transfection system of the present invention can be incorporated into a number of suitable backbone vectors to facilitate manipulation of the expression vectors and constructs. For example, incorporation of the components into a vector containing means that allow replication in a microorganism greatly facilitates propagation and isolation of the constructs (i.e., creating shuttle vectors). Exemplary backbone vectors include, but are not limited to, the following: pCMV6a and pUC19 (Example 1).
A variety of such backbone vectors are available for appropriate host systems. These systems include, but are not limited to, the following: baculovirus {Reilly, P. R., et al., B
Introduction of Expression Vectors and Constructs
The vectors and constructs described herein can be introduced into suitable host cells by a variety of methods. In particular, the mammalian cells that are modified by the process of this invention can be obtained by transfection or infection with a vector. Transfection can be carried out by well known techniques, such as calcium phosphate transfection, DEAE-dextran mediated transfection, electroporation, liposome mediated transfection, or microinjection (Ausubel, et al., supra). Exemplary methods are also described in the Examples. Transfection can be employed with DNA fragments that are unable to replicate, or with DNA that is not readily packaged in viral vectors, or where infection of the mammalian cells with viral DNA is to be avoided.
Vectors can be derived from viral genomes that yield virions or virus-like particles, which may or may not replicate independently as extrachromosomal elements. Virion particles containing the DNA for the high expression locus can be introduced into the host cells by infection. The viral vector may become integrated into the cellular genome. Examples of viral vectors for transformation of mammalian cells are SV40 vectors, and vectors based on papillomavirus, adenovirus, Epstein-Barr virus, vaccinia virus, and retroviruses, such as Rous sarcoma virus, or a mouse leukemia virus, such as Moloney murine leukemia virus. for mammalian cells, electroporation or viral-mediated introduction can be used. Further useful delivery systems and vehicles we described herein (see, for example, section 2.3.1.).
Appropriate transformation transfection conditions can be determined by those skilled in the art in view of the teachings herein.
The cells (e.g., host cells) employed in this invention include all mammalian cells, cell lines, and cell cultures. The cells can be derived from mammals, such as mice, rats, or other rodents, or from primates, such as humans or monkeys. Mammalian germ cells or somatic cells can be employed for this purpose. It will be understood that primary cell cultures or immortalized cells can be employed in carrying out the techniques of this invention.
The mammalian cells are typically grown in cell culture for transformation by the DNA. The cells can be fixed to a solid surface or grown in suspension in appropriate nutrient media.
In the present invention, it is preferred that permanent (i.e., stable) transformation occurs. This is accompanied by integration of the transforming DNA into the cellular genome by recombination. Insertional transformation, which results in the high expression locus being tagged, usually takes place by non-homologous recombination of the DNA construct containing the tag into a random genomic position, although it will be understood that homologous recombination can occur.
It will also be understood that no attempt has been made to determine whether the antibiotic resistance gene and the second selectable marker integrate in a single high expression locus in chromosomal DNA or whether there are multiple sites of integration to form multiple high expression loci in a given cell in any event, the mammalian cells of this invention contain at least one high expression locus.
The transformed cells obtained by the method of this invention can be employed for the preparation of continuous cell lines in which the cells are essentially immortal, or for the preparation of established cell lines that have the potential to be subcultured in vitro. Continuous cell lines and established cell lines can be obtained from a variety of organisms and organs, such as rodent embryos; primate kidneys; rodent and human tumors; and fibroblast, epithelial, or lymphoid cells. Cells exhibiting the highest levels of expression can be cloned, if desired.
Examples of established cell lines that can be transformed by the techniques of this invention are HeLa cells, CV-1 cells, CHO cells, 3T3 cells, L cells, and TC7 cells. All of these cells are sensitive to aminoglyconide antibiotics, such as G418, and are capable of harboring kanamycin or neomycin resistance genes for expression therein.
Moreover, while this invention has been described with reference to expression of a desired functional polypeptide, it will be understood that the polypeptide need not be the object of the invention. This invention is useful for the production of eucaryotic gene transcription and expression products in general, including RNA.
This invention will be more fully described in the following Examples.
pESN1dhfr was constructed as follows. PCR Primers D182E (M1) #1: 5′-GGGTCACGACGAGATCATCGCCGT-3′ (SEQ ID NO:1) and D182 (M1) #2: 3′-CGCATGCCCGACGGCGATGATCT-5′ (SEQ ID NO:2) were used to make pcDNA3M 1, which contains a mutated neo gene, from pcDNA3 (Invitrogen, Inc.), which contains wild-type neo. Throughout the application, where appropriate, it is noted where primers arc presented in 3′ to 5′ orientation. The PCR fragments were cloned into pCRII for TA cloning. The TA plasmids containing each separate mutation were digested with BssHII and SfuI to remove the region of the neo gene that contained the mutation at amino acid residue 182 (Asp instead of wild-type Glu). pcDNA3M1, containing the neo mutation at residue 182, was created by ligation of the M1 PCR fragment to pcDNA3 digested with BssHII and SfuI. The CMV IE promoter was obtained by digesting pcMV6c with BalI/EcoRV, isolating the fragment and ligating the fragment into pcDNA3M1 digested with the NruI and EcoRV. The resulting plasmid was terms pESN1. To add the Ad-DHFR gene to the plasmid, pESN1 was blunt-ended digested with Bst1107I. The plasmid pmCSR (Chiron Corp.) was digested with XhoI and BamHI and the Ad-DHFR cassette isolated. The cassette was then treated with Klenow, blunt-ended and ligated into the blunt-cut pESN1 to create pESN1.dhfr. pcDNA3M2 was created by ligation of the M2 PCR fragment into pcDNA3 digested with BssHII and SfuI. pcDNA3M3 was created by digestion of pcDNA
pESN2dhfr was constructed as follows. Primers D261N (M2) #1: 5′TCCCGCTCAGAAGAACTCGTTAAGAA-3′ (SEQ ID NO:3)and D261N (M2) #2: 3′-CTATCGCCTTCTAACGAGTTCT-5′ (SEQ ID NO:4) were used to make pcDNA3M2, which contains a mutation in the neo gene at amino acid residue 261 (Asn instead of wild-type Asp). The PCR fragments were cloned into pCRII for TA cloning. The TA plasmids containing each separate mutation were digested with BssHII and SfuI to remove the region of the neo gene that contained the mutation at amino acid residue 182 (Asp instead of wild-type Glu). pcDNA3M2, containing the nco mutation at residue 261, was created by ligation of the M2 PCR fragment to pcDNA3 digested with BssHII and SfuI. The CMV IE promoter was obtained by digesting pcMV6c with BalI/EcoRV, isolating the fragment and ligating the fragment into pcDNA3M2 digested with the NruI and EcoRV. The resulting plasmid was terms pESN2. To add the Ad-DHFR gene to the plasmid, pESN2 was blunt-ended digested with Bst1107I. The plasmid pmCSR (Chiron Corp.) was digested with XhoI and BamHI and the Ad-DHFR cassette isolated. The cassette was then treated with Klenow, blunt-ended and ligated into the blunt-cut pENS1 to create pESN2.dhfr.
pESN3dhfr was constructed as follows. pcDNA3M3 was created by digesting pcDNA3M1 and pcDNA3M2 with SmaI and RsrII. The small fragment from pcDNA3M1 was then ligated to the large fragment of pcDNA3M2 to create pcDNA3M3, which contains mutations at residues 182 and 261 of the neo gene. pESN3.dhfr was created as described above by inserting the CMV IE promoter and DHFR genes.
pneo*dhfr*1 and pneo*dhfr*2 were created as follows. A murine DHFR gene was amplified from pSV2 using primers that create BstBI and StuI ends. The wild type Neor gene was removed from pcDNA3 and replaced with the wild-type murine DHFR gene to create pcDNA3dhfr. This plasmid was then used in PCR to create mutant DHFR genes. For pneo*dhfr*l, two adjacent codons in the 5′ region of dhfr (using pSV2dhfr) are replaced with 2 stop codons thereby forming a 5′-mutated DHFR gene. For pneo*dhfr*2, two adjacent codons in the 3′ region are replaced with stop codons. The mutations were introduced by PCR amplification of pcDNA3dhfr using the primers shown in Table 1:
dhfr primer
Mutations in the 5′ end of the dhfr gene were accomplished using primers #5, #6, #7 and #8. Mutations in the 3′ end were accomplished using primers #6, #8, #9 and #10 These new vectors were referred to as pXdhfr*1 and pXdhfr*2, where X refers to any transgene. Mutant forms of the neo resistance gene from pESN1, pESN2 or pESN3 are then inserted into pdhfr*1 or pdhfr*2 to create pneo*dhfr*1 and pneo*dhfr*2. These constructs can be used, as described below, to create a high expression site in a host cell.
pdhfr3′del was constructed as follows. pcDNA3DHFR3′ was digested with KpnI/EcoRV. Secreted alkaline protease (SeAP) was obtained by digesting pSeAP-Basic (Clontech) with ClaI, followed by Klenow treatment and digestion with KpnI. The fragment was then ligated into pcDNA3DHFR3′ under blunt/sticky conditions. pXdhfr3′del refers to this plasmid containing any transgene "X".
pneo*dhfr5′del was constructed as follows. Plasmid pFC55 (Chiron Corp), which contains the same neo resistance gene from pcDNA3, was digested with BssHII and SfuI to insert the neo mutation fragments obtained as described above. The BssHII/SfuI M1, M2 or M3 neo fragments were ligated into pFC55 to create pRSVM1neo, pRSVM2neo or pRSVM3neo, respectively. pCDNA3dhfr5′ (courtesy F. Randazzo) was digested with NruI/KpnI to remove the CMV promoter upstream of the polylinker region. pRSVM1neo was digested with NruI/KpnI and the resulting fragment inserted into pcDNA3dhfr5′, creating pRSVM1neodhfr5′. The resulting plasmid was digested with BssHII and KpnI to insert either RSVM2neo or RSVM3neo fragments.
pCMVKm2: The pCMVKm2 vector was derived from pCMV6a (Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986) and comprises a kanamycin selectable marker, a ColEl origin of replication, a CMV promoter enhancer and Intron A, followed by an insertion site for the synthetic sequences described below followed by a polyadenylation signal derived from bovine growth hormone—the pCMVKm2 vector differs from the pCMV-link vector only in that a polylinker site was inserted into pCMVKm2 to generate pCMV-link (polylinker at positions 1646 to 1697); pESN2dhfr and pCMVPLEdhfr, for expression in Chinese Hamster Ovary (CHO) cells; and, pAcC13, a shuttle vector for use in the Baculovirus expression system (pAcC13, was derived from pAcC12 which was described by Munemitsu S., et al., Mol Cell Biol. 10(1 1):5977-5982, 1990).
pCMV-link contains the CMV promoter (CMV IE ENH/PRO), bovine growth hormone terminator (BGH pA), kanamycin selectable marker (kan), and a ColEI origin of replication (ColEI ori). A polycloning site is also present following the CMV promoter sequences.
A restriction map for vectors pESNdhfr, pESN1dhfr, pESN2dhfr, pESN3dhfr is presented in FIG. 2. In the figure, the CMV promoter (pCMV, hCMVIE), bovine growth hormone terminator (BGHpA), SV40 origin of replication (SV40Ori), neomycin selectable marker (Nco), SV40 polyA (SV4OpA), Adenovirus 2 late promoter (Ad2VLP), and the murine dhfr gene (mu dhfr) are indicated. A polycloning site is also indicated in the figure following the CMV promoter sequences.
Briefly, construction of pCMVPLEdhfr was as follows. To construct a DHFR cassette, the EMCV IRES (internal ribosome entry site) leader was PCR-amplified from pCite-4a+(Novagen, Inc., Milwaukee, Wis.) and inserted into pET-23d (Novagen, Inc., Milwaukee, Wis.) as an Xba-Nco fragment to give pET-EMCV. The dhfr gene was PCR-amplified from pESN2dhfr to give a product with a Gly-Gly-Gly-Ser spacer in place of the translation stop codon and inserted as an Nco-BamH1 fragment to give pET-E-DHFR. Next, the attenuated neo gene was PCR amplified from a pSV2Neo (Clontech, Palo Alto, Calif.) derivative and inserted into the unique BamH1 site of pET-E-DHFR to give pET-E-DHFR/Neo(m2). Finally the bovine growth hormone terminator from pCDNA3 (Invitrogen, Inc., Carlsbad, Calif.) was inserted downstream of the neo gene to give pET-E-DHFR/Neo(m2)BGHt. The EMCV-dhfr/neo selectable marker cassette fragment was prepared by cleavage of pET-E-DHFR/Neo(m2)BGHt.
The CMV enhancer/promoter plus Intron A was transferred from pCMV6a (Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986) as a HindIII-Sa/l fragment into pUC 19 (New England Biolabs, Inc., Beverly, Mass.). The vector backbone of pUC 19 was deleted from the Ndel to the Sap1 sites. The above described DHFR cassette was added to the construct such that the EMCV IRES followed the CMV promoter. The vector also contained an Amp1 gene and an SV40 origin of replication.
pCMVKm2 vectors containing synthetic Gag expression cassettes have been designated as follows: pCMVKm2.GagMod.SF2, pCMVKm2.GagprotMod.SF2, and pCMVKm2.GagpolMod.SF2, as described in co-pending application Atty Docket Number 1621.002.
CAB constructs: All of the constructs described were made and cloned into pAcC13 in the following manner. Since the CAB2 portion of all constructs was generated by PCR all were sequenced confirmed by the ABI system of dye terminator sequence analysis. Initially all constructs were cloned into the intermediate cloning vectors PCRII by INVITROGEN or pBluescript SK+ by Stratagene. This was done for ease of sequencing and further subcloning.
All CAB2 constructs were generated in two parts. First the MCP portion of the CAB2 molecule from the KpnI site at the 5′ end to the HindIII site at amino acid 321 was generated and cloned into PCRII and analyzed to confirm correct sequence. This portion of CAB2 was then cloned into pBluescript SK+. Then into this SK+construct the remaining portion of CAB2, from the HindIII site at amino acid 321 to the 5′ end EcoRI site, including the various 3′ end tails was cloned. The primers used to generate the HindIII 5′ and 3′ ends were: CAB2H3-1 CAGAAAGCTTTCTTCACATTTGTACGTTATTAC (SEQ ID NO:11) DAFTH5-1 CAGAAAGCTTTGTGAAAATTCCTGGCGAGAAGGAC (SEQ ID NO:12)
The original HindIII to EcoRI PCR products containing the Short Heparin consensus, the TFPI heparin consensus, and the GPI tail were initially cloned into PCRII for sequence analysis. The original HindIII to EcoRI PCR products containing the Long Heparin consensus, the two fibronectin tails, and the control sequence with no tail were cloned into pBluescript SK+for sequence analysis. After combining the two halves to make the complete CAB2 in SK+for each of the variants the KpnI to EcoRI fragments were then subcloned into pAcCI3. All were expressed in insect cells using the Baculovirus system.
In addition to insect cell expression some of these constructs were generated for expression in mammalian systems. They include CAB2 with the Long Heparin Consensus sequence, the TFPI Heparin binding tail, and the control with no targeting sequence. All three of these CAB variants were again generated with PCR in order to change the 5′ and 3′ restriction sites required for cloning into mammalian system vectors. All variants were generated with 5′ end Not I and 3′ end Xba I sites and cloned into the intermediate vector PCRII for sequence confirmation. The PCR primers used to generate Not I to Xba I clones were identical to those used to generate the KpnI to EcoRI fragments except for the change in restriction site. Confirmed variants were then cloned into pGEM9Z (Promega). These were then transferred to a mammalian vector for expression.
Cab2 is a chimeric, 110 kDa protein that combines features of membrane cofactor protein (MCP) and decay accelerating factor (DAF) to inhibit complement activation. Higgins et al., supra. Initially, a CHO cell line (N2107) was established after transfection of a pcDNA3 vector (Invitrogen) carrying a sequence encoding CAB2.1. However, the productivity of this cell line was low and, further, declined over time.
Analysis of the polypeptides present in the supernatant of the N2107 cell line revealed a 42 kDa protein having unexplained CAB2.1-related sequence. Poly (A+) mRNA was isolated from cell pellets of cells transiently transfected with CAB2-containing plasmids using Fast Track (InVitrogen). RT-PCR analysis was conducted as described in the Perkin-Elmer RT-PCR kit using primers which flank the coding region. The predominant PCR product encoded a predicted protein of 42 kDa. Also present were several intermediate PCR products and a small amount of full-length (110 kDa) PCR product. Thus, RT-PCR revealed that the 42 kDa contaminant was caused by aberrant mRNA splicing between the chimeric domains of MCP and DAF in CAB2.1.
Aberrant splicing was corrected by overlapping PCR to produce splice-corrected sequences of CAB2.1, CAB4.2 and CAB4.3, by removing donor and acceptor sites for RNA splicing which gave alternate protein side processed product from mammalian cell in such a way as to preserve the amino acid so that it no longer resembled the consensus splicing. The changes were as follows:
Original Codons
New Codons
Donor Sites:
amino acids 47-48
aa 152 153 154 155
Acceptor Sites:
amino acids 372 373 374
(Tyr-Phe-Pro SEQ ID
aa 459 460 461 462
463 464
(Leu Ile Ser Gly Ser Ser
The changes were generated using overlapping PCR with the following primers. To change the Donor site starting at amino acid 47:
To change the Donor site starting at amino acid 152:
To change the Acceptor site starting at amino acid 372:
To Change the Acceptor site starting at amino acid 459:
The 5′ Kpn primer used was:
The 3′ XbaI primer used was;
Also used were the Hind III 5′ and 3′ primers named above.
Splice site deleted constructs included the complete CAB2, CAB2 with the TFPI Heparin binding tail, CAB2 with the partial and complete deletion of the Serine Threonine region to amino acid 570 and 561 respectively, and complete deletion of the serine threonine region with addition of the TFPI heparin consensus sequence.
The primers used for generating the Serine Threonine deletions at the 3′ end of the molecule were:
The constructs were made in two parts in the intermediate vector pBluescript SK+as was done with the original CAB variants. All sequences were confirmed in SK+before completion of the final constructs in mammalian expression vectors. mRNA analyzed by RT-PCR and by Northern blotting showed no aberrant splicing and bands of the correct size.
Splice-corrected CAB2.1 and CAB4.2 were transiently transfected into COS-7 cells using the LipofectAMINE reagent (BRL) for two hours. RT-PCR showed only the correct full-length PCR product (110 kDa). Levels of CAB protein expressed in these cells are shown in Table 2.
Expression Plasmid
CAB 2 conc. (ng/mL)
The following four transfection protocols of plasmids pESN1.dhfr.CAB2.1; pESN3.dhfr.CAB2.1, pESN1.dhrf.CAB42. or pESN3.dhfr.CAB4.2 were conducted: (1) electroporation; (2) calcium phosphate (CaPo4) (Life Technologies, Gaithersburg, Md.); (3) LipofectAMINE (Life Technologies, Gaithersburg, Md.); and (4) TransIT LT-1 reagent (PanVera Corp., Madison, Wis.). Transfections were conducted according to the manufacturer's instructions for all methods except electroporation. Approximately 3�106 cells were used per electroporation, and 50-80% confluent cells (5-8�105 cells) for the three other transfection methods.
Electroporation was conducted as follows. CHO-DHFR− cells were tyrpsinized with STV and media with 10% FBS was added to quench the reaction. Cells were washed twice with DMEM (with L-glutamine) and resuspended to approximately 6�106 cells/mL. 500 μl of cells was added to a sterile cuvette and between about 1-30 μg of DNA added. The cells were electroporated at 330 volts and 975 μFarads capacitance. The cells were plated into 15 mls of α-MEM/nonselective media (containing G418) in a 100 mm dish, incubated for 18-24 hours at 37� C. Media was then replaced with α-MEM selective medium for DHFR and the cells grown until colonies formed. Cells are replated (T75 flasks, 96 well plates or 100 mm dish) and cultured in 20 nM methotrexate for 2-4 weeks at which point the concentration of methotrexate was raised to 40 nM methotrexate.
Approximately 10 μg of plasmid DNA was used per transfection. Transfected cells were COS (UCSF) or DG44 DHFR- CHO cells (Chiron Corp). After transfection, the cells were allowed to recover in non-selective media (500 mL of non-selective media contains 431.5 mL of α-MEM, 50 mL FBS, non-dialyzed, 5 mL penicillin-streptomycin, 0.5 mL gentamicin, 10 mL glutamine, 1 mL thymidine, 1 mL adenosine and 1 mL deoxyadenosine) for 1-2 days. The cells from one transfection were trypsinized as described above, diluted into 20 mL of selective medium (500 mL of selective media contains 434.5 α-MEM, 10 mL glutamine, 0.5 mL gentamicin, 5 mL penicillin-streptomycin, and 50 mls FBS non-dialyzed) and plated into one 96-well culture plate in appropriate selection media, either 250 μg G418 (Gibco-BRL), α-MEM without nucleosides, 10% FBS (DHFR selection) or both media. Cells were maintained in the selective media for approximately 2-3 weeks, at which time untransfected or transiently transfected cells were dead. Surviving colonies were trypsinized and replaced into 24-well culture plates containing 1 mL of selective media.
Growth-positive wells were tested for expression of CAB2.1 or CAB4.2 using ELISA. CAB2.1 or CAB4.2 expressing mini-pools were scaled-up for DHFR selection and amplification with methotrexate (MTX). Results obtained indicated that CaPO4 was the most effective in obtaining stable expression of the trans gene.
The expression of CAB2.1 or CAB4.2 in DHFR- CHO cells is shown in Table 3.
(in pESN3dhfr)
(in pESN3dhfr)
No. of transfections
No. wells screened
No. selected in 24 wells/
range of expression
<0.50-4.10 μg/mL
0.035-5.50 μg/mL
No. amplified in T75 flasks/
range of expression
0.015-210 μg/mL
0.494-2.33 μg/mL
MTX amplification/
range of expression
50.0-106.0 μg/mL
20.0-75.0 μg/mL
Selected for cloning
not determined
In addition to CAB2, expression of uPAR and VEGF-D has also been achieved by calcium phosphate transfection of host cells with pESN2dhfr containing a transgene coding for uPAR or VEGF-D. Expression levels between about 250 mg to about 1 mg/liter have been achieved.
Deposits of Strains Useful in Practicing the Invention
A deposit of biologically pure cultures of the following strains was made with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. The accession number indicated was assigned after successful viability testing, and the requisite fees were paid. The deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of viable cultures for a period of thirty (30) years from the date of deposit and at least five (5) years after the most recent request for the furnishing of a sample of the deposit by the depository. The organisms will be made available by the ATCC under the terms of the Budapest Treaty, which assures permanent and unrestricted availability of the cultures to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. �122 and the Commissioner's rules pursuant thereto (including 37 C.F.R. �1.12). Upon the granting of a patent, all restrictions on the availability to the public of the deposited cultures will be irrevocably removed.
These deposits are provided merely as convenience to those of skill in the art, and are not an admission that a deposit is required under 35 U.S.C. �112. The nucleic acid sequences of these plasmids, as well as the amino acid sequences of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with the description herein. A license may be required to make, use, or sell the deposited materials, and no such license is hereby granted.
ATCC Accession
Chiron Deposit Number
Date Sent to ATCC
pneo3dhfr5′del, CMCC #5090
November 30, 1999
pneoldhfr5′del, CMCC #5089
November 30, 1999
pESN2dhfr, CMCC #5086
November 30, 1999
pdfr3′del, CMCC #5093
November 30, 1999
pneo2dhfr5′del, CMCC #5088
November 30, 1999
pESN1dhfr, CMCC #5085
November 30, 1999
pESN3dhfr, CMCC #5087
November 30, 1999
The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. The invention to be protected herein, however, is not to be construed as limited to the particular forms disclosed, since these are to be regarded as illustrative rather than restrictive. Variations and changes may be made by one of ordinary skill in the art without departing from the spirit of the invention.
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|U.S. Classification
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|International Classification
|Cooperative Classification
|European Classification
|May 8, 2000
Owner name: CHIRON CORPORATION, CALIFORNIA
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