CA2265251A1 - Cell-based drug screens for regulators of gene expression - Google Patents
Cell-based drug screens for regulators of gene expression Download PDFInfo
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- CA2265251A1 CA2265251A1 CA002265251A CA2265251A CA2265251A1 CA 2265251 A1 CA2265251 A1 CA 2265251A1 CA 002265251 A CA002265251 A CA 002265251A CA 2265251 A CA2265251 A CA 2265251A CA 2265251 A1 CA2265251 A1 CA 2265251A1
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- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
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- C12N2510/00—Genetically modified cells
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Abstract
The invention provides methods and compositions for screening for pharmacological agents which regulate gene expression in mammals. An exemplary assay involves (a) contacting a mammalian cell comprising a knock-in mutant of a targeted native allele encoding a reporter of gene expression, as shown in the figure, wherein the expression of the reporter is under the control of the gene expression regulatory sequences of the native allele, with a candidate agent under conditions whereby but for the presence of the agent, the reporter is expressed at a first expression level; and (b) measuring the expression of the reporter to obtain a second expression level, wherein a difference between the first and second expressions levels indicates that the candidate agent modulates gene expression.
Description
1015202530W0 98/ 10059CA 02265251 1999-03-03PCT/U S97/ 15454Cell-Based Drug Screens for Regulators of Gene ExpressionWTRODUCTIONField of the InventionThe field of this invention is knock-in cell-based drug screens for regulators oftargeted gene expression.BackgroundTranscriptional regulation provides an ideal target for therapeutic intervention. Anumber of techniques are available for screening for drugs active at the level of genetranscription. Including in vitro assays such as binding assays (e.g. US Patent No.5,563,036) and RNA polymerase assays (e.g. US Patent No. 5,635,349), cell-based assayssuch as cotransfection assays and Northem-blot analyses.Inherent drawbacks of these in vitro and transfection-based assays include theirlimited recreation or modeling of the natural transcriptional process. On the other hand,Northern-blot analyses of natural transcripts are time and resource demanding and lessamendable to high-throughput drug development programs.SUMMARY OF THE INVENTIONThe invention provides methods and compositions for screening for agents whichregulate the level of targeted gene expression in a natural context. Such agents ï¬nd use inmodulating a wide variety of physiological manifestations of gene expression.The subject assays are cell-based and generally involve contacting a mammaliancell comprising a mutant of a native allele encoding a reporter of the targeted geneexpression, wherein the expression of the reporter is under the control of the native geneexpression regulatory sequences of the native targeted allele, with a candidate agent underconditions whereby but for the presence of the agent, the reporter is expressed at a firstexpression level; and, measuring the expression of the reporter to obtain a secondexpression level, wherein a difference between the first and second expression levelsindicates that the candidate agent modulates the expression of the targeted gene.The mutant generally results from replacement of a portion of the native allele with1015202530W0 98/10059CA 02265251 1999-03-03PCT/US97/15454a sequence encoding the reporter. For example, the cell may be a progeny of, a clone or,or genetically identical to a genetic knock-in cell made by homologous recombination ofthe native allele with a transgene comprising a sequence encoding the reporter ï¬anked byï¬anking sequences capable of effecting the homologous recombination of the transgenewith the native allele, a positive selectable marker positioned inside the ï¬anking sequencesand optionally, a negative selectable marker positioned outside the ï¬anking sequences.The cell may be a primary cell residing in or isolated from an animal transgenic in themutant or derive from a cultured cell line transgenic in the mutant.The invention also encompasses mammalian cells and mammals transgenic in amutant of a native allele encoding a reporter of gene expression, wherein the expression ofthe reporter is under the control of the gene expression regulatory sequences of the nativeallele, genetic knock in vectors for making such animals and cells and methods of makingand using such vectors, cells and animals.DESCRIPTION OF THE FIGURESFig. 1 shows the structures of the mouse ob gene and an exemplary âknock in" targetingvector.Fig. 2 shows the structure of a human LDLR-luciferase knock-in construct and the LDLRgene structure showing the vector insertion site.Fig. 3 shows the structure of a human CYP7-luciferase knock-in construct and the CYP7gene structure showing the vecter insertion site.DETAILED DESCRIPTION OF THE INVENTIONThe general assays involve contacting a cell having a reporter for targeted geneexpression with a candidate agent and monitoring reporter expression to determine if theagent has a speciï¬c effect on targeted gene expression. To accurately reï¬ect targeted geneexpression, the reporter gene is positioned within the targeted gene, resident at its nativelocus in the genome, such that effects of the mutation on transcription and translation atthe locus, as compared with the corresponding wild-type allele, are minimized. Generally,all the native gene sequences 5' and 3' to the native transcriptional start and stop sites areretained in the mutant allele, as are preferably all the native sequences 5' and 3' to thenative translational start and termination sites, where the targeted gene has translational1015202530W0 98/ 10059CA 02265251 1999-03-03PCT/US97/15454stop and strart sites. Hence, the transcriptional and any translational start and terminationsites of the native gene are preferably retained and used for the reporter. The reporter maybe encoded at the translational start site or in frame within the structural gene. Hence, thereporter may be expressed free or as a fusion product with N- and/or C-terminal sequencesof the targeted gene encoded polypeptide, e.g. the reporter gene may be an insertion orpartial replacement of coding sequence.Preferred reporter genes are readily expressed by the host cells and provideproducts that are readily detected and quantified. Exemplary reporter genes include'[3âgalactosidase, CAT, GFP and, preferably, luciferase. The mutated locus may alsocomprise a positive selection marker such as an antibiotic resistance gene, e.g. neomycin,residual from the initial construction of the mutation. Alternatively, such residualsequences may be lost or removed, e.g. using a LoxpâCRE recombination system. in thecourse of cell passage or animal reproduction.The assayed cells are clones of, genetically identical to, or, preferably progeny of agenetic knockâin cell made by homologous recombination, e.g. recombination of thenative targeted allele with a transgene comprising a sequence encoding the reporterï¬anked by ï¬anking sequences capable of effecting the homologous recombination of thetransgene with the native allele, a positive selectable marker positioned inside the ï¬ankingsequences and, optionally, a negative selectable marker positioned outside the ï¬ankingsequences (see Experimental Section, below). The cells used in the assays are preferablydifferentiated which refers to cells which demonstrate at least some cell-type specific geneexpression. Such cells are identified by any of cell-type specific gene expression (e.g. obgene expression), cell-type specific markers, or cell-type specific morphology. A widevariety of cell types are amenable to the methods. Exemplary cell types include ï¬uid (e.g.blood) borne cell types such as lymphocytes, macrophages, etc., and preferably,structurally associated cells types such as adipocytes, hepatocytes, muscle cells, neurons,etc. The cells may be mature cell types or precursor cells, such as preadipocytes, culturedES cells (see, e.g. Pedersen (1994) Reprod. Fertil. Dev., 6, 543-552), etc. The cells maybe primary cells isolated from an animal transgenic in the mutant or derive from a culturedcell line transgenic in the mutant. Primary cells are preferably rodent, more preferablymouse or rat cells and are preferably dissociated and substantially isolated from other celltypes prior to use. Cell lines may be made by gene targeting into established cell lines1015202530W0 98/ 10059CA 02265251 1999-03-03PCT/US97/15454such as 3T3-based cells (e.g. -L1 and -F442A), derived from the transgenic animaldisclosed herein and immortalized, etc.The targeted gene generally encodes transcript which effects, directly or through atranslation product, a cellular function. A wide variety of genes may be targeted.Exemplary genes suitable for targeting include genes encoding cell signaling proteins suchas leptin, receptor such as LDLR, growth factors such as TNFot, transcription factors suchas STAT6, mitochondrial proteins such as UCP-2, enzymes such as CDKs, etc., andnontranslated genes such as epsilon.An exemplary assay involves (a) contacting a mammalian cell (ex vivo for highthroughput applications) comprising a mutant of a native (i.e. otherwise naturally presentin the host cell or animal) allele encoding a reporter of gene expression, wherein theexpression of the reporter is under the control of the native gene expression regulatorysequences (preferably both transcription regulatory elements) of the native allele, with acandidate agent under conditions whereby but for the presence of the agent, the reporter isexpressed at a first expression level; and, (b) measuring the expression of the reporter toobtain a second expression level, wherein a difference between the ï¬rst and secondexpression levels indicates that the candidate agent modulates gene expression.EXAMPLES1. Targeted gene replacement of human cholesterol 7âoc hydroxylasea. Isolation of the human CYP7 genomic cloneA genomic library was constructed in the AGEM11 vector using HepG2 DNA thathad been partially digested with Sau3A restriction endonuclease. Recombinant phagecontaining the CYP7 gene were identified by hybridization to a radiolabeled CYP7 cDNAprobe. From these clones, a 14 kilobasepajr fragment containing sequences fromapproximately -10 kb to +3.6.kb (relative to transcriptional initiation) was isolated foradditional characterization and modification. See Fig. 3.b. Preparation of the CYP7:luciferase targeting constructThe 14 kb fragment of the CYP7 gene was modiï¬ed by siteâdirected mutagenesisin order to eliminate the authentic ATG codon used for translational initiation and tointroduce restriction sites at this position for the insertion of the luciferase reporter gene.The final targeting construct incorporates the coding sequences for ï¬reï¬y luciferase fused1015202530CA 02265251 1999-03-03W0 98/ 10059 PCTlUS97/l5454to the polyadenylation signal from the SV40 early gene, as well as the G418 resistancecartridge containing the Tn5 neomycin resistance gene under the transcriptional control ofthe HSV thymidine kinase promoter. This luciferase-neo cassette was inserted at position+57 in exon 1 of the CYP7 gene, positioning the reporter gene under the transcriptionalcontrol of the CYP7 promoter. The chimeric CYP7:1uciferase:neo gene was subsequentlycloned into the pBluescript-MC1-HSVTk plasmid to allow dual selection with G418 andgancyclovir (see Fig. 3).c. Isolation of CYP7 âknock-in" cell lines: transfection and selection of stably-transformed cell linesThe CYP7 targeting construct was introduced into HepG2 cells by electroporation.Following recovery, the cells were maintained in media supplemented 1 mg/ml G418 and2 pM gancyclovir for 3-4 weeks until isolated colonies were observed. These G418â-GancR cells were isolated and propagated as individual cultures for furthercharacterization.d. Identiï¬cation of gene targeting eventsIndividual clones were expanded into 24 well tissue culture plates, and genomicDNA prepared and analyzed by restriction digestion and Southern hybridization. Onecolony exhibited the pattern predicted for cells that have undergone homologousrecombination and targeted gene replacement, and this culture was expanded for furthercharacterization and use as a reporter cell line for CYP7 gene expression.2. Targeted gene replacement of mouse ob (leptin) genea. Transgenic primary adipocytesApproximately 20 ug of a pBluescript gene-targeting construct containing about 2kb of 5' ï¬anking sequence and about 7 kb of 3' ï¬anking sequence of the second exon ofthe ob gene was electroporated into D3 ES cells using a Bio-Rad gene pulser set at 25uF/350 V. The construct effects the replacement of the second exon with a cassettecontaining the luciferase reporter and the neomycin resistance genes (Fig. 1). Theconstruct also contained a thymidine kinase negative selection marker outside the cassette.After 9 days selection in 180 ug/ml G418 and 2 uM gancyclovir, drugâresistant cloneswere placed into 24-well plates and expanded in culture. Screening for correctly targetedclones was done by Southern analysis and suitable clones were injected into 3.5 day1015202530WO 98/10059CA 02265251 1999-03-03PCT/U S97/ 15454postcoital BALB/c blastocysts to generate chimeras. Heterozygote gerrnline transmissionsare identified by Southern analysis and bred as a stable strain. These are also intercrossedto yield homozygotes for the ob mutation.To isolate primary adipocytes for screening assays, epididymal fat tissue is excisedfrom two month old mice and prepared for cell culture by collagenase digestion asdescribed in Rolland et al. (1995) J Biol Chem 270, 1102-2206. After digestion, primaryadipocytes are isolated by filtration through 180 um sieves, also as described by Rolland etal. (supra).b. Transgenic cultured adipocyte cell linesThe pBluescript gene-targeting construct described above and shown in Figure 1 iselectroporated into 3T3-F442A cells using a Bio-Rad gene pulser set at 25 uF/350 V.After approximately 14 days of positive/negative selection in 180 ug/ml G418 and 2 uMgancyclovir, drug-resistant clones were placed into 6-well plates and expanded in culture.Screening for correctly targeted clones was done by Southern analysis.To obtain differentiated cells for screening assays, grow initial cultures of 50%confluent cells to 100% conï¬uence, then continue incubation 10-14 days to obtainmaximal differentiation.3. Targeted gene replacement of human LDL receptor genea. Targeting construct: Isolation of the human LDLR genomic cloneA genomic library was constructed in the GEM1 1 vector using HepG2 DNA thathad been partially digested with Sau3A restriction endonuclease. Recombinant phagecontaining the LDLR gene were identified by hybridization to a radiolabeled LDLR CDNAprobe. A 13.5 kilobasepair fragment containing sequences from approximately -4 kb to+9.5 kb (relative to transcriptional initiation) was isolated for preparation of the targetingconstruct. See Fig. 2.b. Targeting construct: Preparation of the LDLR:luciferase targeting constructThe 13.5 kb fragment of the LDLR gene was modified by site-directed mutagenesisin order to eliminate the authentic ATG codon used for translational initiation and tointroduce restriction sites at this position for the insertion of the luciferase reporter gene.The ï¬nal targeting construct incorporates the coding sequences for ï¬reï¬y luciferase fusedto the polyadenylation signal from the SV40 early gene, as well as the Tn5 neomycin1015202530CA 02265251 1999-03-03W0 98/10059 PCT/US97/15454resistance gene under the transcriptional control of the HSV thyrnidine kinase promoter.This luciferaseâneo cassette was inserted at the altered ATG site in exon 1 of the LDLRgene, positioning the reporter gene under the transcriptional control of the LDLRpromoter. The chimeric LDLR:luciferase:neo gene was subsequently cloned into thepBluescript-MCI-HSVTk plasmid to allow dual selection with G418 and gancyclovir.c. Isolation of LDLR "knock-in" cell lines: Transfection and selection ofstablyâtransformed cell linesThe LDLR targeting construct was introduced into HepG2 cells by electroporation.Following recovery, the cells were maintained in media supplemented 1 mg/ml G418 and2 uM gancyclovir for 3-4 weeks until individual colonies were observed. TheseG418R-GancR cells were isolated and propagated as individual cultures for furthercharacterization.d. Isolation of LDLR "knock-in" cell lines: Identification of gene targeting eventsIndividual clones were expanded into 24 well tissue culture plates, and genomicDNA was prepared and analyzed by restriction digestion and Southern hybridization. Oneclone exhibited the pattern predicted for cells that have undergone homologousrecombination. The Southern analysis also indicated that one of the two allele of LDLRgene has been replaced by the luciferase-neo cassette. This clone was expanded for furthercharacterization and use as a reporter cell line for LDLR gene expression.e. Characterization of the "knock-in cell lineTo determine whether the expression of luciferase gene is regulated as the sameway as the endogenous LDLR gene, RNA was prepared and analyzed by Northernhybridization. The results indicated that the luciferase mRNA was elevated when cellswere cultured in lipid-depleted media and was suppressed in the presence of25-hydroxycholesterol. The cholesterol-dependent regulation is the hallmark of LDLRgene expression. As indicated, the LDLR mRNA was regulated by in the presence orabsence of cholesterol, albeit in this case the LDLR mRNA level was reduced to half dueto the loss of one allele, which was replaced by the luciferase gene. The use of theluciferase knock-in cell line for LDLR gene expression was further validated by theluciferase activity, which was also regulated by cholesterol as predicted.4. Targeted gene replacement of human TNFoc gene.1015202530WO 98/10059CA 02265251 1999-03-03PCT/U S97/ 15454a. Isolation of the human TNFOL genomic cloneA genomic library is constructed in the AGEMII vector using Thpl DNA partiallydigested with Sau3A restriction endonuclease. Recombinant phage containing the TNFotgene are identiï¬ed by hybridization to a radiolabeled TNFot CDNA probe and from theseclones, a fragment encompassing the transcriptional and translational initiation sitesisolated for additional characterization and modification.b. Preparation of the TNFoc:luciferase targeting constructThe fragment of the TNFoc gene is modiï¬ed by site-directed mutagenesis in orderto eliminate the authentic ATG codon used for translational initiation and to introducerestriction sites at this position for the insertion of the luciferase reporter gene. The finaltargeting construct incorporates the coding sequences for ï¬reï¬y luciferase fused to thepolyadenylation signal from the SV40 early gene, as well as the G418 resistance cartridgecontaining the Tn5 neomycin resistance gene under the transcriptional control of the HSVthyrnidine kinase promoter. This luciferase-neo cassette is inserted in exon 1 of the TNFotgene, positioning the reporter gene under the transcriptional control of the TNFot promoter.The chimeric TN Foczluciferasezneo gene was subsequently cloned into the pBluescript-MC1âI-ISVTk plasmid to allow dual selection with G418 and gancyclovir.c. Isolation of TNFoc âknock-in" cell lines: transfection and selection of stably-transformed cell linesThe TNFOL targeting construct is introduced into Thpl cells by electroporation.Following recovery, the cells are maintained in media supplemented 1 mg/rnl G418 and 2pM gancyclovir for 3-4 weeks until isolated colonies are observed. These G418â-GancRcells are isolated and propagated as individual cultures for further characterization.(1. Identification of gene targeting eventsIndividual clones are expanded into 24 well tissue culture plates, and genomicDNA prepared and analyzed by restriction digestion and Southern hybridization. Coloniesexhibiting the pattern predicted for cells that have undergone homologous recombinationand targeted gene replacement are expanded for further characterization and use asreporter cell lines for TNFot gene expression.5. Targeted gene replacement of human E-selectin gene.a. Isolation of the human E-selectin genomic clone1015202530CA 02265251 1999-03-03W0 98/ 10059 PCT/US97/15454A genomic library is constructed in the AGEM11 vector using ECV304 endothelialcell line DNA partially digested with Sau3A restriction endonuclease. Recombinant phagecontaining the E-selectin gene are identiï¬ed by hybridization to a radiolabeled E-selectinCDNA probe and from these clones, a fragment encompassing the transcriptional andtranslational initiation sites isolated for additional characterization and modification.b. Preparation of the Eâselectin:1uciferase targeting constructThe fragment of the Eâselectin gene is modiï¬ed by site-directed mutagenesis inorder to eliminate the authentic ATG codon used for translational initiation and tointroduce restriction sites at this position for the insertion of the luciferase reporter gene.The ï¬nal targeting construct incorporates the coding sequences for ï¬reï¬y luciferase fusedto the polyadenylation signal from the SV40 early gene, as well as the G418 resistancecartridge containing the Tn5 neomycin resistance gene under the transcriptional control ofthe HSV thymidine kinase promoter. This luciferase-neo cassette is inserted in exon 1 ofthe E-selectin gene, positioning the reporter gene under the transcriptional control of theE-selectin promoter. The chimeric Eâselectin:luciferase:neo gene was subsequently clonedinto the pBluescript-MC 1-HSVTk plasmid to allow dual selection with G418 andgancyclovir.c. Isolation of E-selectin âknock-inâ cell lines: transfection and selection of stably-transformed cell linesThe E-selectin targeting construct is introduced into ECV304 cells byelectroporation. Following recovery, the cells are maintained in media supplemented 1mg/rnl G418 and 2 uM gancyclovir for 3-4 weeks until isolated colonies are observed.These G418â-Gameâ cells are isolated and propagated as individual cultures for furthercharacterization.(1. Identification of gene targeting eventsIndividual clones are expanded into 24 well tissue culture plates, and genomicDNA prepared and analyzed by restriction digestion and Southern hybridization. Coloniesexhibiting the pattern predicted for cells that have undergone homologous recombinationand targeted gene replacement are expanded for further characterization and use asreporter cell lines for E-selectin gene expression.6. Targeted gene replacement of human UCP-2 gene1015202530W0 98I10059CA 02265251 1999-03-03PCT/US97/15454a. Isolation of the human UCP-2 genomic cloneA genomic library is constructed in the /\GEM1l vector using RD(Rhabdomyosarcoma) cell line DNA partially digested with Sau3A restrictionendonuclease. Recombinant phage containing the UCP-2 gene are identified byhybridization to a radiolabeled UCP-2 cDNA probe and from these clones, a fragmentencompassing the transcriptional initiation site isolated for additional characterization andmodification.b. Preparation of the UCPâ2:luciferase targeting constructThe fragment of the UCP-2 gene is modified by site-directed mutagenesis in orderto eliminate the authentic ATG codon used for translational initiation and to introducerestriction sites at this position for the insertion of the luciferase reporter gene. The finaltargeting construct incorporates the coding sequences for fireï¬y luciferase fused to thepolyadenylation signal from the SV40 early gene, as well as the G418 resistance cartridgecontaining the Tn5 neomycin resistance gene under the transcriptional control of the HSVthyrnidine kinase promoter. This luciferase-neo cassette is inserted in exon 1 of the UCP-2gene, positioning the reporter gene under the transcriptional control of the UCP-2promoter. The chimeric UCP-2:luciferase:neo gene was subsequently cloned into thepBluescriptâMC l-HSVTk plasmid to allow dual selection with G418 and gancyclovir.c. Isolation of UCP-2 "knock-in" cell lines: transfection and selection of stably-transformed cell linesThe UCP-2 targeting construct is introduced into RD cells by electroporation.Following recovery, the cells are maintained in media supplemented 1 mg/ml G418 and 2uM gancyclovir for 3-4 weeks until isolated colonies are observed. These G418R-GancRcells are isolated and propagated as individual cultures for further characterization.d. Identification of Gene Targeting EventsIndividual clones are expanded into 24 well tissue culture plates, and genomicDNA prepared and analyzed by restriction digestion and Southern hybridization. Coloniesexhibiting the pattern predicted for cells that have undergone homologous recombinationand targeted gene replacement are expanded for further characterization and use asreporter cell lines for UCP-2 gene expression.7. Targeted gene replacement of mouse UCP-2 gene.101015202530WO 98/10059CA 02265251 1999-03-03PCT/U S97/ 15454a. Transgenic primary cellsApproximately 20 ug of a pBluescript gene-targeting construct containing about 2kb of 5' ï¬anking sequence and about 5 kb of 3' ï¬anking sequence of the first exon of themouse UCP-2 gene is electroporated into D3 ES cells using a Bio-Rad gene pulser set at25 uF/350 V. The construct effects the replacement of the first exon with a cassettecontaining the luciferase reporter and the neomycin resistance genes. The construct alsocontains a thyrnidine kinase negative selection marker outside the cassette. After 9 daysselection in 180 ug/ml G418 and 2 uM gancyclovir, drugâresistant clones are placed into24-well plates and expanded in culture. Screening for correctly targeted clones is done bySouthern analysis and suitable clones injected into 3.5 day postcoital BALB/c blastocyststo generate chimeras. Heterozygote germline transmissions are identified by Southernanalysis and bred as a stable strain. These are also intercrossed to yield homozygotes forthe UCP-2 mutation.Primary muscle and/or adipoctyes are used. For example, to isolate primaryadipocytes for screening assays, epididymal fat tissue is excised from two month old miceand prepared for cell culture by collagenase digestion as described in Rolland et al. (1995)J Biol Chem 270, 1102-2206. After digestion, primary adipocytes are isolated by ï¬ltrationthrough 180 um sieves.b. Transgenic cultured adipocyte cell linesThe pBluescript gene-targeting construct described above is electroporated into3T3-F442A cells using a Bio-Rad gene pulser set at 25 uF/350 V. After approximately 14days of positive/negative selection in 180 ug/ml G418 and 2 uM gancyclovir. drug-resistant clones were placed into 6âwell plates and expanded in culture. Screening forcorrectly targeted clones was done by Southern analysis.To obtain differentiated cells for screening assays, grow initial cultures of 50%conï¬uent cells to 100% conï¬uence, then continue incubation 10-14 days to obtainmaximal differentiation.8. Targeted gene replacement of mouse Ob receptor gene.a. Transgenic primary cellsApproximately 20 ug of a pBluescript gene-targeting construct containing about 2kb of 5' ï¬anking sequence and about 5 kb of 3' ï¬anking sequence of the first exon of the111015202530WO 98/10059CA 02265251 1999-03-03PCT/US97/15454Ob receptor gene is electroporated into D3 ES cells using a Bio-Rad gene pulser set at 25uF/350 V. The construct effects the replacement of the first exon with a cassettecontaining the luciferase reporter and the neomycin resistance genes. The construct alsocontains a thyrnidine kinase negative selection marker outside the cassette. After 9 daysselection in 180 ug/ml G418 and 2 uM gancyclovir, drug-resistant clones are placed into24-well plates and expanded in culture. Screening for correctly targeted clones is done bySouthern analysis and suitable clones injected into 3.5 day postcoital BALB/c blastocyststo generate chimeras. Heterozygote germline transmissions are identified by Southernanalysis and bred as a stable strain. These are also intercrossed to yield homozygotes forthe Ob receptor gene mutation. Several primary cell types may be used. For example, toisolate primary thypothalamic neurons for screening assays, hypothalamic tissue is excisedfrom two month old mice and âprepared for cell culture by collagenase digestion andprimary thypothalamic neurons are isolated.b. Transgenic cultured GT1 hypothalamic neuronal cell linesThe pBluescript gene-targeting construct described above is electroporated intoGT1 cells using a Bio-Rad gene pulser set at 25 uF/350 V. After approximately 14 days ofpositive/negative selection in 180 ug/ml G418 and 2 uM gancyclovir, drug-resistant cloneswere placed into 6-well plates and expanded in culture. Screening for correctly targetedclones was done by Southern analysis. To obtain differentiated cells for screening assays,grow initial cultures of 50% conï¬uent cells to 100% conï¬uence, then continue incubation10-14 days to obtain maximal differentiation.9. Targeted gene replacement of human 6 (epsilon) gene.a. Isolation of the human e genomic cloneA genomic library is constructed in the }tGEM1l vector using BJ AB cell line DNApartially digested with Sau3A restriction endonuclease. Recombinant phage containingthe 6 gene are identiï¬ed by hybridization to a radiolabeled 6 cDNA probe and from theseclones, a fragment encompassing the transcriptional initiation site isolated for additionalcharacterization and modiï¬cation.b. Preparation of the ezluciferase targeting constructThe fragment of the 6 gene is modified by site-directed mutagenesis in order tointroduce restriction sites at this position for the insertion of the luciferase reporter gene.121015202530W0 98/10059CA 02265251 1999-03-03PCT/US97/ 15454The final targeting construct incorporates the coding sequences for fireï¬y luciferase fusedto the polyadenylation signal from the SV40 early gene, as well as the G418 resistancecartridge containing the Tn5 neomycin resistance gene under the transcriptional control ofthe HSV thymidine kinase promoter. This luciferase-neo cassette is inserted in the 6 gene,positioning the reporter gene under the transcriptional control of the 6 promoter. Thechimeric e:luciferase:neo gene was subsequently cloned into the pB1uescriptâMCl-HSVTkplasmid to allow dual selection with G418 and gancyclovir.c. Isolation of e âknock-inâ cell lines: transfection and selection of stably-transformed celllinesThe e targeting construct is introduced into BJ AB cells by electroporation.Following recovery, the cells are maintained in media supplemented 1 mg/ml G418 and 2uM gancyclovir for 3-4 weeks until isolated colonies are observed. These G4l8RâGancRcells are isolated and propagated as individual cultures for further characterization.d. Identiï¬cation of Gene Targeting EventsIndividual clones are expanded into 24 well tissue culture plates, and genomicDNA prepared and analyzed by restriction digestion and Southern hybridization. Coloniesexhibiting the pattern predicted for cells that have undergone homologous recombinationand targeted gene replacement are expanded for further characterization and use asreporter cell lines for 6 gene expression.10. Targeted gene replacement of mouse STAT6 gene.a. Transgenic primary cellsApproximately 20 ug of a pBluescript gene-targeting construct containing about 5kb of 5' ï¬anking sequence and about 5 kb of 3' ï¬anking sequence of the first exon of theSTAT6 gene is electroporated into D3 ES cells using a Bio-Rad gene pulser set at 25uF/350 V. The construct effects the replacement of the first exon with a cassettecontaining the luciferase reporter and the neomycin resistance genes. The construct alsocontains a thymidine kinase negative selection marker outside the cassette. After 9 daysselection in 180 ug/ml G418 and 2 uM gancyclovir. drug-resistant clones are placed into24-well plates and expanded in culture. Screening for correctly targeted clones is done bySouthern analysis and suitable clones injected into 3.5 day postcoital BALB/c blastocyststo generate chimeras. Heterozygote germline transmissions are identified by Southern131015202530W0 98/ 10059CA 02265251 1999-03-03PCTIUS97/1 5454analysis and bred as a stable strain. These are also intercrossed to yield homozygotes forthe STAT6 gene mutation. Several primary cell types may be used for screening assays,e.g. primary B-lymphocytes isolated from peripheral blood.11. Targeted gene replacement of human EPAS-1 gene.a. Isolation of the human EPAS-1 genomic cloneA genomic library is constructed in the AGEM11 vector using ECV304 endothelialcell line DNA partially digested with Sau3A restriction endonuclease. Recombinant phagecontaining the EPAS-1 gene are identified by hybridization to a radiolabeled EPAS-1CDNA probe and from these clones, a fragment encompassing the transcriptional initiationsite isolated for additional characterization and modification.b. Preparation of the EPAS-1 :luciferase targeting constructThe fragment of the EPAS-1 gene is modified by site-directed mutagenesis in orderto eliminate the authentic ATG codon used for translational initiation and to introducerestriction sites at this position for the insertion of the luciferase reporter gene. The finaltargeting construct incorporates the coding sequences for fireï¬y luciferase fused to thepolyadenylation signal from the SV40 early gene, as well as the G418 resistance cartridgecontaining the Tn5 neomycin resistance gene under the transcriptional control of the HSVthymidine kinase promoter. This luciferase-neo cassette is inserted in exon 1 of the EPAS-I gene, positioning the reporter gene under the transcriptional control of the EPAS-1promoter. The chimeric EPAS-1 zluciferasezneo gene was subsequently cloned into thepBluescript-MCl-HSVTk plasmid to allow dual selection with G418 and gancyclovir.c. Isolation of EPAS-1 âknock-in" cell lines: transfection and selection of stably-transformed cell linesThe EPAS-1 targeting construct is introduced into ECV304 cells byelectroporation. Following recovery, the cells are maintained in media supplemented 1mg/ml G418 and 2 uM gancyclovir for 3-4 weeks until isolated colonies are observed.These G418â-GancR cells are isolated and propagated as individual cultures for furthercharacterization.d. Identification of gene targeting eventsIndividual clones are expanded into 24 well tissue culture plates, and genomicDNA prepared and analyzed by restriction digestion and Southern hybridization. Colonies141015202530WO 98110059CA 02265251 1999-03-03PCT/US97/15454exhibiting the pattern predicted for cells that have undergone homologous recombinationand targeted gene replacement are expanded for further characterization and use asreporter cell lines for EPAS-1 gene expression.12. Targeted gene replacement of human utrophin gene.a. Isolation of the human utrophin genomic cloneA genomic library is constructed in the ?tGEMll vector using RD(Rhabdomyosarcoma) cell line DNA partially digested with Sau3A restrictionendonuclease. Recombinant phage containing the utrophin gene are identified byhybridization to a radiolabeled utrophin cDNA probe and from these clones. a fragmentencompassing the transcriptional and translational initiation sites isolated for additionalcharacterization and modiï¬cation. See, e.g. Tinsley et al. (1996) Nature 384, 349-353.b. Preparation of the utrophin:luciferase targeting constructThe fragment of the utrophin gene is modified by siteâdirected mutagenesis inorder to eliminate the authentic ATG codon used for translational initiation and tointroduce restriction sites at this position for the insertion of the luciferase reporter gene.The ï¬nal targeting construct incorporates the coding sequences for fireï¬y luciferase fusedto the polyadenylation signal from the SV40 early gene, as well as the G418 resistancecartridge containing the Tn5 neomycin resistance gene under the transcriptional control ofthe HSV thymidine kinase promoter. This luciferase-neo cassette is inserted in exon 1 ofthe utrophin gene, positioning the reporter gene under the transcriptional control of theutrophin promoter. The chimeric utrophin:luciferase:neo gene was subsequently clonedinto the pBluescript-MC 1-HSVTk plasmid to allow dual selection with G418 andgancyclovir.c. Isolation of utrophin "knock-inâ cell lines: transfection and selection of stably-transformed cell linesThe utrophin targeting construct is introduced into RD cells by electroporation.Following recovery, the cells are maintained in media supplemented 1 mg/ml G418 and 2pM gancyclovir for 3-4 weeks until isolated colonies are observed. These G418â-GancRcells are isolated and propagated as individual cultures for further characterization.d. Identiï¬cation of gene targeting eventsIndividual clones are expanded into 24 well tissue culture plates. and genomic151015202530WO 98/10059CA 02265251 1999-03-03PCT/U S97/ 15454DNA prepared and analyzed by restriction digestion and Southern hybridization. Coloniesexhibiting the pattern predicted for cells that have undergone homologous recombinationand targeted gene replacement are expanded for further characterization and use asreporter cell lines for utrophin gene expression.13. Targeted gene replacement of African green monkey HNF-lot gene.a. Isolation of the African green monkey HNF-lot genomic cloneA genomic library is constructed in the AGEMII vector using CV-1 cell line DNApartially digested with Sau3A restriction endonuclease. Recombinant phage containingthe African green monkey HNF-lot gene are identified by hybridization to a radiolabeledmonkey HNFâ1oL cDNA probe and from these clones, a fragment encompassing thetranscriptional and translational initiation site isolated for additional characterization andmodification.b. Preparation of the HNF-loczluciferase targeting constructThe fragment of the HNF-lot gene is modiï¬ed by site-directed mutagenesis inorder to eliminate the authentic ATG codon used for translational initiation and tointroduce restriction sites at this position for the insertion of the luciferase reporter gene.The ï¬nal targeting construct incorporates the coding sequences for ï¬reï¬y luciferase fusedto the polyadenylation signal from the SV40 early gene, as well as the G418 resistancecartridge containing the Tn5 neomycin resistance gene under the transcriptional control ofthe HSV thyrnidine kinase promoter. This luciferase-neo cassette is inserted in exon 1 ofthe HNF-lot gene, positioning the reporter gene under the transcriptional control of theHNF-lot promoter. The chimeric HNF-loaluciferasezneo gene was subsequently clonedinto the pBluescript-MClâHSVTk plasmid to allow dual selection with G418 andgancyclovir.c. Isolation of HNF-loc âknock-inâ cell lines: transfection and selection of stably-transformed cell linesThe HNF-lot targeting construct is introduced into CV-1 cells by electroporation.Following recovery, the cells are maintained in media supplemented 1 mg/ml G418 and 2pM gancyclovir for 3-4 weeks until isolated colonies are observed. These G418â-Ganckcells are isolated and propagated as individual cultures for further characterization.(1. Identification of gene targeting events16101520CA 02265251 1999-03-03W0 98/10059 PCT/US97/15454Individual clones are expanded into 24 well tissue culture plates, and genomicDNA prepared and analyzed by restriction digestion and Southern hybridization. Coloniesexhibiting the pattern predicted for cells that have undergone homologous recombinationand targeted gene replacement are expanded for further characterization and use asreporter cell lines for HNF~loc gene expression.14. Cell-based transcription assay.- Aliquot 100 ul cell suspension (104-105 primary or cultured, differentiated cells)into each well of 96-well plate under sterile conditions- Add compound or extract to 10 uM final concentration- Incubate 6-18 hrs at 37 °C- Remove incubation media- Add 100 ul lysis/luciferin buffer containing 1.0% non-ionic detergent (Triton x-100), 530 uM ATP, 270 uM coA and 470 uM luciferin (Promega).- Measure luciferase-derived luminescence on automated Torcon AMLâ34 and7710 Microplate Luminometer (Cambridge Technologies, Inc.) luminometers.All publications and patent applications cited in this specification are hereinincorporated by reference as if each individual publication or patent application werespeciï¬cally and individually indicated to be incorporated by reference. Although theforegoing invention has been described in some detail by way of illustration and examplefor purposes of clarity of understanding, it will be readily apparent to those of ordinaryskill in the art in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit or scope of theappended claims.17
Claims (6)
1. An isolated genetic knock-in mammalian cell, wherein said cell is, or is a progeny of, a genetic knock-in cell made by homologous recombination of a targeted native allele with a transgene comprising a sequence encoding a reporter flanked by flanking sequences which effect the homologous recombination of said transgene with said native allele, wherein the expression of said reporter is under the control of native gene expression regulatory sequences of said native allele.
2. The genetic knock-in mammalian cell of claim 1, wherein said knock-in cell results from the replacement of a portion of said native allele with a sequence encoding said reporter.
3. The genetic knock-in mammalian cell of claim 1, wherein said reporter is luciferase.
4. The genetic knock-in mammalian cell of claim 1, wherein said cell is a progeny of a transgenic cell line.
5. A cell-based method for screening for modulators of a targeted gene expression, said method comprising steps:
a) determining a first reporter expression level in a first isolated mammalian cell according to claim 1;
(b) contacting a second isolated mammalian cell according to claim 1 with a candidate agent under conditions whereby but for the presence of said agent, said reporter is expressed at said first reporter expression level;
(c) determining a second reporter expression level in said second isolated mammalian cell;
(d) comparing said first expression level with said second expression level, wherein a difference between said first and second expression levels indicates that said candidate agent modulates the targeted gene expression.
a) determining a first reporter expression level in a first isolated mammalian cell according to claim 1;
(b) contacting a second isolated mammalian cell according to claim 1 with a candidate agent under conditions whereby but for the presence of said agent, said reporter is expressed at said first reporter expression level;
(c) determining a second reporter expression level in said second isolated mammalian cell;
(d) comparing said first expression level with said second expression level, wherein a difference between said first and second expression levels indicates that said candidate agent modulates the targeted gene expression.
6. The method of claim 5, wherein said second cell is in a suspension of identical cells, said reporter is luciferase, said contacting step comprises depositing aliquots of said suspension into wells of a plate under sterile conditions, adding said agent to said aliquots and incubating said aliquots 6-18 hours, and said measuring step comprises adding a lysing detergent, ATP, coenzyme A and luciferin to said aliquots and measuring luciferase-derived luminescence.
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US08/707,408 | 1996-09-04 | ||
US08/707,408 US5780258A (en) | 1996-09-04 | 1996-09-04 | Drug screens for regulators of the expression of the obese gene |
US08/866,942 | 1997-05-31 | ||
US08/866,942 US6566089B1 (en) | 1996-09-04 | 1997-05-31 | Cell-based drug screens for regulators of gene expression |
PCT/US1997/015454 WO1998010059A1 (en) | 1996-09-04 | 1997-09-02 | Cell-based drug screens for regulators of gene expression |
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CA2265251A1 true CA2265251A1 (en) | 1998-03-12 |
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CA002265251A Abandoned CA2265251A1 (en) | 1996-09-04 | 1997-09-02 | Cell-based drug screens for regulators of gene expression |
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US (2) | US6566089B1 (en) |
EP (1) | EP0929665A4 (en) |
JP (1) | JP2000517193A (en) |
AU (1) | AU726014B2 (en) |
CA (1) | CA2265251A1 (en) |
WO (1) | WO1998010059A1 (en) |
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US6566089B1 (en) * | 1996-09-04 | 2003-05-20 | Tularik Inc. | Cell-based drug screens for regulators of gene expression |
US6458574B1 (en) * | 1996-09-12 | 2002-10-01 | Transkaryotic Therapies, Inc. | Treatment of a α-galactosidase a deficiency |
SE9702457D0 (en) * | 1997-06-26 | 1997-06-26 | Pharmacia & Upjohn Ab | screening |
US7270998B2 (en) * | 2000-06-01 | 2007-09-18 | Japan Science And Technology Corporation | Method of concentrating and separating dopaminergic neurons |
WO2002040990A1 (en) * | 2000-11-15 | 2002-05-23 | Akiko Itai | Method of profiling protein |
JP5570677B2 (en) * | 2002-04-25 | 2014-08-13 | シャイアー ヒューマン ジェネティック セラピーズ インコーポレイテッド | Treatment of α-galactosidase A deficiency |
CN100439500C (en) * | 2002-05-17 | 2008-12-03 | 中国人民解放军军事医学科学院生物工程研究所 | Mouse model of hipatitis B virus gene positioning integration to result in liver cell callcer |
ATE488595T1 (en) * | 2003-05-06 | 2010-12-15 | Nat Inst Of Advanced Ind Scien | SYSTEM FOR DETERMINING THE ACTIVITY OF MULTIPLE GENE TRANSCRIPTIONS |
KR102424119B1 (en) * | 2015-03-16 | 2022-07-25 | 리제너론 파마슈티칼스 인코포레이티드 | Non-human animals exhibiting reduced upper and lower motor neuron function and sensory perception |
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US5665543A (en) * | 1989-07-18 | 1997-09-09 | Oncogene Science, Inc. | Method of discovering chemicals capable of functioning as gene expression modulators |
WO1991001379A1 (en) * | 1989-07-18 | 1991-02-07 | Oncogene Science, Inc. | Method of transcriptionally modulating gene expression and of discovering chemicals capable of functioning as gene expression modulators |
CA2045175C (en) * | 1989-11-06 | 2003-03-18 | Arthur I. Skoultchi | Production of proteins using homologous recombination |
US5401629A (en) * | 1990-08-07 | 1995-03-28 | The Salk Institute Biotechnology/Industrial Associates, Inc. | Assay methods and compositions useful for measuring the transduction of an intracellular signal |
GB9113097D0 (en) * | 1991-06-18 | 1991-08-07 | Boehringer Ingelheim Int | Chimeric gene |
US5358866A (en) * | 1991-07-03 | 1994-10-25 | The United States Of America As Represented By The Department Of Health And Human Services | Cytosine deaminase negative selection system for gene transfer techniques and therapies |
ES2204899T3 (en) * | 1992-01-07 | 2004-05-01 | Elan Pharmaceuticals, Inc. | MODELS OF TRANSGENIC ANIMALS FOR ALZHEIMER'S DISEASE. |
JP2002515730A (en) * | 1995-10-16 | 2002-05-28 | カイロン コーポレイション | Method for screening a factor that modulates gene expression |
US6566089B1 (en) * | 1996-09-04 | 2003-05-20 | Tularik Inc. | Cell-based drug screens for regulators of gene expression |
US5780258A (en) * | 1996-09-04 | 1998-07-14 | Tularik, Inc | Drug screens for regulators of the expression of the obese gene |
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- 1997-09-02 WO PCT/US1997/015454 patent/WO1998010059A1/en not_active Application Discontinuation
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WO1998010059A1 (en) | 1998-03-12 |
JP2000517193A (en) | 2000-12-26 |
US6566089B1 (en) | 2003-05-20 |
EP0929665A4 (en) | 2003-03-12 |
AU4246597A (en) | 1998-03-26 |
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