WO1992022647A1 - Detection precoce d'embryons transgeniques - Google Patents

Detection precoce d'embryons transgeniques Download PDF

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Publication number
WO1992022647A1
WO1992022647A1 PCT/US1992/005097 US9205097W WO9222647A1 WO 1992022647 A1 WO1992022647 A1 WO 1992022647A1 US 9205097 W US9205097 W US 9205097W WO 9222647 A1 WO9222647 A1 WO 9222647A1
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embryo
transgene
cell
restriction site
dna
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PCT/US1992/005097
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English (en)
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Paul Krimpenfort
Sang He Lee
Rein Strijker
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Genpharm International, Inc.
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Publication of WO1992022647A1 publication Critical patent/WO1992022647A1/fr

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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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
    • 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
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • transgenic livestock High costs and long gestation periods of large animals make it difficult, time consuming and expensive to produce transgenic livestock.
  • Current techniques to produce transgenic animals involve transfer of all microinjected embryos to recipients and identification of transgenic animals among newborns. Only 1.3% and 10% of the newborns were transgenic in sheep and pigs, respectively (Hammer et al., Nature 315:680-683 (1985), incorporated herein by reference), and 5% of bovine fetuses were transgenic (Biery et al., Theriogenology 29:224 (1988), incorporated herein by reference) .
  • transgene integration e.g.,in the preimplantation embryo
  • Methods for early detection of transgene integration would significantly decrease the number of pregnancies required to produce a transgenic animal and substantially increase the likelihood that an implanted embryo will produce a transgenic animal. These methods would thus allow for far more efficient production of transgenic offspring, providing significant savings of time and resources. Such methods would be especially important for those animals for which very low or non-existent frequencies of transgenesis have been obtained, e.g. bovine species.
  • the present invention fulfills these and other needs.
  • the present invention provides methods for the early detection of integrated transgenes in the nuclear genome of animal embryos.
  • such integrated transgenes are detected by performing in situ hybridization on at least one cell from an embryo with a nucleic acid probe substantially complementary to the transgene.
  • the embryo cell(s) used for Ln situ hybridization are in interphase (G 0 or G ) or metaphase, but preferably metaphase.
  • the presence of such an integrated transgene is detectable as a discrete hybridization signal on both sister chromatids of a chromosome in a metaphase spread and is detectable in the nucleus of interphase cells as discrete intense hybridization signals against a diffuse background of non-specific staining.
  • Such a method is preferably nondestructive: a biopsy of at least one cell is removed from the embryo to be analyzed by in situ hybridization, and the resulting biopsied cell is viable.
  • the probe is typically labeled by biotinylation, and is preferably long, at least 2 kilobases in length to provide optimum sensitivity.
  • nuclear DNA from at least one cell from said embryo is treated with a restriction enzyme, such as Dpn I, capable of cleaving a methylated restriction site and incapable of cleaving said unmethylated restriction site, thereby producing restriction fragments of said nuclear DNA.
  • a restriction enzyme such as Dpn I
  • These restriction fragments are amplified by PCR with PCR primers substantially complementary to sequences on different strands of the transgene and flanking the unmethylated restriction site.
  • the amplified transgene may be detected only if the restriction enzyme has not cleaved its restriction sequence.
  • the embryos tested by these methods are typically bovine orulas at the 8 to 16 cell stage or are compacted embryos that have developed past the 16 cell stage.
  • clone the animal embryo prior to testing for an integrated transgene, such as by nuclear transfer or propagation of cloned cleaved embryos (e.g., hemi-embryos, infra) .
  • Methods are also provided which additionally determine the sex of the embryo tested by the methods above described. Also embraced are embryos which have been produced by any of the methods of the present invention.
  • the present invention provides methods for early detection of transgene integration in embryos which permit implantation and development to maturity of transgenic embryos so analyzed.
  • Two basic approaches are provided: the first is based on the polymerase chain reaction (PCR) , the second on the in situ hybridization of nuclear DNA from embryonic cells with labeled probes. Each is described below in detail. These not only allow the early and rapid detection of transgene sequences in the cells of embryos into which transgene DNA has been introduced, but allow one to determine whether such DNA is truly integrated into the nuclear genome, giving rise to a transgenic animal, or is nonintegrated DNA which will eventually be lost. False positives, arising when nonintegrated DNA is mistakenly identified as integrated transgenes, would otherwise demand a significant expenditure of time and resources.
  • the power of these techniques is enhanced when they are used in concert, preferably using PCR as an initial detection method, followed by in situ hybridization as a confirmatory method.
  • Transgene sequences may be introduced into host cells in a variety of ways well known in the art, including: electroporation (Thomas and Capecchi, Cell 51:503-512 (1987) or microinjection of the transgene into the pronuclei of fertilized oocytes or nuclei of ES cells of the animal (Zimmer and Gruss, Nature 338:150-153 (1989); transfection of ES cells in culture by calcium phosphate precipitation (Gossler et al., Proc. Natl. Acad. Sci. USA 83:9065-9069 (1986); and infection of zygotes with a retrovirus containing the transgene (Jaenisch, Proc. Natl. Acad. Sci. USA. 73. 1260-1264 (1976) ; Robertson et al., Nature 323:445-448 (1986); all incorporated herein by reference) .
  • pre-implantation embryos preferably contain approximately 16 to 150 cells.
  • the 8 to 32 cell stage of an embryo is commonly referred to as a morula.
  • blastocysts Those pre-implantation embryos containing more than 32 cells are commonly referred to as blastocysts.
  • Blastocysts are generally characterized as demonstrating the development of a blastocoel cavity, typically at the 64 cell stage.
  • Methods for culturing fertilized oocytes to the pre-implantation stage include, for example, those described by Gordon et al., Meth. Enzymol.
  • a biopsy is obtained, i.e., at least one cell is removed from the embryo.
  • the embryo is preferably not cultivated past the morula stage (32 cells) .
  • Division of the pre-implantation embryo may result in two "hemi-embryos" (hemi-morula or hemi-blastocyst) , at least one of which is viable, i.e., capable of subsequent development after implantation into the appropriate female to develop in utero to term.
  • an embryo may be either equally or unequally divided into two hemi-embryos which are not necessarily of equal cell number. Indeed, only one or two cells may constitute a biopsy from a single embryo; for that reason, one may more accurately speak of a "biopsied embryo” resulting from the removal of a biopsy of one or more cells from an embryo for analysis by the early detection methods provided herein. Essentially, all that is required is that one of the embryos which is not analyzed as hereinafter described be of sufficient cell number to develop to full term in utero.
  • the biopsied embryo if shown to be transgenic, may be implanted in a female recipient animal to develop to term.
  • the methods for detecting transgenesis in pre-implantation embryos provided herein are combined with embryonic cloning steps to generate a population of transgenic embryos having the same genotype, several of which may provide cell biopsies for transgenesis detection.
  • embryonic cloning steps to generate a population of transgenic embryos having the same genotype, several of which may provide cell biopsies for transgenesis detection.
  • such cloned embryos may thereafter be implanted into recipient females to produce a population of transgenic animals also having the same genotype.
  • embryonic cloning ensures not only that enough biopsied cells are available to provide a conclusive test for transgenesis, but also that at least one of the cloned embryos shown to be transgenic will produce a transgenic animal.
  • embryo cloning may be performed by several different approaches. In one cloning method, the transgenic hemi-e bryo is cultured in the same or in a similar media as used to culture individual oocytes to the pre-implantation stage.
  • transgenic embryo so formed (preferably a transgenic morula) is then divided into "transgenic hemi- embryos" which can then be implanted into a recipient female to form a clonal population of two transgenic animals.
  • transgenic hemi-embryos obtained may be again cultivated to the pre-implantation stage, divided, and recultivated to the transgenic embryo stage. This procedure is repeated until the desired number of clonal transgenic embryos having the same genotype are obtained.
  • Such transgenic embryos may then be implanted into recipient females to produce a clonal population of transgenic animals.
  • the transgenic embryo is cloned by nuclear transfer according to the techniques of Prather et al. (Biol. Reprod. 37:59-86 (1988), incorporated herein by reference) and Roble et al. (J. Anim. Sci. 64:642-664 (1987) ; incorporated herein by reference) .
  • nuclei from individual cells of the transgenic embryo are transplanted into enucleated oocytes, each of which is thereafter cultured to the blastocyst stage.
  • the transgenic embryos may be subjected to another round of cloning or may be transferred to a recipient parent for production of transgenic offspring having the same genotype.
  • one may produce multiple, genetically identical embryos.
  • One may obtain one or a small number of cells from each of these identical embryos to test for transgenesis by the methods of the present invention.
  • transgenesis is understood herein as meaning the integration of a transgene into the nuclear genome of a host organism, and its subsequent replication and stable maintenance as part of the nuclear genome.
  • integration is meant that the transgene is located on a chromosome, continuous with and linked by covalent bonds to the double stranded DNA which constitutes the chromosome. This situation may be contrasted with that of non-integrated extra-chromosomal DNA elements such as plasmids, episomes, and double minutes.
  • transgenic embryos and/or transgenic animals having the same "genotype” means that the genomic DNA is substantially identical among the individuals of the embryo and/or transgenic animal population, having derived from the same genetic source. During mitosis, however, various somatic mutations may occur which may produce some variations in the genotype of one or more cells and/or animals. Thus, a population considered to have the same genotype may demonstrate individual or subpopulation variation.
  • PCR is a cyclical process for the amplification of a nucleic acid template comprising the steps of: (a) denaturation of the template (generally by heating to 95°C to 100°C) , (b) hybridization of dual oligodeoxynucleotide primers to the denatured template, and (c) template replication, consisting of an extension of these primers by a DNA polymerase (generally a thermostable polymerase such as Taq polymerase) .
  • a DNA polymerase generally a thermostable polymerase such as Taq polymerase
  • dNTPs deoxynucleotide triphosphates
  • a buffer to provide, for example, the salt and pH conditions necessary for optimal polymerase activity.
  • the primers are so designed that they hybridize to sequences flanking the region to be amplified, one on each strand, and that the single strand generated by extension of each primer can act as a template for the extension of the other primer.
  • each cycle of replication provides a two-fold amplification of the template.
  • PCR can be thus be used to amplify target DNA sequences several million-fold.
  • the amplified DNA can then be analyzed by restriction enzymes and electrophoresis.
  • oligonucleotides primers are synthesized that preferably hybridize specifically to sequences in the transgene, or, if present, to sequences in accompanying vector sequences. Upon amplification of the target sequence a DNA fragment of specific size will be generated.
  • a typical PCR buffer contains 10 to 50 mM Tris-HCl (between pH 8.3 and 8.8) when measured at 20°C, 50 mM KC1 and 1.5 mM MgCl 2 . Modifications of this buffer may be necessary for certain combinations of target nucleic acids and primers, as will be readily appreciated by those skilled in the art.
  • a number of suitable commercially available DNA polymerases are appropriate for performing PCR.
  • Thermostable polymerases are preferred, such as those obtained from Thermus aquaticus (Taq polymerase) or T. flavis.
  • Teq polymerase Thermus aquaticus
  • other polymerases may be appropriate, including E. coli DNA polymerase I or its Klenow fragment, reverse transcriptase, phage T4 or T7 polymerases, or structural variants and modified forms of these and other polymerases.
  • the sensitivity of PCR is particularly useful for analyzing small DNA samples (e.g. from a small number of cells) . This sensitivity can, however, be problematic unless stringent precautions are taken to avoid specific contamination. DNA contamination from aerosol particles, pipettes, primers, etc.
  • PCR analyses in a completely isolated lab equipped with a flow hood with filter; special equipment (pipettes, microfuges, etc.) dedicated to PCR; all reagents (primers, buffers, Taq polymerase) supplied in kits that can be opened and used only once.
  • the problem of discriminating integrated from non- integrated DNA constructs may be solved by taking advantage of the difference in methylation patterns between DNA replicated in prokaryotes and DNA replicated in eukaryotes.
  • DNA constructs which have been cloned in prokaryotes and/or methylated in vitro will retain the prokaryotic methylation profile, unless they integrate in and, consequently, are replicated as part of the host, genome.
  • the A of the sequence GATC the recognition site for the restriction enzyme Dpn I, is methylated in Darn "1" strains of E. coli; it is unmethylated in eukaryotic cells, which lack the dam methylase.
  • Eukaryotic DNA is methylated at G in the sequence GC. After several cleavages, the integrated transgene, having been replicated along with the nuclear genome, will be methylated in a eukaryotic fashion.
  • restriction endonucleases capable of cutting methylated restriction sites but not nonmethylated restriction sites (e.g., Dpn I)
  • Dpn I nonmethylated restriction sites
  • methylation sensitive enzymes which can cut sequences in the transgene which display the eukaryotic methylation pattern but which fail to cut the same sequences when they display a prokaryotic methylation pattern.
  • inverse PCR An alternative method for detecting integrated transgenes, "inverse PCR", makes use of primers which have opposite (reverse) orientation (Triglia et al. , Nucl. Acids Res. 16:8186 (1988); Och an et al., Genetics 120:621-623 (1988) , both incorporated herein by reference) .
  • the DNA isolated from biopsies is cut with restriction enzymes and the fragments are circularized by ligation.
  • DNA fragments of expected length are amplified (derived from concatamers)
  • DNA from transgenic embryos will also give rise to fragments of varying size. These fragments contain host sequences which are present between the reverse primers in the religated circles.
  • PCR primers are preferably designed to be complementary to sequences within the transgene itself.
  • a preferred method for detecting transgenesis by PCR at an early stage in the embryo ⁇ s development employs a methylation sensitive restriction endonuclease such as Dpn I.
  • Dpn I recognizes and cleaves the sequence GATC in double stranded DNA only when the adenine in each strand within this sequence is methylated at N-6.
  • the transgene containing the sequence GATC is methylated prior to microinjection either by transferring the transgene on an appropriate plasmid to a Dam + strain of a microorganism such as E. coli MM294, or by directly methylating the transgene in vitro with dam methylase (commercially available from a number of vendors, including New England Biolabs) .
  • the methylated transgene (preferably without any exogenous sequences such as plasmid vector) is then microinjected into fertilized oocytes (approximately 10 to 500 copies per pronucleus, more preferably 50 to 100 copies per pronucleus) .
  • fertilized oocytes so obtained are cultured in vitro to the pre-implantation stage. During this early growth and cell division phase, the genomic DNA is replicated. Accordingly, those copies of the methylated transgene integrated into the genome of the fertilized oocyte are unmethylated after replication, whereas any non-integrated transgenes which may still exist after replication will remain methylated (Lacks et al., J. Mol. Biol.
  • This differential methylation pattern for an integrated versus a non-integrated transgene permits the identification of which fertilized oocytes have integrated the transgene into the genome.
  • the identification of the pre-implantation embryos containing the integrated transgene is achieved by analyzing the DNA from each of the embryo biopsies (or the pooled biopsies from several genetically identical embryos arising from cloning a single embryo) .
  • Such DNA is typically obtained by lysing the biopsied cells and analyzing the released DNA after treatment as described by Nino iya et al., Molecular Reproduction and Development 1:242-248 (1989), incorporated herein by reference. Each of the DNA samples is treated with Dpn I.
  • PCR is used to amplify all or part of the transgene.
  • two extension primers each complementary to opposite strands at opposing ends of the transgene are used.
  • such extension primers are chosen such that the amplified gene product spans the Dpn I site in the transgene. That is, the primer binding sites for the two PCR primers flank the Dpn I site. If Dpn I cleavage has not occurred, PCR amplification will result in amplified sequences having a predicted size, whereas no amplification product will result for those transgenes which have been cleaved.
  • the Dpn I digested and PCR amplified DNA from the biopsy is subjected to electrophoresis.
  • electrophoresis is preferably followed by hybridization with labeled probe complementary to the region of the transgene between the two extension primers.
  • labeled probe complementary to the region of the transgene between the two extension primers.
  • the remaining untreated transgenic biopsied embryo is transplanted into a recipient parent.
  • the transgenic animal having the desired phenotype conferred by the integrated transgene is identified by an appropriate method in utero or after birth.
  • the use of Dpn I requires that the sequence GATC be present in the transgene of interest. In those cases when such a sequence is not present, it may be readily introduced into the transgene by site directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci.
  • transgenic embryos and/or transgenic animals having the same "genotype" means that the genomic DNA is substantially identical between the individuals of the embryo and/or transgenic animal population. It is to be understood, however, that during mitosis various somatic mutations may occur which may produce some variations in the genotype of one or more cells and/or animals. Thus, a population having the same genotype may demonstrate individual or subpopulation variations.
  • a sample is electrophoresed on an agarose or polyacrylamide gel.
  • Smaller DNA fragments resulting from amplification are preferably analyzed by polyacrylamide gel electrophoresis.
  • DNA bands are visualized by ethidium bromide staining, or, to increase sensitivity, gel electrophoresis can be followed by Southern blotting or dot blotting and hybridization with a labeled probe by techniques well known in the art.
  • labeled PCR primers which are incorporated into the amplified DNA product, may be employed by methods well known in the art to assist in visualization of the amplification product and thus increase sensitivity.
  • nucleic acid probes are used to locate specific complementary nucleic acid sequences, e.g., specific DNA sequences on intact chromosomes, in situ, a procedure called "in situ hybridization".
  • Labeled nucleic acid probes of a predetermined nucleotide sequence are hybridized to the chromosomes of sample cells (or tissues) , the DNA strands of which have been denatured to a substantially single-stranded character by a brief exposure to a very high pH condition.
  • the chromosomal regions that bind the probe(s) during the hybridization step are examined by microscopy to determine whether they hybridize to the probe(s) and thus contain the specific nucleic acids of interest.
  • the direct methods the reporter molecule is bound to the nucleic acid probe so that the molecular hybrids between probe and target sequences can be visualized microscopically immediately after the in situ hybridization procedure.
  • Such methods include the terminal fluorochrome labeling procedure of RNA probes (Baumann et al. , 1980, 1984, which are incorporated herein by reference) and the direct enzyme labeling procedure of nucleic acids (Renz and Kurz (1984, which is incorporated herein by reference) .
  • the probe For indirect procedures, the probe must contain an element, introduced chemically or enzymatically, that renders it detectable by affinity cytochemistry, hence the term indirect. Again, the presence of such an element should not, or only in a limited way, interfere with the actual hybridization reaction and the stability of the resulting hybrid.
  • hapten modifications including acetylaminofluorene, mercury, biotin and transamination procedures, have been described (Langer et al. , Proc. Natl. Acad. Sci. USA 78:6633-6637 (1981); Leary et al., Proc. Natl. Acad. Sci. USA 80:4045-4049 (1983); Landegent et al., Exp. Cell Res.
  • oligonucleotides containing functional groups e.g. primary aliphatic amines or sulfhydryl groups
  • haptens e.g., biotin or digoxygenin
  • reporters like fluorochromes or enzymes
  • the probes are synthesized with nucleotides that contain a biotin side chain, and the hybridized probes are detected by staining with a network of streptavidin and some type of marker molecule.
  • this technique it is possible to detect specific, single copy nucleic acid sequences in individual cells and on individual chromosomes (See for review: Raap et al., In Techniques in Immunocytochemistrv. vol. IV, ed. G. Bullock and P. Petrusz: New York, Academic Press (1990) , incorporated herein by reference) .
  • metaphase chromosomes for in situ hybridization is that it facilitates the discrimination between integrated and non-integrated DNA by localizing specific hybridization signal(s) indicative of integrated transgene DNA on adjacent chromatids.
  • in situ hybridization can be performed on cells which are not in metaphase (e.g., interphase cells from an embryo biopsy, which are typically not synchronized) and integrated transgene DNA can be identified by the presence of discrete hybridization signal(s) which can be differentiated from the non-specific hybridization background.
  • a discrete hybridization signal is identified as one or more localized intense staining positions which are differentiated from non-specific hybridization by visual inspection, autoradiographic analysis, or computerized imaging methods wherein background noise is subtracted by one or more imagen analysis algorithms known in the art.
  • Nonintegrated DNA molecules will probably be present in high numbers, fused together in one or a few large concatamers, yet diffused randomly throughout the nucleus and not associated directly with a chromosome.
  • Integrated DNA will be visualized as discrete spots in the nucleus of cells (generally fixed cells) , and in metaphase cells can be visualized as discrete spots on one or-both sister chromatids at the same location for each integration site.
  • in situ hybridization methods used DNA or RNA probes labeled directly with a terminal fluorochrome.
  • the sensitivity of these direct methods was low. Therefore, immunocytochemical amplification procedures have been developed to increase detection sensitivity.
  • Several chemical and enzymatic nucleic acid hapten modifications are now available for the amplification on DNA or RNA probes for in situ hybridization (Raap et al., 1989). All modifications have in common that the hapten does not affect the hybridization properties of the probes. Most haptenized probes are detected, after hybridization, with antibodies specific for the hapten.
  • PCR is employed to provide an initial determination whether the transgene is incorporated into the genome of an embryo, while in situ hybridization is used to confirm the results of the initial testing. Alternatively, PCR is used to confirm the results obtained by an initial test using in situ hybridization.
  • the sex of the embryos so tested can further be sexed by PCR or in situ hybridization using probes specific for sex-determining sequences, such as sequences characteristic of the Y chromosome or other sequences involved in sex determination (e.g., sex steroid receptor genes, etc.) which are not linked to the Y chromosome and which are known in the art.
  • in utero analysis is performed by several techniques.
  • transvaginal puncture of the amniotic cavity is performed under echoscopic guidance (Bowgso et al. (1975) Bet. Res. 96:124-127; Rumsey et al. (1974) J. Anim. Sci. 39:386-391; both incorporated herein by reference) .
  • This involves recovering about 15 to 20 milliliters of amniotic fluid between about day 35 and day 100 of gestation.
  • This volume of amniotic fluid contains about 1000 to 12,000 cells per ml originating from the urogenital tract, the skin, and possibly the lungs of the developing embryo. Most of these cells are dead. Such cells, however, contain genomic DNA which is subjected to PCR analysis for the transgene as an indication of a successful transgenesis. Alternatively, fetal cells may be recovered by chorion puncture. This method also may be performed transvaginally and under echoscopic guidance. In this method, a needle is used to puncture the recipient animal's placenta, particularly the placentonal structures, which are fixed against the vaginal wall. Such sampling may be performed around day 60 of gestation. Chorion cells, if necessary, are separated from maternal tissue and subjected to PCR analysis for the transgene as an indication of successful transgenesis.
  • Transgenesis may also be detected after birth.
  • transgene integration can be detected by taking an appropriate tissue biopsy such as from the ear or tail of the putative transgenic animal. About one to two centimeters of tail or about five to ten square millimeters of ear are obtained, followed by Southern blotting with a probe for the transgene according to the method of Hogan et al. (1986) Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory, incorporated herein by reference.
  • recombinant DNA techniques employed when using the present invention are well established and constitute recognized methods, e.g., for the use of restriction endonucleases; for the preparation of predetermined nucleotides in sequence as hybridization probes; and for the various methods of labelling such DNA or RNA probes using a variety of labels, such as radionuclides. Accordingly, it is presumed that one practicing the present invention is familiar with the applications and limits of the various techniques and will recognize that minor changes in reagents, concentrations, temperature, reaction times and similar alterations of known methods are merely obvious variations of choice.
  • transgene refers to a DNA sequence which is capable of producing a desirable phenotype when contained in the genome of cells of a transgenic animal.
  • transgene often comprises a recombinant DNA sequence encoding a "recombinant polypeptide".
  • the transgene is capable of being expressed to produce the recombinant polypeptide.
  • the term also is used to embrace vector sequences and non-coding flanking sequences which may accompany such polypeptide-encoding sequences.
  • Specific hybridization is defined herein as the formation of hybrids between a probe polynucleotide (e.g., a polynucleotide of the invention which may include substitutions, deletion, and/or additions as compared to a target sequence) and a specific target polynucleotide (e.g., a polynucleotide having a predetermined sequence, such as a transgene sequence) , wherein the probe preferentially hybridizes to the specific target such that, for example, a single band corresponding to a transgene restriction fragment can be identified on a Southern blot of DNA prepared from a cell having an integrated copy of the transgene DNA.
  • a probe polynucleotide e.g., a polynucleotide of the invention which may include substitutions, deletion, and/or additions as compared to a target sequence
  • a specific target polynucleotide e.g., a polynucleotide having a pre
  • PCR primers used in the methods of the present invention are oligonucleotides, whether occurring naturally as in a purified restriction digest, or produced synthetically, which are capable of hybridizing specifically to a known sequence in a target gene.
  • a PCR primer especially the terminal 3 ' nucleotide of the primer, has hybridized, it acts as a point of initiation of synthesis under conditions in which synthesis of a primer extension product is favored.
  • Such conditions typically include the presence of four different nucleotide triphosphates and a thermostable polymerase in an appropriate buffer and at a suitable temperature.
  • primers are preferably single stranded for maximum efficiency and amplification, although double stranded primers may be employed if denatured to separate the complementary strands before use.
  • the oligonucleotides employed as primers may contain naturally-occurring nucleotides or their analogs, such as 7-deazaguanosine or inosine, and may be either DNA or RNA.
  • Such an oligonucleotide will preferably contain at least one or preferably more than one region of eight or more consecutive nucleotides having perfect complementarity with the target sequence. They will also usually have one or more nucleotides at the 3 • end displaying perfect complementarity (i.e., base pairing) with a single-stranded region of a target nucleic acid. However, longer oligonucleotides (e.g., more than 20 nucleotides) can anneal with the desired specificity, even though no such region of consecutive nucleotides with perfect complementarity may be present. In general, oligonucleotides that specifically hybridize to a predetermined sequence (unique or repetitive) or sequence motif are suitable for use as primers.
  • the primer sequence need not, however, reflect the exact sequence of the template, and may include sequences in addition to those allowing the primer to hybridize with specificity to the template. Alternatively, noncomplementary bases or longer sequences can be interspersed into the primer provided that the primer sequence has sufficient complementarity with the sequence of the target sequence to hybridize with it and thereby allow synthesis of the extension product.
  • the primer may include nucleotides which have been substituted, e.g., with biotin. As a consequence of amplification by PCR, the sequence and substituents of the primer are introduced into the amplified product.
  • oligonucleotide is an oligodeoxyribonucleotide.
  • Oligonucleotide primers typically are from about 8 to 50, usually about 12 to 50, and preferably 16 to 30 nucleotides in length, although longer or shorter primers may be appropriate.
  • Suitable probes may be RNA, DNA, or an analogous polymer (e.g., a polyamide nucleis acid or a methylphosphonate oligonucleotide) and may be either double-stranded or single-stranded.
  • an analogous polymer e.g., a polyamide nucleis acid or a methylphosphonate oligonucleotide
  • In situ hybridization probes will typically be at least about 100 to 1000 bases long, preferably greater than 2 kb in overall length to increase hybridization signal as long as possible (greater than 2 kb is preferable) to increase sensitivity of detection (e.g., signal-to-noise ratio), although shorter probes may be used, and will preferably be complementary to unique sequences in a transgene (except where the transgene comprises repetitive sequences that may be distinguished from sequences in the host genome by specific hybridization) to reduce the chance for nonspecific hybridization.
  • Probes for Southern blots may be shorter probes substantially complementary to sequences in a transgene.
  • the nucleic acid probes may be derived from genomic DNA or cDNA, prepared by chemical or enzymatic synthesis (e.g., as an RNA transcribed from a template sequence comprising a sequence substantially complementary to a transgene) , or may be a hybrid of the various combinations. Recombinant nucleic acids comprising sequences otherwise not naturally occurring may also be employed. They may contain naturally occurring nucleotides or their analogs, such as 7-deazaguanosine or inosine.
  • probes may be labeled by any of the methods commonly used in the art, such as nick translation or random hexamer labeling.
  • nucleic acid probes will include an isolated nucleic acid attached to a label or reporter molecule.
  • Probes may be prepared by nick translation, Klenow fill-in reaction, random hexamer priming, or other methods known in the art.
  • For isolating nucleic acids, choosing label or reporter molecules, labeling probes, and other aspects of probe preparation see, inter alia, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989) , Current Protocols in Molecular Biology, F. Ausubel et al., ed. Greene publishing and Wiley- Interscience, New York (1987) , incorporated herein by reference.
  • An oligonucleotide primer or a probe is "substantially homologous" or “substantially complementary” to a target nucleic acid when it preferentially anneals or hybridizes to a desired sequence position on a strand of the nucleic acid being targeted or its complementary strand such that stable and specific binding occurs between the primer and the target nucleic acid under selective conditions (See. M. Kanehisa, Nucleic Acids Res. 12:203 (1984), incorporated herein by reference) . Selectivity of hybridization exists when hybridization occurs which is more selective than total lack of specificity, and is generally marked, in the methods of the present invention, by the formation of a desired extension product.
  • Proper annealing conditions depend, for example, upon an oligonucleotide*s length, base composition, and the number of mismatches and their position on an oligonucleotide, or on the melting temperature (T m of the oligonucleotide) , salt concentration of the medium, and other conditions, and generally are determined according to formulae and methods known in the art, which can include generation of a standardization of calibration hybridization temperature curve.
  • T m of the oligonucleotide melting temperature
  • salt concentration of the medium and other conditions
  • Stringent primer annealing conditions will vary with the specific application, but typically include salt concentrations of less than about 1 M, more usually less than about 500 mM and preferably less than about 200 mM. Temperature conditions will typically be greater than 22°C, more typically greater than about 30°C and preferably in excess of about 37°C.
  • the combination of parameters is more important than the absolute measure of any one alone.
  • an oligonucleotide primer or a probe is considered “substantially homologous" or substantially complementary" to a target sequence whenever the primer and target sequence, or their complementary strands, when optimally aligned and compared, are identical with appropriate nucleotide insertions or deletions, generally in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98 to 99.5% of the nucleotides.
  • substantially similarity or “substantial complementarity” as used herein denotes a characteristic of a polypeptide sequence or nucleic acid sequence, wherein the polypeptide sequence has at least 50 percent sequence identity compared to a reference sequence, and the nucleic acid sequence has at least 70 percent sequence identity compared to a reference sequence. The percentage of sequence identity is calculated excluding small deletions or additions which total less than 25 percent of the reference sequence.
  • the reference sequence may be a subset of a larger sequence, such as a portion of a transgene construct (e.g., selectable marker sequence, homologous recombination sequence, structural gene sequence) ; however, the reference sequence is at least 18 nucleotides long in the case of polynucleotides, and typically is longer than about 30 nucleotides or more.
  • a transgene construct e.g., selectable marker sequence, homologous recombination sequence, structural gene sequence
  • the reference sequence is at least 18 nucleotides long in the case of polynucleotides, and typically is longer than about 30 nucleotides or more.
  • EXPERIMENTAL EXAMPLES Although the following examples are described in detail for embryonic cells, it is believed that the method(s) are generally applicable to detection of transgenesis in other eukarotic cell types, particularly various types of somatic mammalian cells such as nucleated cells of the lymphohematopoietic lineage, fibroblasts, basal epithelial cells, and others.
  • the method(s) may also be used for sex determination of embryos by using probes that detect the presence of the Y chromosome and/or other non-Y-linked sequences that are responsible for or correlate with sex determination.
  • Immature oocytes are obtained in large quantity (400- 600/day) by aspirating follicles of ovaries obtained at abbatoirs. Immature oocytes are cultured for a period in vitro before they are competent to be fertilized. Once "matured", oocytes are fertilized with sperm which has also been matured, or "capacitated” in vitro. The pronuclei of the fertilized oocyte is then injected with the transgene encoding for the expression and secretion of human lactoferrin.
  • Zygotes resulting from this in vitro fertilization and microinjection are then cultured to the late morula or blastocyst stage in media with somatic tisssue, or in medium "conditioned” by somatic tissue. Blastocysts are then transferred non- surgically to recipient cattle for the balance of gestation or analyzed for integration of the transgene as described herein.
  • In vitro maturation IVM1. Ovaries are obtained immediately after slaughter at local abbatoirs and oocytes are recovered. Alternatively, oocytes are obtained from living cattle by surgical, endoscopic, or transvaginal ultrasonic approaches. In all cases, oocytes are aspirated from ovarian follicles (2-10 mm diameter) .
  • oocytes After washing, oocytes are placed in a maturation medium capable of supporting nuclear and cytoplasmic maturation of bovine oocytes. Examples of such media are given by Sirard et al. (Biol. Reprod. 39:546-552 (1988) , incorporated herein by reference) .
  • IVF In vitro fertilization
  • Matured oocytes are fertilized with either fresh or frozen thawed sperm.
  • Sperm are then added to a fertilization media consisting of a modified Tyrode's solution (Parrish et al. (1986) supra. , incorporated herein by reference) supplemented with heparin to induce sperm capacitation (Parrish et al., Biol. Reprod. 38:1171-1180 (1988) , incorporated herein by reference) .
  • Capacitation constitutes the final sperm maturation process which is essential for fertilization.
  • Sperm and oocytes are co-cultured for 18 hours.
  • a useful feature of this IVF method is that (in the case of frozen sperm) consistent, repeatable results are obtained once optimal fertilization conditions for a particular ejaculate have been defined (Parrish et al. (1986) supra.. incorporated herein by reference) .
  • IVC In vitro culture
  • Conventional culture systems which support development of mouse, rabbit, or human ova, do not support development of bovine embryos past the 8-16 cell stage. This problem has been overcome by pre-conditioning culture media with oviductal tissue. Oviduct-conditioned medium will support bovine embryos past the 8-16 cell stage to the blastocyst stage in vitro (Eyestone and First, J. Reprod. Fert. 85:715-720 (1989), incorporated herein by reference). Bovine embryos generally have proven refractory to in vitro culture. This in part stems from the existence of a "block" to cleavage in vitro at the 8-16 cell stage.
  • This block may be alleviated by culturing embryos in the oviducts of rabbits (reviewed by Boland, Theriogenology 21:126-137 (1982), incorporated herein by reference) or sheep (Willadeen in: Mammalian Egg Transfer (E. Adams, ed.), pp. 185-210 (1982); Eyestone et al. , Theriogenology 28:1-7 (1987); both incorporated herein by reference) .
  • Bovine embryos did not yield to attempts to culture them in vitro past the 8-16 cell "block” until Camous et al., J. Reprod. Fert. 72:479-485 (1984), incorporated herein by reference) demonstrated cleavage to 216 cells when embryos were co-cultured with trophoblastic tissue.
  • the co-culture procedure was extended to oviductal tissue, based on the ability of homo- or hetero-oviducts to support development from zygote to blastocyst.
  • bovine embryos co-cultured with oviductal tissue, or in medium conditioned by oviductal tissue developed from zygote to blastocyst in vitro (Eyestone and First, J. Reprod. Fert.
  • Blastocysts have been produced in this system after superovulation and artificial insemination, or by in vitro maturation (IVM) , and fertilization (IVF) of immature oocytes. Blastocysts produced in this fashion resulted in pregnancies and live calves after transfer to recipient animals. The results obtained were as follows:
  • Oviduct Tissue Preparation of Oviduct Tissue and Use For Co-Culture and Conditioned Medium.
  • Ovine oviducts are obtained after slaughter or by salpingectomy.
  • the lumenal tissue is harvested by scraping an intact oviduct gently with a glass slide and washed five times in 10 ml modified Tyrode's-Hepes solution (Parrish et al. , Biol. Reprod. 38:1171-1180 (1988), incorporated herein by reference) .
  • the final tissue pellet is suspended in M199 + 10% fetal calf serum at a ratio, of 1 volume tissue to 50 volumes of media.
  • the tissue suspension can be used for embryo-co- culture.
  • media may be conditioned for 48 h, and after centrifuging the suspension, the supernatant may be used as embryo culture medium.
  • Conditioned medium may be stored at -70°C, if desired.
  • Conditioned medium should be used at full strength for embryo culture (no dilution) (Eyestone (1989) ) .
  • hLF Transgene into Bovine Pronuclei
  • the DNA fragment containing the hLF expression unit is excised from the vector by digestion with the appropriate restriction enzyme(s) and separated by agarose gel electrophoresis. The fragment is purified by electroelution, phenol and chloroform extraction and ethanol precipitation. The DNA fragment is dissolved in and dialyzed in 10 mM Tris, 0.1 mM EDTA (pH 7.2) at a concentration of 1 to 2 ⁇ g/ml. Microinjection needles are filled with the dialyzed DNA solution. Before in vitro fertilization, cumulus cells are removed from the egg by either vortexing at maximal speed for two minutes or pipetting up and down several times.
  • Bovine pronuclei are injected in the same manner as murine pronuclei (Hogan et al., in: Manipulating the Mouse Embryo. Cold Spring Harbor Laboratory (1986) , incorporated herein by reference) with an additional centrifugation step in order to visualize the pronuclei.
  • the injection takes place 18-24 hours after fertilization. The time varies depending on the bull used as a source of semen. Nuclei become visible at different times in different batches of semen.
  • Bovine oocytes matured and fertilized in vitro, are spun in a microfuge tube (Eppendorf) in 1 ml of Tyrode's-Hepes solution (Parrish (1987) , incorporated herein by reference) at 14500xg for eight minutes (Wall et al., Biol. Reprod. 32:645- 651 (1985) , incorporated herein by reference) .
  • the embryos are transferred to a drop of Tyrode•s-Hepes solution on a microscope slide covered with paraffin oil.
  • Using a hydraulic system the oocytes are fixed to the egg holder in such a way that both the pronuclei are visible (using interference- contrast or phase contrast optics) .
  • the oocytes are rolled to change their position on the egg holder to visualize the pronuclei.
  • the injection needle is brought into the same sharp focus as one of the pronuclei.
  • the needle is then advanced through the zona pellucida and cytoplasm into the pronucleus.
  • a small volume, 1-3 pi, is injected (containing 20-100 DNA copies) into the pronucleus either by using a constant flow or a pulse flow (using a switch) of DNA solution out of the needle.
  • two cell stage embryos are spun as described and the nuclei of both blastomeres are injected as described.
  • the injected embryos are then transferred to a drop of co-culture medium as described in Example 1 in order to develop to the morula or blastocyst stage.
  • oviduct cell conditioned medium was compared with oviduct epithelial cell coculture system as systems for culturing in vitro matured and fertilized oocytes up to the blastocyst stage.
  • oviduct epithelial cell coculture system embryos are cultured on a monolayer of bovine oviduct epithelial cells (BOEC) . Using this system, 30% of the fertilized oocytes develop to the blastocyst stage.
  • BOEC bovine oviduct epithelial cells
  • the isolation of single or multiple blastomeres from a preimplantation embryo requires penetration of the zona pellucida (ZP) .
  • ZP zona pellucida
  • the murine ZP can easily be dissolved at a precise, limited area using a constant narrow flow of acidic Tyrode (pH 2.3) solution, although the ZP of bovine embryos cannot be dissolved by acidic Tyrode, even with a pH lower than 2.1.
  • a slit is made in the ZP which can be opened with a beveled glass pipet with an opening large enough to subsequently aspirate blastomeres. After withdrawing the aspiration pipet the ZP will reclose.
  • This method is more laborious but can be more precisely controlled and causes the least disturbance of normal development. Little damage is caused to the embryo and to the blastomeres that are removed.
  • an opening can be made in the ZP with a sharp glass needle and one or more blastomeres can then be aspirated out using a hollow micropipette which is inserted into the opening made by the glass needle.
  • Biopsies obtained as described herein are also useful for multiplication of embryos proven to be transgenic.
  • the oocyte Upon the microinjection of a construct, the oocyte is cultured, preferably at least to the 8-cell stage, even though analysis of embryos before that time is possible.
  • Each embryo is cleaved and subjected to lysis (King et al., Molecular Reprod. and Devel. 1:57-62 (1988); proteolysis (Higuchi, "Amplifications (A Forum for PCR Users” 2:1-3 (1989); both incorporated herein by reference) and Dpn I digestion.
  • PCR is performed as described previously (Ninomiya et al., Molecular Reprod. and Devel. 1:242-248 (1989), incorporated herein by reference) with sets of two primers which flank a Dpn I site in the construct.
  • One such set of primers are the forward primer ATG AAA CTT ATC CTC ACC TGT CTT GTG (in the ⁇ Sl portion) and the reverse primer GGG TTT TCG AGG GTG CCC CCG AGG ATG GAT (in the hLF portion) of an hLF transgene disclosed in the commonly owned co-pending application U.S.S.N. 619,131, incorporated herein by reference.
  • the following buffer is added to a total volume of 50 ⁇ l in a 500 ⁇ l microfuge (Eppendorf) tube: 50 mM KC1, 10 mM Tris-HCl (pH 9.0 at 25°C) , 1.5 mM MgCl 2 , 0.01% gelatin, 0.1% Triton X-100, 50 ⁇ M each of dATP, dGTP, dCTP and dTTP, 50 pmol forward primer, 50 pmol reverse primer, 0.8 U Dpnl (Bethesda Research Laboratories) , and 2 U Taq polymerase (Promega) .
  • the mixture is overlaid with a drop of light mineral oil to prevent evaporation.
  • the mixture is incubated at 37°C for 20 min, followed by 93°C for 5 min. 50 cycles of PCR follow with each cycles consisting of: denaturation for 1 min at 93°C; annealing for 1 min at 55°C; and extension for 1.3 min at 72°C. After the 50 cycles are complete, a further 10 min incubation at 72°C follows. The reaction may be held at 4°C at this stage until further analysis is undertaken.
  • PCR amplified products about one- third to one-half of the PCR reaction volume is electrophoresed on an agarose or polyacrylamide gel. DNA bands are visualized by ethidium bromide staining, or, if additional sensitivity is required, by Southern blotting of the PCR products followed by probing with a labeled probe substantially complementary to the transgene.
  • ISH in situ hybridization
  • pronuclear injection of bovine embryos is used to introduce a transgene
  • the microinjected embryo is cultured and develops to a morula or blastocyst stage, generally to at least the eight-cell stage and preferably to the sixteen-cell stage.
  • a biopsy of cells from the cultured embryo is taken, usually the biopsy sample consists of about two or three cells of the embryo.
  • the detection of one or more integrated transgene(s) in the biopsy sample is informative for the biopsied embryo, which can be propagated further (e.g., for producing cloned hemi-embryos and/or for implantation) .
  • Two or three cell biopsy of 16 cell-atage morulae generally result in about 70 to 90 percent survival rates of the biopsied morulae.
  • Transgenesis can also be detected by performing in situ hybridization on cloned cleavage bovine embryos (i.e., embryos which have been cleaved and cloned subsequent to pronuclear transgene microinjection) .
  • a cloned embryo is synchronized in metaphase (e.g., by incubation in a medium containing colcemid) .
  • An entire cloned cleavage embryo, or a biopsy thereof, that is substantially synchronized in metaphase (at least about 50 percent of the cells are in metaphase) can be fixed and used for ISH analysis.
  • Metaphase synchronization can also be performed on biopsy material, wherein the biopsy cells are incubated in a suitable culture medium containing a mitotic arrest agent such as colcemid.
  • the metaphase stage is typically preferred because the integrated transgene DNA is easily discriminated from non ⁇ integrated DNA because it is represented by hybridization signals at the same location on both sister chromatids.
  • metaphase-synchronization of biopsied blastomeres requires additional time and effort and the ISH procedure also can be practiced on interphase cells, which are more easily obtained.
  • the criterion on which a cell, such as a biopsied embryo cell, is judged to be transgenic on the basis of ISH is that essentially the majority, usually all, of the biopsied cells show the same general hybridization pattern, i.e., one or two (depending on the stage of the cell cycle, Gl or G2) distinct hybridization signals which are distinguishable over a less intense diffuse background (if present) .
  • Unintegrated DNA gives rise to a random staining pattern which is indicated by a preponderance of the biopsy cells showing an indistinct hybridization pattern.
  • Embryo Biopsy Procedure Transgene DNA is introduced into bovine embryos, typically by microinjection of pronuclei, and the resultant putative transgenic embryos are cultured under suitable culturing conditions.
  • a biopsy of the embryo is taken, usually at the 16-cell stage (morula) or from compacted embryos (later than the 16-cell stage) , and the biopsy comprises at least one cell, typically at least two cells, optionally at least three cells, sometimes more than three cells but always less than the number of cells comprising the embryo, typically less than about half the cells comprising the embryo, and most usually less than about 25 percent * of the cells comprising the embryo.
  • the biopsy is generally performed by forming a slit or perforation in the zona pellucida (supra) and extracting the cell(s) composing the biopsy sample from the embryo.
  • the biopsy cell(s) can then be cultured for metaphase synchronization and spreading, or can be used directly for in situ hybridization essentially regardless of the stage of the cell cycle, but usually are interphase cell(s).
  • the embryo(s) are incubated for about 15 to 30 minutes in medium depleted in calcium and magnesium (e.g., Ca +2 -Mg +2 -free medium) prior to manipulation.
  • the biopsy cells are removed as described, and biopsy cell(s) are then washed in TL-Hepes and placed directly on a microscope slide with a minimum volume of TL-Hepes.
  • the TL-Hepes is carefully aspirated away, leaving the biopsy essentially free of TL-Hepes.
  • a drop of approximately 5 ⁇ l of fixative is added to the biopsy immediately and the cells are preferably spread before the fixative is completely evaporated.
  • Approximately l ⁇ l of softening solution is added at about room temperature (i.e., about 15 to 30° C, usually 20 to 25° C) and additional softening solution is typically added continuously until the biopsy is well-spread and clean. Once spread, prehybridization and subsequent procedures are essentially as described hereinbelow.
  • Embryo cells either from whole embryos, cloned cleaved embryos, or biopsies, may optionally be cultured in a suitable medium containing a cell cycle blocking agent, such as colcemid, to produce metaphase-synchronized cells.
  • a cell cycle blocking agent such as colcemid
  • the resultant metaphase-synchronized cells can then be analyzed for transgenesis by ISH.
  • the procedure is generally applicable to cultured biopsy cells and to cleavage embryos which develop from a cultured biopsy sample.
  • Fixation 3:1 methanol/glacial acetic acid
  • Fixative 3:1 methanol/glacial acetic acid
  • the fixation time is approximately 5 minutes at -20°C in cases where the zona pellucida is not intact (e.g., has been perforated with a glass needle) , but may be longer if the ZP is intact, usually not more than about 20 to 30 minutes.
  • RNAse treatment Each slide is incubated with approximately 100 ⁇ l RNAse (0.1 mg/ml) under a coverslip for about 60 minutes at 37°C in a slide-jar. At the end of incubation, the coverslip is removed by the addition of 100 ml of 2xSSC (Sambrook et al., 1989) and gentle shaking of the siide-jar for 5 minutes on the top of an automatic shaker. The slides are further washed three times with 2xSSC at room temperature. Throughout the entire procedure, all the washings require typically 5 minutes incubation at room temperature with constant gentle shaking unless specified otherwise. Slides are dehydrated by sequential washing with 70%, 90%, and 100% ethanol.
  • Denaturation Chromosomes are denatured by incubation of the slides for 3 minutes at 80°C after application of 100 ⁇ l of denaturation solution (a mixture of 70 ⁇ l of 100 formamide, 10 ⁇ l of 20xSSC and 10 ⁇ l of 0.5 M phosphate, pH 7.0 and 10 ⁇ l of H 2 0) per each slide and covered with coverslips. AFter denaturation, 100 ml of chilled (-20°C) 70% ethanol was poured into the slide-jar and the slides are dehydrated and air-dried at room temperature.
  • denaturation solution a mixture of 70 ⁇ l of 100 formamide, 10 ⁇ l of 20xSSC and 10 ⁇ l of 0.5 M phosphate, pH 7.0 and 10 ⁇ l of H 2 0
  • Probe (20 ng per slide) and sheared bovine genomic DNA (10 ⁇ g per slide) is dissolved in a desired volume of hybridization solution (a mixture of 2.5 ⁇ l 100% formamide, 0.5 ⁇ l 20XSSC, 0.5 ⁇ l 0.5 M phosphate (pH 7.0), 1.5 ⁇ l H 2 0 and 5 ⁇ l 20% dextran sulfate.
  • the probe solution is then denatured by heating at 75°C for 5 minutes followed by quick chilling to 4°C.
  • the probe is further incubated for at least 2 hours at 37°C to block out repetitive sequences before application to slides (10 ⁇ l per slide) .
  • washing solution A 50% formamide/2xSSC, adjusted to pH 7.0 with HCl and prewarmed to 45°C
  • washing solution B O.lxSSC, 60°C
  • each washing takes 5 minutes with shaking of the slide-jar. It is important that the temperature is maintained either at 45°C or 60°C throughout the washing procedure.
  • washing solution C (4xSSC/0.05% Tween 20) at room temperature.
  • the third immunocytochemical reaction is carried out with fluorescein avidin-D FITC solution as described above.
  • the slides are dehydrated by washing with 100 ml of 70%, 90%, and 100% ethanol.
  • Embedding Embedding solution is made by dissolving 2 gm of 1,4-
  • DABCO Diazabicyclo-(2,2,2)-octane
  • Experiment 1 This experiment compares the results obtained by ISH of about one-half of a split embryo to results obtained from PCR analysis of the remainder of the biopsied embryo.
  • Transgenic bovine embryos were produced by pronuclear microinjection. The embryos were cultured and developed to the morula/blastocyst stage, at which time the embryo was split and one half analyzed by in situ hybridization (ISH) and the other by PCR.
  • ISH in situ hybridization
  • the results are shown in Table 1: plus (+) indicates a result that is positive for integrated transgene DNA, minus (-) indicates a negative result indicating the absence of integrated transgene DNA, plus/minus (+/-) indicates an ambiguous result, and "mos.” indicates that the embryo sample was mosaic, with some cells positive and other cells negative. "N.D.” indicates the sample was not determined.
  • Experiment 2 compares the results obtained by ISH of 2-3 cell biopsies of embryos at the morula stage to results obtained from ISH analysis of the remainder of the biopsied embryos which were subsequently expanded to the blastocyst stage.
  • Transgenic bovine embryos were produced by pronuclear microinjection. The embryos were cultured and developed to the morula stage, at which time the embryo was biopsied and the 2-3 cell biopsy samples analyzed by in situ hybridization (ISH) and the remainder of the embryo was cultivated to the blastocyst stage and analyzed by ISH on the entire blastocyst.
  • ISH in situ hybridization
  • Experiment 3 compares the results obtained by ISH of 2-3 cell biopsies of embryos at the morula stage to results obtained from PCR analysis of the remainder of the biopsied embryos which were subsequently expanded to the blastocyst stage.
  • Transgenic bovine embryos were produced by pronuclear microinjection. The embryos were cultured and developed to the morula stage, at which time the embryo was biopsied and the 2-3 cell biopsy samples analyzed by in situ hybridization (ISH) and the remainder of the embryo was cultivated to the blastocyst stage and analyzed by PCR on the entire blastocyst.
  • ISH in situ hybridization
  • the ISH and PCR results correlate, with the ISH method identifying a few positive integrated transgenics that were scored as negative by PCR.
  • Transgenic murine embryos were produced by pronuclear microinjection and developed to the eight-cell stage.
  • Two-cell biopsies were performed and analyzed by ISH. Embryos that were scored as positive or negative for integrated transgene DNA (in blastocyst stage) were transferred to pseudopregnant foster mothers. Of 17 biopsies analyzed, there were 2 positives, 12 negatives, and 3 indeterminates (+/-) scored. DNA from the resultant pups is analyzed by Southern blotting in order to check the prediction by the biopsy ISH.

Abstract

La présente invention se rapporte à des procédés basés sur l'hybridation in situ et l'amplification enzymatique du génome, destinés à la détection précoce de transgènes intégrés dans le génome nucléaire.
PCT/US1992/005097 1991-06-12 1992-06-12 Detection precoce d'embryons transgeniques WO1992022647A1 (fr)

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Cited By (128)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0502976A1 (fr) * 1989-12-01 1992-09-16 Genpharm Int Production de polypeptides recombines par des especes bovines et procedes transgeniques.
WO1993022432A1 (fr) * 1992-05-01 1993-11-11 Genzyme Corporation Procedes d'identification d'embryons de preimplantation transgeniques
WO1995014376A1 (fr) * 1993-11-22 1995-06-01 Ppl Therapeutics (Scotland) Ltd. Detection pre-implantation
WO1995020653A1 (fr) * 1994-01-31 1995-08-03 Institut National De La Recherche Agronomique (Inra) Utilisation du gene de la luciferase de vargula pour la selection d'embryons transgeniques
WO1996036734A1 (fr) * 1995-05-18 1996-11-21 Abbott Laboratories Sondes de peptides polymeres et leurs utilisations
US5633076A (en) * 1989-12-01 1997-05-27 Pharming Bv Method of producing a transgenic bovine or transgenic bovine embryo
US5888733A (en) * 1995-11-16 1999-03-30 Dako A/S In situ hybridization to detect specific nucleic acid sequences in eucaryotic samples
US6168918B1 (en) 1996-01-31 2001-01-02 American Home Products Corp. Method of detecting foreign DNA integrated in eukaryotic chromosomes
US6682736B1 (en) 1998-12-23 2004-01-27 Abgenix, Inc. Human monoclonal antibodies to CTLA-4
WO2004050683A2 (fr) 2002-12-02 2004-06-17 Abgenix, Inc. Anticorps agissant sur le facteur de necrose des tumeurs (tnf) et leurs utilisation
WO2004084823A2 (fr) 2003-03-19 2004-10-07 Abgenix, Inc. Anticorps contre l'antigene de lymphocytes t, du domaine d'immunoglobuline et du domaine 1 de mucine (tim-1) et leurs utilisations
WO2005016111A2 (fr) 2003-08-08 2005-02-24 Abgenix, Inc. Anticorps diriges vers l'hormone parathyroide et leurs utilisations
WO2005092926A2 (fr) 2004-03-19 2005-10-06 Amgen Inc. Reduction du risque d'anticorps humains et anti-humains par la manipulation du gene v
US6984720B1 (en) 1999-08-24 2006-01-10 Medarex, Inc. Human CTLA-4 antibodies
WO2006003179A2 (fr) 2004-07-01 2006-01-12 Novo Nordisk A/S Anticorps anti-kir humains
WO2006055638A2 (fr) 2004-11-17 2006-05-26 Abgenix, Inc. Anticorps monoclonaux entierement humains diriges contre l'il-13
WO2006068975A2 (fr) 2004-12-20 2006-06-29 Abgenix, Inc. Proteines de liaison specifiques de la matriptase humaine
US7109003B2 (en) 1998-12-23 2006-09-19 Abgenix, Inc. Methods for expressing and recovering human monoclonal antibodies to CTLA-4
WO2007059082A1 (fr) 2005-11-10 2007-05-24 Curagen Corporation Methode de traitement du cancer de l'ovaire et du rein utilisant des anticorps diriges contre l'antigene a domaine 1 de mucine et a domaine immunoglobuline des lymphocytes t (tim-1)
WO2007077028A2 (fr) 2005-12-30 2007-07-12 U3 Pharma Ag Anticorps dirigés contre le her-3 et leurs utilisations
US7452535B2 (en) 2002-04-12 2008-11-18 Medarex, Inc. Methods of treatment using CTLA-4 antibodies
WO2009040134A1 (fr) 2007-09-26 2009-04-02 U3 Pharma Gmbh Protéines de liaison avec l'antigène du facteur de croissance de type facteur de croissance épidermique se liant à l'héparine
WO2009054873A2 (fr) 2007-08-02 2009-04-30 Novimmune S.A. Anticorps anti-rantes et leurs procédés d'utilisation
US7605238B2 (en) 1999-08-24 2009-10-20 Medarex, Inc. Human CTLA-4 antibodies and their uses
EP2110434A1 (fr) 2002-02-25 2009-10-21 Genentech, Inc. Recepteur de cytokine de type 1 GLM-R
EP2112166A2 (fr) 1998-12-23 2009-10-28 Pfizer Inc. Anticorps monoclonaux humaines pour CTLA-4
US7625549B2 (en) 2004-03-19 2009-12-01 Amgen Fremont Inc. Determining the risk of human anti-human antibodies in transgenic mice
WO2010054403A1 (fr) 2008-11-10 2010-05-14 Alexion Pharmaceuticals, Inc. Procédés et compositions pour le traitement de troubles associés au complément
WO2010072740A2 (fr) 2008-12-23 2010-07-01 Astrazeneca Ab Agents de liaison ciblés dirigés contre α5β1 et leurs applications
WO2010128407A2 (fr) 2009-05-05 2010-11-11 Novimmune S.A. Anticorps anti-il-17f et leurs méthodes d'utilisation
EP2284194A1 (fr) 2004-12-21 2011-02-16 AstraZeneca AB Anticorps dirigés contre l'Angiopoiétine 2 et leurs utilisations
WO2011057250A1 (fr) 2009-11-09 2011-05-12 Alexion Pharmaceuticals, Inc. Réactifs et procédés destinés à détecter des globules blancs associés à une hpn de type ii et leur identification comme facteurs de risque pour des troubles thrombotiques
WO2011060206A2 (fr) 2009-11-13 2011-05-19 U3 Pharma Gmbh Matière et procédés pour traiter ou prévenir des maladies associées à her-3
WO2011085343A1 (fr) 2010-01-11 2011-07-14 Alexion Pharmaceuticals, Inc Biomarqueurs d'effets immunomodulateurs chez des humains traités par des anticorps anti-cd200
EP2357202A1 (fr) 2006-04-10 2011-08-17 AstraZeneca AB Agents de liaison ciblés, dirigés contre l'uPAR, et utilisations
EP2361933A2 (fr) 2005-01-26 2011-08-31 Amgen Fremont Inc. Anticorps contre l'interleukine-1 bêta
WO2011137395A1 (fr) 2010-04-30 2011-11-03 Rother Russell P Anticorps anti-c5a et méthodes pour utiliser les anticorps
US8071323B2 (en) 2006-04-07 2011-12-06 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Human monoclonal antibodies that bind human insulin like growth factors and their use
EP2404616A2 (fr) 2005-12-13 2012-01-11 AstraZeneca AB Protéines de liaison spécifiques pour facteurs de croissance de type insuline et leurs utilisations
WO2012018404A2 (fr) 2010-08-06 2012-02-09 U3 Pharma Gmbh Utilisation d'agents de liaison her3 dans le traitement de la prostate
EP2420514A1 (fr) 2006-08-03 2012-02-22 MedImmune Limited Agents de liaison ciblés dirigés ciblant PDGFR-alpha et leurs utilisations
EP2457439A1 (fr) 1999-06-10 2012-05-30 Amgen Fremont Inc. Animaux transgeniques servant a produire des isotypes specifiques d'anticorps humains via des regions de transition non parentes
EP2457587A1 (fr) 2003-06-27 2012-05-30 Amgen Fremont Inc. Anticorps dirigés vers les mutants de suppression de récepteur de facteur de croissance épidermique et utilisations associées
EP2463305A1 (fr) 2006-01-12 2012-06-13 Alexion Pharmaceuticals, Inc. Anticorps pour OX-2/CD200 et utilisations associées
WO2012102679A1 (fr) 2011-01-24 2012-08-02 National University Of Singapore Protéines de liaison à l'antigène lipoarabinomannane coiffé d'un mannose provenant de mycobactéries pathogènes
WO2012106634A1 (fr) 2011-02-03 2012-08-09 Alexion Pharmaceuticals, Inc. Utilisation d'un anticorps anti-cd200 pour prolonger la survie d'allogreffes
WO2012138997A1 (fr) 2011-04-07 2012-10-11 Amgen Inc. Nouvelles protéines de liaison d'egfr
WO2012154999A1 (fr) 2011-05-10 2012-11-15 Amgen Inc. Procédés de traitement ou de prévention de troubles associés au cholestérol
EP2527456A1 (fr) 2004-10-22 2012-11-28 Revivicor Inc. Porcs transgéniques déficients en chaîne légère d'immunoglobuline endogène
WO2013012855A1 (fr) 2011-07-18 2013-01-24 Amgen Inc. Protéines de liaison à antigène de l'apéline et leurs utilisations
US8398975B2 (en) 2006-08-03 2013-03-19 Medimmune Limited Antibodies directed to αVβ6 and uses thereof
WO2013067060A1 (fr) 2011-11-01 2013-05-10 Bionomics, Inc. Anticorps anti-gpr49
WO2013067057A1 (fr) 2011-11-01 2013-05-10 Bionomics, Inc. Anticorps anti-gpr49
WO2013067055A1 (fr) 2011-11-01 2013-05-10 Bionomics, Inc. Procédés de blocage de la croissance des cellules souches cancéreuses
WO2013067054A1 (fr) 2011-11-01 2013-05-10 Bionomics, Inc. Anticorps et procédés de traitement du cancer
WO2013075048A1 (fr) 2011-11-16 2013-05-23 Amgen Inc. Procédé de traitement de troubles associés au mutant de délétion viii du récepteur du facteur de croissance épidermique
WO2013166448A1 (fr) 2012-05-03 2013-11-07 Amgen Inc. Formulations stables contenant des anticorps anti-pcsk9
WO2013188448A2 (fr) 2012-06-11 2013-12-19 Amgen Inc. Protéines de liaison à un antigène antagoniste d'un double récepteur et leurs utilisations
WO2014114801A1 (fr) 2013-01-25 2014-07-31 Amgen Inc. Anticorps ciblant cdh19 pour un mélanome
WO2014114800A1 (fr) 2013-01-25 2014-07-31 Amgen Research (Munich) Gmbh Constructions d'anticorps pour cdh19 et cd3
WO2014123580A1 (fr) 2013-02-06 2014-08-14 Inhibrx Llc Anticorps cd47 n'induisant ni l'appauvrissement en globules rouges ni l'appauvrissement en plaquettes
WO2014140368A1 (fr) 2013-03-15 2014-09-18 Amgen Research (Munich) Gmbh Constructions d'anticorps pour m2 et cd3 de grippe
WO2014140358A1 (fr) 2013-03-15 2014-09-18 Amgen Research (Munich) Gmbh Molécules de liaison à chaîne simple comprenant l'abp à l'extrémité n-terminale
WO2014209384A1 (fr) 2013-06-28 2014-12-31 Amgen Inc. Procédés de traitement d'une hypercholestérolémie familiale homozygote
US9045541B2 (en) 2012-02-06 2015-06-02 Inhibrx Llc CD47 antibodies and methods of use thereof
US9062111B2 (en) 2005-12-07 2015-06-23 Medarex, L.L.C. CTLA-4 antibody dosage escalation regimens
EP2918605A1 (fr) 2007-11-12 2015-09-16 U3 Pharma GmbH Anticorps axl
US9150644B2 (en) 2011-04-12 2015-10-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Human monoclonal antibodies that bind insulin-like growth factor (IGF) I and II
EP2927244A1 (fr) 2008-09-19 2015-10-07 MedImmune, LLC Anticorps dirigés contre DLL4 et leurs utilisations
DE202008018562U1 (de) 2007-08-23 2015-11-02 Amgen Inc. Antigenbindende Proteine gegen Proprotein Convertase Subtilisin Kexin Typ 9 (PCSK9)
WO2015195917A1 (fr) 2014-06-18 2015-12-23 Mersana Therapeutics, Inc. Anticorps monoclonaux dirigés contre l'épitope her2 et procédés d'utilisation de ceux-ci
WO2016016415A1 (fr) 2014-07-31 2016-02-04 Amgen Research (Munich) Gmbh Construction d'anticorps monocaténaires bispécifiques avec distribution tissulaire améliorée
WO2016016412A1 (fr) 2014-07-31 2016-02-04 Amgen Research (Munich) Gmbh Constructions d'anticorps pour cdh19 et cd3
WO2016016859A1 (fr) 2014-07-31 2016-02-04 Amgen Research (Munich) Gmbh Constructions optimisées d'anticorps monocaténaires, bispécifiques, spécifiques d'espèces croisées
WO2016166360A1 (fr) 2015-04-17 2016-10-20 Bayer Pharma Aktiengesellschaft Constructions d'anticorps bispécifiques pour cdh3 et cd3
US9546214B2 (en) 2014-04-04 2017-01-17 Bionomics, Inc. Humanized antibodies that bind LGR5
WO2017021362A1 (fr) 2015-07-31 2017-02-09 Amgen Research (Munich) Gmbh Constructions d'anticorps pour flt3 et cd3
WO2017021370A1 (fr) 2015-07-31 2017-02-09 Amgen Research (Munich) Gmbh Constructions d'anticorps bispécifiques se liant à l'egfrviii et à cd3
WO2017021354A1 (fr) 2015-07-31 2017-02-09 Amgen Research (Munich) Gmbh Constructions d'anticorps anti-cd70 et cd3
WO2017021349A1 (fr) 2015-07-31 2017-02-09 Amgen Research (Munich) Gmbh Constructions d'anticorps bispécifiques se liant à dll3 et à cd3
WO2017021356A1 (fr) 2015-07-31 2017-02-09 Amgen Research (Munich) Gmbh Constructions d'anticorps bispécifiques se liant à la mésothéline et à cd3
US9585374B2 (en) 2004-10-22 2017-03-07 Revivicor, Inc. Ungulates with genetically modified immune systems
WO2017160754A1 (fr) 2016-03-15 2017-09-21 Mersana Therapeutics,Inc. Conjugués anticorps-médicament ciblant napi2b et leurs procédés d'utilisation
WO2017182427A1 (fr) 2016-04-19 2017-10-26 Amgen Research (Munich) Gmbh Administration d'une construction bispécifique se liant à cd33 et cd3 destinée à une utilisation dans un procédé de traitement de la leucémie myéloïde
US9815890B2 (en) 2010-06-22 2017-11-14 The Regents Of The University Of Colorado, A Body Corporate Antibodies to the C3d fragment of complement component 3
EP3279215A1 (fr) 2009-11-24 2018-02-07 MedImmune Limited Agents de liaison ciblés contre b7-h1
WO2018049248A1 (fr) 2016-09-09 2018-03-15 Icellhealth Consulting Llc Virus oncolytique équipé de molécules d'engagement bispécifiques
WO2018141910A1 (fr) 2017-02-02 2018-08-09 Amgen Research (Munich) Gmbh Composition pharmaceutique à faible ph comprenant des constructions d'anticorps d'engagement avec les lymphocytes t
US10077304B2 (en) 2013-08-14 2018-09-18 The Governing Council Of The University Of Toronto Antibodies against frizzled receptor
WO2018204907A1 (fr) 2017-05-05 2018-11-08 Amgen Inc. Composition pharmaceutique comprenant des constructions d'anticorps bispécifiques pour un stockage et une administration améliorés
US10149461B2 (en) 2008-10-27 2018-12-11 Revivicor, Inc. Immunocompromised ungulates
US10301391B2 (en) 2016-02-03 2019-05-28 Amgen Research (Munich) Gmbh BCMA and CD3 bispecific T cell engaging antibody constructs
WO2019118426A1 (fr) 2017-12-11 2019-06-20 Amgen Inc. Procédé de fabrication continue pour des produits d'anticorps bispécifiques
WO2019133961A1 (fr) 2017-12-29 2019-07-04 Amgen Inc. Constructions d'anticorps bispécifiques dirigés contre muc17 et cd3
EP3524626A1 (fr) 2007-03-22 2019-08-14 Biogen MA Inc. Protéines de liaison, y compris des anticorps, dérivés et fragments d'anticorps qui se lient spécifiquement au cd154 et leurs utilisations
WO2020025792A1 (fr) 2018-08-03 2020-02-06 Amgen Research (Munich) Gmbh Constructions d'anticorps pour cldn18.2 et cd3
WO2020025532A1 (fr) 2018-07-30 2020-02-06 Amgen Research (Munich) Gmbh Administration prolongée d'une construction d'anticorps bispécifique se liant à cd33 et cd3
WO2020043670A1 (fr) 2018-08-27 2020-03-05 Affimed Gmbh Cellules nk cryoconservées préchargées avec une construction d'anticorps
WO2020077212A1 (fr) 2018-10-11 2020-04-16 Amgen Inc. Traitement en aval de constructions d'anticorps bispécifiques
US10745487B2 (en) 2016-03-22 2020-08-18 Bionomics Limited Method of treating cancer by administering an anti-LGR5 monoclonal antibody
US10781264B2 (en) 2016-02-03 2020-09-22 Amgen Research (Munich) Gmbh PSMA and CD3 bispecific T cell engaging antibody constructs
WO2020252442A1 (fr) 2019-06-13 2020-12-17 Amgen Inc. Commande de perfusion contenant de la biomasse automatisée dans la fabrication de produits biologiques
US10870701B2 (en) 2016-03-15 2020-12-22 Generon (Shanghai) Corporation Ltd. Multispecific fab fusion proteins and use thereof
WO2021021676A1 (fr) 2019-07-26 2021-02-04 Amgen Inc. Protéines de liaison à un antigène anti-il13
WO2021050640A1 (fr) 2019-09-10 2021-03-18 Amgen Inc. Procédé de purification de polypeptides de liaison à un antigène bispécifique présentant une capacité de liaison dynamique de capture de protéine l améliorée
WO2021097344A1 (fr) 2019-11-13 2021-05-20 Amgen Inc. Procédé de réduction de la formation d'agrégats dans le traitement en aval de molécules de liaison à l'antigène bispécifiques
US11013800B2 (en) 2011-05-16 2021-05-25 Evive Biotech Ltd. Multi-specific Fab fusion proteins comprising a CD3-binding Fab fragment with N-terminal fusion to binding domains and methods of use
WO2021127528A1 (fr) 2019-12-20 2021-06-24 Amgen Inc. Constructions d'anticorps multispécifique agoniste de cd40 ciblé par la mésothéline permettant le traitement de tumeurs solides
WO2021130383A1 (fr) 2019-12-27 2021-07-01 Affimed Gmbh Procédé de production de construction d'anticorps bispécifique fcyriii x cd30
WO2021150824A1 (fr) 2020-01-22 2021-07-29 Amgen Research (Munich) Gmbh Combinaisons de constructions d'anticorps et d'inhibiteurs du syndrome de libération de cytokine et leurs utilisations
WO2021183861A1 (fr) 2020-03-12 2021-09-16 Amgen Inc. Méthodes de traitement et de prophylaxie du crs chez des patients, comprenant une association d'anticorps bispécifiques se liant à une cellule tumorale du cds x et d'un inhibiteur du tnf alpha ou de l'il-6
WO2021188851A1 (fr) 2020-03-19 2021-09-23 Amgen Inc. Anticorps contre la mucine 17 et leurs utilisations
WO2021236638A1 (fr) 2020-05-19 2021-11-25 Amgen Inc. Constructions de liaison à mageb2
WO2021243320A2 (fr) 2020-05-29 2021-12-02 Amgen Inc. Administration atténuant des effets indésirables d'une construction d'anticorps bispécifique de liaison à cd33 et cd3
WO2022074206A1 (fr) 2020-10-08 2022-04-14 Affimed Gmbh Lieurs trispécifiques
WO2022096704A1 (fr) 2020-11-06 2022-05-12 Amgen Inc. Domaine de liaison à l'antigène à taux de coupure réduit
WO2022096700A1 (fr) 2020-11-06 2022-05-12 Amgen Research (Munich) Gmbh Constructions polypeptidiques se liant sélectivement à cldn6 et cd3
WO2022096698A1 (fr) 2020-11-06 2022-05-12 Amgen Inc. Constructions polypeptidiques se liant à cd3
WO2022096716A2 (fr) 2020-11-06 2022-05-12 Amgen Inc. Molécules bispécifiques multicibles de liaison à un antigène à sélectivité accrue
US11434302B2 (en) 2016-02-03 2022-09-06 Amgen Research (Munich) Gmbh Bispecific T cell engaging antibody constructs
WO2022212831A1 (fr) 2021-04-02 2022-10-06 Amgen Inc. Constructions de liaison à mageb2
WO2022234102A1 (fr) 2021-05-06 2022-11-10 Amgen Research (Munich) Gmbh Molécules de liaison à l'antigène ciblant cd20 et cd22 destinées à être utilisées dans des maladies prolifératives
WO2023007023A1 (fr) 2021-07-30 2023-02-02 Affimed Gmbh Corps duplex
WO2023079493A1 (fr) 2021-11-03 2023-05-11 Affimed Gmbh Liants de cd16a bispécifiques
WO2023078968A1 (fr) 2021-11-03 2023-05-11 Affimed Gmbh Liants de cd16a bispécifiques
EP4248976A2 (fr) 2007-08-23 2023-09-27 Amgen Inc. Protéines de liaison à un antigène pour proprotéine convertase subtilisine kexine de type 9 (pcsk9)
WO2023218027A1 (fr) 2022-05-12 2023-11-16 Amgen Research (Munich) Gmbh Molécules bispécifiques multicibles à chaînes multiples de liaison à un antigène à sélectivité accrue
WO2024059675A2 (fr) 2022-09-14 2024-03-21 Amgen Inc. Composition de stabilisation de molécule bispécifique

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3717797A (en) * 1996-07-15 1998-02-09 Children's Medical Center Corporation Method to co-detect introduced genes and their products

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DEVELOPMENTAL BIOLOGY, Volume 101, issued 1984, K.H. COX et al., "Detection of mRNAs in Sea Urchin Embryos by in situ Hybridization using Asymmetric RNA Probes", pages 485-502. *
R.B. CHURCH, "Transgenic Models in Medicine and Agriculture", published 1990, by WILEY-LISS, INC., pages 33-45. *

Cited By (234)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5741957A (en) * 1989-12-01 1998-04-21 Pharming B.V. Transgenic bovine
EP0737746A2 (fr) * 1989-12-01 1996-10-16 Pharming B.V. Production de polypeptides recombinants par l'espèce bovine et procédés transgéniques
EP0737746A3 (fr) * 1989-12-01 1996-10-23 Pharming B.V. Production de polypeptides recombinants par l'espèce bovine et procédés transgéniques
US5633076A (en) * 1989-12-01 1997-05-27 Pharming Bv Method of producing a transgenic bovine or transgenic bovine embryo
EP0502976A1 (fr) * 1989-12-01 1992-09-16 Genpharm Int Production de polypeptides recombines par des especes bovines et procedes transgeniques.
US6013857A (en) * 1989-12-01 2000-01-11 Pharming B.V. Transgenic bovines and milk from transgenic bovines
WO1993022432A1 (fr) * 1992-05-01 1993-11-11 Genzyme Corporation Procedes d'identification d'embryons de preimplantation transgeniques
WO1995014376A1 (fr) * 1993-11-22 1995-06-01 Ppl Therapeutics (Scotland) Ltd. Detection pre-implantation
WO1995020653A1 (fr) * 1994-01-31 1995-08-03 Institut National De La Recherche Agronomique (Inra) Utilisation du gene de la luciferase de vargula pour la selection d'embryons transgeniques
FR2715665A1 (fr) * 1994-01-31 1995-08-04 Agronomique Inst Nat Rech Utilisation du gène de la luciférase de Vargula pour la sélection d'embryons transgéniques.
WO1996036734A1 (fr) * 1995-05-18 1996-11-21 Abbott Laboratories Sondes de peptides polymeres et leurs utilisations
US5888733A (en) * 1995-11-16 1999-03-30 Dako A/S In situ hybridization to detect specific nucleic acid sequences in eucaryotic samples
US6168918B1 (en) 1996-01-31 2001-01-02 American Home Products Corp. Method of detecting foreign DNA integrated in eukaryotic chromosomes
US7411057B2 (en) 1998-12-23 2008-08-12 Amgen Fremont Inc. Nucleic acids encoding human monoclonal antibodies to CTLA-4
EP2112166A2 (fr) 1998-12-23 2009-10-28 Pfizer Inc. Anticorps monoclonaux humaines pour CTLA-4
US8143379B2 (en) 1998-12-23 2012-03-27 Amgen Fremont Inc. Human monoclonal antibodies to CTLA-4
US8491895B2 (en) 1998-12-23 2013-07-23 Amgen Fremont Inc. Methods of treating cancer with human monoclonal antibodies to CTLA-4
US8883984B2 (en) 1998-12-23 2014-11-11 Amgen Fremont Inc. Human monoclonal antibodies to CTLA-4
US7824679B2 (en) 1998-12-23 2010-11-02 Amgen Fremont Inc. Human monoclonal antibodies to CTLA-4
US7807797B2 (en) 1998-12-23 2010-10-05 Amgen Fremont Inc. Human monoclonal antibodies to CTLA-4
US9963508B2 (en) 1998-12-23 2018-05-08 Amgen Fremont Inc. Human monoclonal antibodies to CTLA-4
EP3553085A1 (fr) 1998-12-23 2019-10-16 Pfizer Inc Anticorps monoclonaux humains dirigés contre l'antigène ctla-4
US7109003B2 (en) 1998-12-23 2006-09-19 Abgenix, Inc. Methods for expressing and recovering human monoclonal antibodies to CTLA-4
US7132281B2 (en) 1998-12-23 2006-11-07 Amgen Fremont Inc. Methods and host cells for producing human monoclonal antibodies to CTLA-4
US6682736B1 (en) 1998-12-23 2004-01-27 Abgenix, Inc. Human monoclonal antibodies to CTLA-4
EP2457439A1 (fr) 1999-06-10 2012-05-30 Amgen Fremont Inc. Animaux transgeniques servant a produire des isotypes specifiques d'anticorps humains via des regions de transition non parentes
US8017114B2 (en) 1999-08-24 2011-09-13 Medarex, Inc. Human CTLA-4 antibodies and their uses
US7605238B2 (en) 1999-08-24 2009-10-20 Medarex, Inc. Human CTLA-4 antibodies and their uses
US6984720B1 (en) 1999-08-24 2006-01-10 Medarex, Inc. Human CTLA-4 antibodies
EP2110434A1 (fr) 2002-02-25 2009-10-21 Genentech, Inc. Recepteur de cytokine de type 1 GLM-R
US7452535B2 (en) 2002-04-12 2008-11-18 Medarex, Inc. Methods of treatment using CTLA-4 antibodies
WO2004050683A2 (fr) 2002-12-02 2004-06-17 Abgenix, Inc. Anticorps agissant sur le facteur de necrose des tumeurs (tnf) et leurs utilisation
WO2004084823A2 (fr) 2003-03-19 2004-10-07 Abgenix, Inc. Anticorps contre l'antigene de lymphocytes t, du domaine d'immunoglobuline et du domaine 1 de mucine (tim-1) et leurs utilisations
EP3000886A1 (fr) 2003-03-19 2016-03-30 Amgen Fremont Inc. Anticorps contre l'antigene de lymphocytes t, du domaine d'immunoglobuline et du domaine 1 de mucine (tim-1) et leurs utilisations
EP3679951A1 (fr) 2003-06-27 2020-07-15 Amgen Fremont Inc. Anticorps dirigés vers les mutants de suppression de récepteur de facteur de croissance épidermique et utilisations associées
EP2457586A1 (fr) 2003-06-27 2012-05-30 Amgen Fremont Inc. Anticorps dirigés vers les mutants de suppression de récepteur de facteur de croissance épidermique et utilisations associées
EP3037105A1 (fr) 2003-06-27 2016-06-29 Amgen Fremont Inc. Anticorps dirigés vers les mutants de suppression de récepteur de facteur de croissance épidermique et utilisations associées
EP2457587A1 (fr) 2003-06-27 2012-05-30 Amgen Fremont Inc. Anticorps dirigés vers les mutants de suppression de récepteur de facteur de croissance épidermique et utilisations associées
EP3011971A1 (fr) 2003-06-27 2016-04-27 Amgen Fremont Inc. Anticorps dirigés vers les mutants de suppression de récepteur de facteur de croissance épidermique et utilisations associées
WO2005016111A2 (fr) 2003-08-08 2005-02-24 Abgenix, Inc. Anticorps diriges vers l'hormone parathyroide et leurs utilisations
US8597615B2 (en) 2004-03-19 2013-12-03 Amgen Fremont Inc. Methods of monitoring a human anti-human antibody response and inhibitors thereof
WO2005092926A2 (fr) 2004-03-19 2005-10-06 Amgen Inc. Reduction du risque d'anticorps humains et anti-humains par la manipulation du gene v
US8198508B2 (en) 2004-03-19 2012-06-12 Amgen Fremont, Inc. Reducing the risk of human anti-human antibodies through V gene manipulation
US7625549B2 (en) 2004-03-19 2009-12-01 Amgen Fremont Inc. Determining the risk of human anti-human antibodies in transgenic mice
EP2418224A2 (fr) 2004-03-19 2012-02-15 Amgen Inc. Réduction du risque d'anticorps humains et anti-humains par manipulation du gène V
EP2287195A2 (fr) 2004-07-01 2011-02-23 Novo Nordisk A/S Anticorps contre le Pan-KIR2DL NK-récepteur et leur utilisation diagnostique et thérapeutique
WO2006003179A2 (fr) 2004-07-01 2006-01-12 Novo Nordisk A/S Anticorps anti-kir humains
US11085054B2 (en) 2004-10-22 2021-08-10 Revivicor, Inc. Ungulates with genetically modified immune systems
EP2527456A1 (fr) 2004-10-22 2012-11-28 Revivicor Inc. Porcs transgéniques déficients en chaîne légère d'immunoglobuline endogène
US9585374B2 (en) 2004-10-22 2017-03-07 Revivicor, Inc. Ungulates with genetically modified immune systems
WO2006055638A2 (fr) 2004-11-17 2006-05-26 Abgenix, Inc. Anticorps monoclonaux entierement humains diriges contre l'il-13
WO2006068975A2 (fr) 2004-12-20 2006-06-29 Abgenix, Inc. Proteines de liaison specifiques de la matriptase humaine
EP3699191A1 (fr) 2004-12-21 2020-08-26 MedImmune Limited Anticorps dirigés contre angiopoietin-2 et leurs utilisations
EP2284194A1 (fr) 2004-12-21 2011-02-16 AstraZeneca AB Anticorps dirigés contre l'Angiopoiétine 2 et leurs utilisations
EP2361933A2 (fr) 2005-01-26 2011-08-31 Amgen Fremont Inc. Anticorps contre l'interleukine-1 bêta
WO2007059082A1 (fr) 2005-11-10 2007-05-24 Curagen Corporation Methode de traitement du cancer de l'ovaire et du rein utilisant des anticorps diriges contre l'antigene a domaine 1 de mucine et a domaine immunoglobuline des lymphocytes t (tim-1)
EP2548583A2 (fr) 2005-11-10 2013-01-23 Curagen Corporation Methode de traitement du cancer de l'ovaire et du rein utilisant des anticorps diriges contre l'antigene a domaine 1 de mucine et a domaine immunoglobuline des lymphocytes t (tim-1)
US9573999B2 (en) 2005-12-07 2017-02-21 E. R. Squibb & Sons, L.L.C. CTLA-4 antibody dosage escalation regimens
US9062111B2 (en) 2005-12-07 2015-06-23 Medarex, L.L.C. CTLA-4 antibody dosage escalation regimens
EP2404616A2 (fr) 2005-12-13 2012-01-11 AstraZeneca AB Protéines de liaison spécifiques pour facteurs de croissance de type insuline et leurs utilisations
EP2993187A2 (fr) 2005-12-30 2016-03-09 U3 Pharma GmbH Anticorps dirigés contre her-3 et leurs utilisations
US9988462B2 (en) 2005-12-30 2018-06-05 Daiichi Sankyo Europe Gmbh Material and methods for treating or preventing HER-3 associated diseases
WO2007077028A2 (fr) 2005-12-30 2007-07-12 U3 Pharma Ag Anticorps dirigés contre le her-3 et leurs utilisations
US8771695B2 (en) 2005-12-30 2014-07-08 U3 Pharma Gmbh Antibodies directed to HER-3 and uses thereof
EP3196213A2 (fr) 2005-12-30 2017-07-26 Daiichi Sankyo Europe GmbH Anticorps dirigés contre her-3 et leurs utilisations
EP3950715A1 (fr) 2005-12-30 2022-02-09 Amgen Inc. Anticorps dirigés contre her-3 et leurs utilisations
US7705130B2 (en) 2005-12-30 2010-04-27 U3 Pharma Gmbh Antibodies directed to HER-3 and uses thereof
EP2463305A1 (fr) 2006-01-12 2012-06-13 Alexion Pharmaceuticals, Inc. Anticorps pour OX-2/CD200 et utilisations associées
EP3101033A1 (fr) 2006-01-12 2016-12-07 Alexion Pharmaceuticals, Inc. Anticorps pour ox-2/cd200 et utilisations associées
US8071323B2 (en) 2006-04-07 2011-12-06 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Human monoclonal antibodies that bind human insulin like growth factors and their use
EP2357202A1 (fr) 2006-04-10 2011-08-17 AstraZeneca AB Agents de liaison ciblés, dirigés contre l'uPAR, et utilisations
US8398975B2 (en) 2006-08-03 2013-03-19 Medimmune Limited Antibodies directed to αVβ6 and uses thereof
US8894998B2 (en) 2006-08-03 2014-11-25 Medimmune Limited Antibodies directed to αVβ6 and uses thereof
EP2420514A1 (fr) 2006-08-03 2012-02-22 MedImmune Limited Agents de liaison ciblés dirigés ciblant PDGFR-alpha et leurs utilisations
EP2420513A1 (fr) 2006-08-03 2012-02-22 MedImmune Limited Agents de liaison ciblés dirigés ciblant PDGFR-alpha et leurs utilisations
EP3524626A1 (fr) 2007-03-22 2019-08-14 Biogen MA Inc. Protéines de liaison, y compris des anticorps, dérivés et fragments d'anticorps qui se lient spécifiquement au cd154 et leurs utilisations
WO2009054873A2 (fr) 2007-08-02 2009-04-30 Novimmune S.A. Anticorps anti-rantes et leurs procédés d'utilisation
EP3666797A1 (fr) 2007-08-23 2020-06-17 Amgen, Inc Protéines de liaison à un antigène pour proprotéine convertase subtilisine kexine de type 9 (pcsk9)
EP4248976A2 (fr) 2007-08-23 2023-09-27 Amgen Inc. Protéines de liaison à un antigène pour proprotéine convertase subtilisine kexine de type 9 (pcsk9)
DE202008018562U1 (de) 2007-08-23 2015-11-02 Amgen Inc. Antigenbindende Proteine gegen Proprotein Convertase Subtilisin Kexin Typ 9 (PCSK9)
EP2497783A2 (fr) 2007-09-26 2012-09-12 U3 Pharma GmbH Protéines de liaison avec l'antigène du facteur de croissance de type facteur de croissance épidermique se liant à l'héparine
WO2009040134A1 (fr) 2007-09-26 2009-04-02 U3 Pharma Gmbh Protéines de liaison avec l'antigène du facteur de croissance de type facteur de croissance épidermique se liant à l'héparine
EP2918605A1 (fr) 2007-11-12 2015-09-16 U3 Pharma GmbH Anticorps axl
EP2927244A1 (fr) 2008-09-19 2015-10-07 MedImmune, LLC Anticorps dirigés contre DLL4 et leurs utilisations
US10149461B2 (en) 2008-10-27 2018-12-11 Revivicor, Inc. Immunocompromised ungulates
WO2010054403A1 (fr) 2008-11-10 2010-05-14 Alexion Pharmaceuticals, Inc. Procédés et compositions pour le traitement de troubles associés au complément
EP3101031A1 (fr) 2008-11-10 2016-12-07 Alexion Pharmaceuticals, Inc. Procédés et compositions de traitement de troubles associés à un complément
EP2894166A1 (fr) 2008-11-10 2015-07-15 Alexion Pharmaceuticals, Inc. Procédés et compositions pour le traitement de troubles associés à un complément
EP3121197A1 (fr) 2008-11-10 2017-01-25 Alexion Pharmaceuticals, Inc. Procédés et compositions de traitement de troubles associés à un complément
EP3974448A1 (fr) 2008-11-10 2022-03-30 Alexion Pharmaceuticals, Inc. Procédés et compositions de traitement de troubles associés à un complément
EP2894165A1 (fr) 2008-11-10 2015-07-15 Alexion Pharmaceuticals, Inc. Procédés et compositions pour le traitement de troubles associés à un complément
WO2010072740A2 (fr) 2008-12-23 2010-07-01 Astrazeneca Ab Agents de liaison ciblés dirigés contre α5β1 et leurs applications
WO2010128407A2 (fr) 2009-05-05 2010-11-11 Novimmune S.A. Anticorps anti-il-17f et leurs méthodes d'utilisation
WO2011057250A1 (fr) 2009-11-09 2011-05-12 Alexion Pharmaceuticals, Inc. Réactifs et procédés destinés à détecter des globules blancs associés à une hpn de type ii et leur identification comme facteurs de risque pour des troubles thrombotiques
EP3351558A2 (fr) 2009-11-13 2018-07-25 Daiichi Sankyo Europe GmbH Matériau et procédés de traitement ou de prévention de maladies associées à des her-3
US9803025B2 (en) 2009-11-13 2017-10-31 Amgen, Inc. Material and methods for treating or preventing HER-3 associated diseases
US9101760B2 (en) 2009-11-13 2015-08-11 U3 Pharma Gmbh Material and methods for treating or preventing HER-3 associated diseases
WO2011060206A2 (fr) 2009-11-13 2011-05-19 U3 Pharma Gmbh Matière et procédés pour traiter ou prévenir des maladies associées à her-3
EP2719708A2 (fr) 2009-11-13 2014-04-16 U3 Pharma GmbH Matériau et procédés de traitement ou de prévention de maladies associées à des HER-3
EP2896632A2 (fr) 2009-11-13 2015-07-22 U3 Pharma GmbH Matériau et procédés de traitement ou de prévention de maladies associées à des HER-3
EP3670539A1 (fr) 2009-11-13 2020-06-24 Daiichi Sankyo Europe GmbH Matériau et procédés de traitement ou de prévention de maladies associées à des her-3
EP3279215A1 (fr) 2009-11-24 2018-02-07 MedImmune Limited Agents de liaison ciblés contre b7-h1
WO2011085343A1 (fr) 2010-01-11 2011-07-14 Alexion Pharmaceuticals, Inc Biomarqueurs d'effets immunomodulateurs chez des humains traités par des anticorps anti-cd200
US9469690B2 (en) 2010-04-30 2016-10-18 Alexion Pharmaceuticals, Inc. Methods of treating complement-associated disorders with anti-C5a antibodies
US11407821B2 (en) 2010-04-30 2022-08-09 Alexion Pharmaceuticals, Inc. Anti-C5A antibodies
US9221901B2 (en) 2010-04-30 2015-12-29 Alexion Pharmaceuticals, Inc. Methods of treating complement-associated disorders with anti-C5a antibodies
US9963503B2 (en) 2010-04-30 2018-05-08 Alexion Pharmaceuticals, Inc. Methods of producing anti-C5a antibodies
WO2011137395A1 (fr) 2010-04-30 2011-11-03 Rother Russell P Anticorps anti-c5a et méthodes pour utiliser les anticorps
EP2824111A2 (fr) 2010-04-30 2015-01-14 Alexion Pharmaceuticals, Inc. Anticorps anti-C5A et leurs procédés d'utilisation de ces anticorps
US9011852B2 (en) 2010-04-30 2015-04-21 Alexion Pharmaceuticals, Inc. Anti-C5a antibodies
US9434784B1 (en) 2010-04-30 2016-09-06 Alexion Pharmaceuticals, Inc. Nucleic acids encodng anti-C5A antibodies
US9309310B2 (en) 2010-04-30 2016-04-12 Alexion Pharmaceuticals, Inc. Nucleic acids encoding anti-C5a antibodies
US10450370B2 (en) 2010-04-30 2019-10-22 Alexion Pharmaceuticals, Inc. Anti-C5a antibodies
US9371378B1 (en) 2010-04-30 2016-06-21 Alexion Pharmaceuticals, Inc. Anti-C5a antibodies
US9815890B2 (en) 2010-06-22 2017-11-14 The Regents Of The University Of Colorado, A Body Corporate Antibodies to the C3d fragment of complement component 3
USRE49339E1 (en) 2010-06-22 2022-12-20 The Regents Of The University Of Colorado, A Body Corporate Antibodies to the C3D fragment of complement component 3
WO2012018404A2 (fr) 2010-08-06 2012-02-09 U3 Pharma Gmbh Utilisation d'agents de liaison her3 dans le traitement de la prostate
WO2012102679A1 (fr) 2011-01-24 2012-08-02 National University Of Singapore Protéines de liaison à l'antigène lipoarabinomannane coiffé d'un mannose provenant de mycobactéries pathogènes
WO2012106634A1 (fr) 2011-02-03 2012-08-09 Alexion Pharmaceuticals, Inc. Utilisation d'un anticorps anti-cd200 pour prolonger la survie d'allogreffes
WO2012138997A1 (fr) 2011-04-07 2012-10-11 Amgen Inc. Nouvelles protéines de liaison d'egfr
US9676846B2 (en) 2011-04-12 2017-06-13 The United States Of America As Represented By The Secretary, Department Of Health And Human Services Human monoclonal antibodies that bind insulin-like growth factor (IGF) I and II
US9150644B2 (en) 2011-04-12 2015-10-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Human monoclonal antibodies that bind insulin-like growth factor (IGF) I and II
EP3597218A1 (fr) 2011-05-10 2020-01-22 Amgen, Inc Procédés de traitement ou de prévention de troubles associés au cholestérol
WO2012154999A1 (fr) 2011-05-10 2012-11-15 Amgen Inc. Procédés de traitement ou de prévention de troubles associés au cholestérol
US11013800B2 (en) 2011-05-16 2021-05-25 Evive Biotech Ltd. Multi-specific Fab fusion proteins comprising a CD3-binding Fab fragment with N-terminal fusion to binding domains and methods of use
WO2013012855A1 (fr) 2011-07-18 2013-01-24 Amgen Inc. Protéines de liaison à antigène de l'apéline et leurs utilisations
US10598653B2 (en) 2011-11-01 2020-03-24 Bionomics Inc. Methods of blocking cancer stem cell growth
WO2013067054A1 (fr) 2011-11-01 2013-05-10 Bionomics, Inc. Anticorps et procédés de traitement du cancer
WO2013067055A1 (fr) 2011-11-01 2013-05-10 Bionomics, Inc. Procédés de blocage de la croissance des cellules souches cancéreuses
US10196442B2 (en) 2011-11-01 2019-02-05 Bionomics Inc. Methods of inhibiting growth of a colon cancer tumor in a subject by administering monoclonal antibodies to G protein-coupled receptor 49 (GPR49)
WO2013067057A1 (fr) 2011-11-01 2013-05-10 Bionomics, Inc. Anticorps anti-gpr49
WO2013067060A1 (fr) 2011-11-01 2013-05-10 Bionomics, Inc. Anticorps anti-gpr49
US9221907B2 (en) 2011-11-01 2015-12-29 Bionomics Inc. Anti-GPR49 monoclonal antibodies
US9220774B2 (en) 2011-11-01 2015-12-29 Bionomics Inc. Methods of treating cancer by administering anti-GPR49 antibodies
US9221906B2 (en) 2011-11-01 2015-12-29 Bionomics Inc. Methods of inhibiting solid tumor growth by administering GPR49 antibodies
WO2013075048A1 (fr) 2011-11-16 2013-05-23 Amgen Inc. Procédé de traitement de troubles associés au mutant de délétion viii du récepteur du facteur de croissance épidermique
US9663575B2 (en) 2012-02-06 2017-05-30 Inhibrx, Lp CD47 antibodies and methods of use thereof
EP3578569A1 (fr) 2012-02-06 2019-12-11 Inhibrx, Inc. Anticorps anti-cd47 et leurs méthodes d'utilisation
US9045541B2 (en) 2012-02-06 2015-06-02 Inhibrx Llc CD47 antibodies and methods of use thereof
WO2013166448A1 (fr) 2012-05-03 2013-11-07 Amgen Inc. Formulations stables contenant des anticorps anti-pcsk9
EP3656399A1 (fr) 2012-05-03 2020-05-27 Amgen, Inc Formulations stables contenant des anticorps anti-pcsk9
EP3540070A1 (fr) 2012-06-11 2019-09-18 Amgen Inc. Protéines de liaison à un antigène antagoniste d'un double récepteur et leurs utilisations
WO2013188448A2 (fr) 2012-06-11 2013-12-19 Amgen Inc. Protéines de liaison à un antigène antagoniste d'un double récepteur et leurs utilisations
EP3498857A1 (fr) 2012-06-11 2019-06-19 Amgen, Inc. Protéines de liaison à un antigène antagoniste d'un double récepteur et leurs utilisations
US11498964B2 (en) 2013-01-25 2022-11-15 Amgen Research (Munich) Gmbh Antibody constructs for CDH19 and CD3
EP3699194A1 (fr) 2013-01-25 2020-08-26 Amgen Research (Munich) GmbH Constructions d'anticorps pour cdh19 et cd3
WO2014114801A1 (fr) 2013-01-25 2014-07-31 Amgen Inc. Anticorps ciblant cdh19 pour un mélanome
WO2014114800A1 (fr) 2013-01-25 2014-07-31 Amgen Research (Munich) Gmbh Constructions d'anticorps pour cdh19 et cd3
EP4137518A1 (fr) 2013-02-06 2023-02-22 Inhibrx, Inc. Anticorps cd47 n'induisant ni l'appauvrissement en globules rouges ni l'appauvrissement en plaquettes
WO2014123580A1 (fr) 2013-02-06 2014-08-14 Inhibrx Llc Anticorps cd47 n'induisant ni l'appauvrissement en globules rouges ni l'appauvrissement en plaquettes
EP3653642A1 (fr) 2013-03-15 2020-05-20 Amgen Research (Munich) GmbH Molécules de liaison à chaîne unique comprenant une abp n-terminale
WO2014140358A1 (fr) 2013-03-15 2014-09-18 Amgen Research (Munich) Gmbh Molécules de liaison à chaîne simple comprenant l'abp à l'extrémité n-terminale
WO2014140368A1 (fr) 2013-03-15 2014-09-18 Amgen Research (Munich) Gmbh Constructions d'anticorps pour m2 et cd3 de grippe
WO2014209384A1 (fr) 2013-06-28 2014-12-31 Amgen Inc. Procédés de traitement d'une hypercholestérolémie familiale homozygote
US10077304B2 (en) 2013-08-14 2018-09-18 The Governing Council Of The University Of Toronto Antibodies against frizzled receptor
EP3705494A2 (fr) 2013-08-14 2020-09-09 Sachdev Sidhu Anticorps contre les protéines frizzled et leurs méthodes d'utilisation
US10689442B2 (en) 2013-08-14 2020-06-23 Sachdev Sidhu Antibodies against Frizzled receptor
US10358500B2 (en) 2014-04-04 2019-07-23 Bionomics Inc. Humanized antibodies that bind LGR5
US9546214B2 (en) 2014-04-04 2017-01-17 Bionomics, Inc. Humanized antibodies that bind LGR5
WO2015195917A1 (fr) 2014-06-18 2015-12-23 Mersana Therapeutics, Inc. Anticorps monoclonaux dirigés contre l'épitope her2 et procédés d'utilisation de ceux-ci
EP4285917A2 (fr) 2014-06-18 2023-12-06 Mersana Therapeutics, Inc. Anticorps monoclonaux dirigés contre l'épitope her2 et procédés d'utilisation de ceux-ci
WO2016016859A1 (fr) 2014-07-31 2016-02-04 Amgen Research (Munich) Gmbh Constructions optimisées d'anticorps monocaténaires, bispécifiques, spécifiques d'espèces croisées
WO2016016412A1 (fr) 2014-07-31 2016-02-04 Amgen Research (Munich) Gmbh Constructions d'anticorps pour cdh19 et cd3
WO2016016415A1 (fr) 2014-07-31 2016-02-04 Amgen Research (Munich) Gmbh Construction d'anticorps monocaténaires bispécifiques avec distribution tissulaire améliorée
US9765157B2 (en) 2014-07-31 2017-09-19 Amgen Research (Munich) Gmbh Antibody constructs for CDH19 and CD3
US11661462B2 (en) 2014-07-31 2023-05-30 Amgen Research (Munich) Gmbh Optimized cross-species specific bispecific single chain antibody contructs
US11028171B2 (en) 2015-04-17 2021-06-08 Amgen Research (Munich) Gmbh Bispecific antibody constructs for CDH3 and CD3
EP4276116A2 (fr) 2015-04-17 2023-11-15 Amgen Research (Munich) GmbH Constructions d'anticorps bispécifiques pour cdh3 et cd3
US11926666B2 (en) 2015-04-17 2024-03-12 Amgen Research (Munich) Gmbh Bispecific antibody constructs for CDH3 and CD3
WO2016166360A1 (fr) 2015-04-17 2016-10-20 Bayer Pharma Aktiengesellschaft Constructions d'anticorps bispécifiques pour cdh3 et cd3
EP4327885A2 (fr) 2015-07-31 2024-02-28 Amgen Research (Munich) GmbH Constructions d'anticorps pour msln et cd3
EP4219562A2 (fr) 2015-07-31 2023-08-02 Amgen Research (Munich) GmbH Constructions d'anticorps pour flt3 et cd3
US11447567B2 (en) 2015-07-31 2022-09-20 Amgen Research (Munich) Gmbh Antibody constructs for FLT3 and CD3
WO2017021362A1 (fr) 2015-07-31 2017-02-09 Amgen Research (Munich) Gmbh Constructions d'anticorps pour flt3 et cd3
WO2017021370A1 (fr) 2015-07-31 2017-02-09 Amgen Research (Munich) Gmbh Constructions d'anticorps bispécifiques se liant à l'egfrviii et à cd3
US10519241B2 (en) 2015-07-31 2019-12-31 Amgen Research (Munich) Gmbh Antibody constructs for EGFRVIII and CD3
WO2017021354A1 (fr) 2015-07-31 2017-02-09 Amgen Research (Munich) Gmbh Constructions d'anticorps anti-cd70 et cd3
WO2017021349A1 (fr) 2015-07-31 2017-02-09 Amgen Research (Munich) Gmbh Constructions d'anticorps bispécifiques se liant à dll3 et à cd3
WO2017021356A1 (fr) 2015-07-31 2017-02-09 Amgen Research (Munich) Gmbh Constructions d'anticorps bispécifiques se liant à la mésothéline et à cd3
US10851170B2 (en) 2015-07-31 2020-12-01 Amgen Research (Munich) Gmbh Antibody constructs for CD70 and CD3
US10683351B2 (en) 2015-07-31 2020-06-16 Amgen Research (Munich) Gmbh Antibody constructs for DLL3 and CD3
US11591396B2 (en) 2015-07-31 2023-02-28 Amgen Research (Munich) Gmbh Antibody constructs for DLL3 and CD3
US11884720B2 (en) 2015-07-31 2024-01-30 Amgen Research (Munich) Gmbh Antibody constructs for MSLN and CD3
EP3912999A1 (fr) 2015-07-31 2021-11-24 Amgen Research (Munich) GmbH Constructions d'anticorps bispécifiques se liant à egfrviii et cd3
US10294300B2 (en) 2015-07-31 2019-05-21 Amgen Research (Munich) Gmbh Antibody constructs for DLL3 and CD3
US11155629B2 (en) 2015-07-31 2021-10-26 Amgen Research (Munich) Gmbh Method for treating glioblastoma or glioma with antibody constructs for EGFRVIII and CD3
EP3865514A1 (fr) 2015-07-31 2021-08-18 Amgen Research (Munich) GmbH Constructions d'anticorps pour dll3 et cd3
US10301391B2 (en) 2016-02-03 2019-05-28 Amgen Research (Munich) Gmbh BCMA and CD3 bispecific T cell engaging antibody constructs
EP4206228A1 (fr) 2016-02-03 2023-07-05 Amgen Research (Munich) GmbH Constructions d'engagement de lymphocytes t bispécifiques psma et cd3
US11434302B2 (en) 2016-02-03 2022-09-06 Amgen Research (Munich) Gmbh Bispecific T cell engaging antibody constructs
EP4039709A1 (fr) 2016-02-03 2022-08-10 Amgen Research (Munich) GmbH Constructions d'anticorps engageant les lymphocytes bispécifiques bcma et cd3
US10781264B2 (en) 2016-02-03 2020-09-22 Amgen Research (Munich) Gmbh PSMA and CD3 bispecific T cell engaging antibody constructs
US11352433B2 (en) 2016-02-03 2022-06-07 Amgen Research (Munich) Gmbh BCMA and CD3 bispecific T cell engaging antibody constructs
US10870701B2 (en) 2016-03-15 2020-12-22 Generon (Shanghai) Corporation Ltd. Multispecific fab fusion proteins and use thereof
WO2017160754A1 (fr) 2016-03-15 2017-09-21 Mersana Therapeutics,Inc. Conjugués anticorps-médicament ciblant napi2b et leurs procédés d'utilisation
EP4302782A2 (fr) 2016-03-15 2024-01-10 Mersana Therapeutics, Inc. Conjugués anticorps-médicament ciblant napi2b et leurs procédés d'utilisation
US10745487B2 (en) 2016-03-22 2020-08-18 Bionomics Limited Method of treating cancer by administering an anti-LGR5 monoclonal antibody
WO2017182427A1 (fr) 2016-04-19 2017-10-26 Amgen Research (Munich) Gmbh Administration d'une construction bispécifique se liant à cd33 et cd3 destinée à une utilisation dans un procédé de traitement de la leucémie myéloïde
US11053318B2 (en) 2016-04-19 2021-07-06 Amgen Research (Munich) Gmbh Administration of a bispecific construct binding to CD33 and CD3 for use in a method for the treatment of myeloid leukemia
WO2018049248A1 (fr) 2016-09-09 2018-03-15 Icellhealth Consulting Llc Virus oncolytique équipé de molécules d'engagement bispécifiques
WO2018049261A1 (fr) 2016-09-09 2018-03-15 Icellhealth Consulting Llc Virus oncolytique exprimant des modulateurs du point de contrôle immunitaire
WO2018141910A1 (fr) 2017-02-02 2018-08-09 Amgen Research (Munich) Gmbh Composition pharmaceutique à faible ph comprenant des constructions d'anticorps d'engagement avec les lymphocytes t
US11918650B2 (en) 2017-05-05 2024-03-05 Amgen Inc. Pharmaceutical composition comprising bispecific antibody constructs for improved storage and administration
WO2018204907A1 (fr) 2017-05-05 2018-11-08 Amgen Inc. Composition pharmaceutique comprenant des constructions d'anticorps bispécifiques pour un stockage et une administration améliorés
WO2019118426A1 (fr) 2017-12-11 2019-06-20 Amgen Inc. Procédé de fabrication continue pour des produits d'anticorps bispécifiques
WO2019133961A1 (fr) 2017-12-29 2019-07-04 Amgen Inc. Constructions d'anticorps bispécifiques dirigés contre muc17 et cd3
WO2020025532A1 (fr) 2018-07-30 2020-02-06 Amgen Research (Munich) Gmbh Administration prolongée d'une construction d'anticorps bispécifique se liant à cd33 et cd3
WO2020025792A1 (fr) 2018-08-03 2020-02-06 Amgen Research (Munich) Gmbh Constructions d'anticorps pour cldn18.2 et cd3
US11692031B2 (en) 2018-08-03 2023-07-04 Amgen Research (Munich) Gmbh Antibody constructs for CLDN18.2 and CD3
WO2020043670A1 (fr) 2018-08-27 2020-03-05 Affimed Gmbh Cellules nk cryoconservées préchargées avec une construction d'anticorps
WO2020077212A1 (fr) 2018-10-11 2020-04-16 Amgen Inc. Traitement en aval de constructions d'anticorps bispécifiques
WO2020252442A1 (fr) 2019-06-13 2020-12-17 Amgen Inc. Commande de perfusion contenant de la biomasse automatisée dans la fabrication de produits biologiques
WO2021021676A1 (fr) 2019-07-26 2021-02-04 Amgen Inc. Protéines de liaison à un antigène anti-il13
WO2021050640A1 (fr) 2019-09-10 2021-03-18 Amgen Inc. Procédé de purification de polypeptides de liaison à un antigène bispécifique présentant une capacité de liaison dynamique de capture de protéine l améliorée
WO2021097344A1 (fr) 2019-11-13 2021-05-20 Amgen Inc. Procédé de réduction de la formation d'agrégats dans le traitement en aval de molécules de liaison à l'antigène bispécifiques
WO2021127528A1 (fr) 2019-12-20 2021-06-24 Amgen Inc. Constructions d'anticorps multispécifique agoniste de cd40 ciblé par la mésothéline permettant le traitement de tumeurs solides
WO2021130383A1 (fr) 2019-12-27 2021-07-01 Affimed Gmbh Procédé de production de construction d'anticorps bispécifique fcyriii x cd30
WO2021150824A1 (fr) 2020-01-22 2021-07-29 Amgen Research (Munich) Gmbh Combinaisons de constructions d'anticorps et d'inhibiteurs du syndrome de libération de cytokine et leurs utilisations
WO2021183861A1 (fr) 2020-03-12 2021-09-16 Amgen Inc. Méthodes de traitement et de prophylaxie du crs chez des patients, comprenant une association d'anticorps bispécifiques se liant à une cellule tumorale du cds x et d'un inhibiteur du tnf alpha ou de l'il-6
WO2021188851A1 (fr) 2020-03-19 2021-09-23 Amgen Inc. Anticorps contre la mucine 17 et leurs utilisations
WO2021236638A1 (fr) 2020-05-19 2021-11-25 Amgen Inc. Constructions de liaison à mageb2
WO2021243320A2 (fr) 2020-05-29 2021-12-02 Amgen Inc. Administration atténuant des effets indésirables d'une construction d'anticorps bispécifique de liaison à cd33 et cd3
WO2022074206A1 (fr) 2020-10-08 2022-04-14 Affimed Gmbh Lieurs trispécifiques
WO2022096716A2 (fr) 2020-11-06 2022-05-12 Amgen Inc. Molécules bispécifiques multicibles de liaison à un antigène à sélectivité accrue
WO2022096704A1 (fr) 2020-11-06 2022-05-12 Amgen Inc. Domaine de liaison à l'antigène à taux de coupure réduit
WO2022096700A1 (fr) 2020-11-06 2022-05-12 Amgen Research (Munich) Gmbh Constructions polypeptidiques se liant sélectivement à cldn6 et cd3
WO2022096698A1 (fr) 2020-11-06 2022-05-12 Amgen Inc. Constructions polypeptidiques se liant à cd3
WO2022212831A1 (fr) 2021-04-02 2022-10-06 Amgen Inc. Constructions de liaison à mageb2
WO2022234102A1 (fr) 2021-05-06 2022-11-10 Amgen Research (Munich) Gmbh Molécules de liaison à l'antigène ciblant cd20 et cd22 destinées à être utilisées dans des maladies prolifératives
WO2023007023A1 (fr) 2021-07-30 2023-02-02 Affimed Gmbh Corps duplex
WO2023078968A1 (fr) 2021-11-03 2023-05-11 Affimed Gmbh Liants de cd16a bispécifiques
WO2023079493A1 (fr) 2021-11-03 2023-05-11 Affimed Gmbh Liants de cd16a bispécifiques
WO2023218027A1 (fr) 2022-05-12 2023-11-16 Amgen Research (Munich) Gmbh Molécules bispécifiques multicibles à chaînes multiples de liaison à un antigène à sélectivité accrue
WO2024059675A2 (fr) 2022-09-14 2024-03-21 Amgen Inc. Composition de stabilisation de molécule bispécifique

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