JPH10507637A - Interleukin-1β deficient transgenic animal - Google Patents

Abstract

  (57) [Summary] By introducing a gene encoding a modified IL-1β gene into a host animal, a transgenic animal having a modification in the IL-1β gene is produced.

Description

DETAILED DESCRIPTION OF THE INVENTION       Interleukin-1β deficient transgenic animal Field of the invention   The present invention relates to a transgenic strain having a mutation in the interleukin-1β gene. For non-human animals.Background of the Invention   Mammalian interleukin-1 (IL-1) is part of the general inflammatory response. Are immunoregulatory proteins secreted by certain cell types. IL-1 The first cell type that produces is peripheral blood monocytes. But other non-transf A cell type that releases or contains IL-1 or an IL-1-like molecule Has been reported. These cell types include epithelial cells (Luger et al., J. Immun ol. 127: 1493-1498 (1981); Le et al., J. Immunol. 138: 2520-2526 (1987); Lovett and And Larsen, J. Clin. Invest. 82: 115-122 (1988)), connective tissue cells (Ollivierre et al. Biochem. Biophys. Res. Comm. 141: 904-911 (1986); Le et al., J. Immunol. 138: 2520-2526 (1987)), cells of neuronal origin (Giulian et al., J. Exp. Med. 164: 594-6). 04 (1986), and leukocytes (Pistoia et al., J. Immunol. 136: 1688-1692 (1986); Acres Et al., Mol. Immuno. 24: 479-485 (1987); Acres et al., J. Immunol. 138: 2132-2136 (1987); L indemann et al., J. Immunol. 140: 837-839 (1988)). Transform cells The line was also found to produce IL-1. These cell lines include The leukemia series P388D, J774, THP. 1. U-937 (Krakauer and And Oppenheimer, Cell. Immunol. 80: 223-229 (1983); Matsushima et al., Biochem. 25: 3242-3429 (1986)), EBV-transformed human B lymphoblastoid lineage (Acr es et al., J. Immunol. 138: 2132-2136 (1987)), and transform mouse epidermis There are keratinocytes (Luger et al., J. Immunol. 125: 2147-2152 (1982)).   Biologically active IL-1 exists as two different forms, of which IL- 1α has an isoelectric point of about 5.2, IL-β has an isoelectric point of about 7.0, and both types have The molecular weight is about 17,500 (Bayne et al., J. Exp. Med. 163: 1267-1280 (1986); Schmidt, J. Exp. Med. 160: 772 (1984)). Both polypeptides are evolutionarily conserved And have about 27-33% homology at the amino acid level (Clark et al. Nucleic Acids Res. 14: 7897-7914 (1986)).   Mammalian IL-1β is a cell-associated precursor polypeptide of about 31.5 kDa. (Limjuco et al., Proc. Natl. Acad. Sci. USA 83: 3972-3976 (1986)). . The precursor IL-1β cannot bind to the IL-1 receptor and is biologically inactive (Mo sley et al., J. Biol. Chem. 262: 2941-2944 (1987)). Biological activity is a proteolytic It appears to depend on some type of processing, but its processing The 31.5 kDa form converts to mature 17.5 kDa. Pre-IL-1β translation product The fully human nucleotide sequence and deduced amino acid sequence of are known, March et al., Nat. ure315: 641-647 (1985).   According to recent studies, processing and secretion of IL-1β has been demonstrated in monocyte and monocyte cell lines. Column specific (Matsusima et al., J. Immunol. 135: 1132 (1985)). For example, Fibroblasts and epidermal keratinocytes synthesize IL-1β precursors but activate them It has not been demonstrated that it processes anything or secretes mature IL-1β. (Young et al., J. Cell Biol. 107: 447 (1988); Corbo et al., Eur. J. Biochem. 16). 9: 669 (1987)).   Processing and secretion of IL-1β is used by normal secreted proteins It is performed in a unique path different from the one Several observations support the hypothesis. IL-1β molecules from five different species are hydrophobic Does not contain a sex signal sequence (Lomedico et al., Nature 312: 458 (1984); Auron et al., Proc USA. 81: 7907 (1984); Gray et al., J. Immunol. 137: 3644 (1986); Malisz ewski et al., Mol. Immunol. 25: 429 (1988), Mori et al., Biochem. Biophys. Res. Commun. . 150: 1237 (1988); Furutani et al., Nucleic Acid Res. 13: 5869 (1985)). Furthermore, When both human and mouse IL-1β precursors are synthesized in vitro, Do not translocate through competent microsomal membranes and Despite the presence of hydrate addition sites, it does not contain N-linked carbohydrates. Most Later, according to light microscopy and electron microscopy studies, immunological observation showed that IL-1β Demonstrating the presence of IL-1β in organelles that are localized in quality and involved in normal secretion Not available (Bayne et al., J. Exp. Med. 163: 1267-1280 (1986); Singer et al., J. Exp. Med. 1). 67: 389 (1988)).   In activated monocytes, IL-1β secretion was inhibited from pulse-chase experiments. It is suggested to be associated with processing. According to these experiments, processing The intracellular pool of unpurified precursors is converted to extracellular mature IL-1β (Haz uda et al. J. Biol. Chem. 263: 847381989). IL-1β precursors are sometimes found extracellularly But high concentrations in vivo, excess trypsin, chymotrypsin or collagena Contributes to the formation of 17 kDa IL-1β unless incubated in the presence of (Hazuda et al., J. Biol. Chem. 264: 1689 (1989)). But these All of the proteinases¹¹⁷Mature IL-1β having the amino terminal as Seems unable to produce.   Proteolytic maturation of precursor IL-1β to 17 kDa IL-1β Is apparently Asp¹¹⁶And Ala¹¹⁷By cutting between. Interleukin-1 An endoproteinase named β-converting enzyme (ICE) is an Asp¹¹⁶-Ala^{1 17} And Asp²⁷-Gly²⁸Can cleave the IL-1β precursor at the homologous site of , Ala¹¹⁷Production of mature IL-1β with the appropriate amino terminus of ing. Asp at position 116 has been found to be essential for cleavage, Replaces Asp with Ala (Kostura et al., Proc. Natl. Acad. Sci. 86: 5227-5231 (198 9) or substitution with another amino acid (Howard et al., J. Immunol., 147, 2964-9, 1991) This is because this cleavage event is inhibited.   Precise role of IL-1β in normal tissue development and maintenance, and embryo and fetal development The percent is currently not fully understood. Biology of IL-1β in acute and chronic inflammation It is important to assess whether IL-1β is a suitable target drug due to its critical importance It is.   Generation of IL-1β Deficient Transgenic Mice Shows the Normal Role of IL-1β Animal models of IL-1β deficiency, It can be used to design and evaluate different approaches to regulation. like this Transgenic IL-1β modified mice can also be used as a cell source in cell culture. Can be used.Summary of the Invention   IL-1β is a site thought to be a major mediator of chronic and acute inflammation Cain. Trans having a modified copy of the endogenous native IL-1β gene Create a transgenic animal. These transgenic animals have been tested for IL-1β. It is useful for analyzing modulators of in vivo activity and IL-1β activity, And IL-1β mediated diseases including acute inflammation.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows a genomic map of the mouse IL-1β gene and the replacement vector p1284. Mouse chromosome IL-1β by targeted recombination using 9-316-1 A predicted alteration of the gene.   FIG. 2 shows two types of target embryos having inactivated IL-1β (knockout) gene. It is a Southern blot analysis of a stem (ES) clone.   FIG. 3 shows tails from transgenic mice with IL-1β knockout. 3 is a Southern blot analysis of the DNA of FIG. Geno from heterozygous x heterozygous crosses Southern analysis of DNA homologues in mice homozygous for the disrupted IL-1β allele Gives the expected number of   FIG. 4 shows IL-1 in knockout mice and wild-type control mice after LPS induction. It is Northern hybridization analysis of (beta) RNA.Detailed description of the invention   Generating transgenic animals having a partially deleted IL-1β gene You. Possible alterations of natural genes In addition, there are modifications, deletions and substitutions of nucleotides and amino acids. Modifications and deletions are natural Gene is rendered non-functional, producing "knock-out" animals. These tran Sgenic animals were used to generate animal models of human disease, and It is important for the ultimate treatment of a linked disease or condition. “Knockout of IL-1β Transgenic animals having a non-human model of disease including IL-1β Useful for establishing and distinguishing the activities of various interleukins in in vivo systems It is.   Disruption of mouse IL-1β gene by isolation of mouse genomic IL-1β gene Of targeting solids can be constructed. Mouse genome IL-1β The gene was isolated using mouse IL-1β cDNA (Telford, J.L. et al., Nuc l.Acids Res. 14: 9955-9963, 1986).   The term "animal" as used herein includes all vertebrates except humans. And individual animals of all stages of development or development, including embryonic and fetal stages. No. “Transgenic animals” are targeted recombinant or microinjected Or by deliberate genetic manipulation at the subcellular level, such as infection with a recombinant virus. Genetic information directly or indirectly altered or accepted Any animal that contains one or more cells with a report. “Transgenic animals The term "is intended to include normal crossbreeding or in vitro fertilization. And one or more cells are modified by the recombinant DNA molecule or It is intended to include animals that receive NA molecules. This molecule binds a specific locus Targeted specifically, integrated randomly into the chromosome, or replicated extrachromosomally DNA to be produced. The term “germline transgenic animal” Genetic modification or genetic information is introduced into the germline, thereby transferring the genetic information to progeny Refers to a transgenic animal that confers the ability to transmit to Such offspring are If they have some or all of the genetic modification or genetic information at the time, It is a transgenic animal.   Genetic modification or genetic information refers to the animal species to which the recipient animal (or recipient) belongs. May be foreign to the individual, or only to a particular individual recipient. Or genetic information already possessed by the recipient animal. In the last example The modified or introduced gene may be expressed differently than the native gene Absent.   Generally, the modified IL-1β gene is naturally occurring in the host animal. Should not fully encode the same IL-1β as the type, and the expression product It should be slightly or significantly modified or completely absent. But, More moderately modified TL-1β genes are within the scope of the invention.   Genes used to modify target genes can be obtained by various techniques However, the techniques include isolation from genomic sources, preparation of cDNA from isolated mRNA templates. , Direct synthesis, or a combination thereof, but is not limited thereto.   The type of target cells for transgene (or transgene) introduction is embryonic stem cells (E S). ES cells are obtained from pre-implanted embryos cultured in vitro and fused with embryos (M.J.Evans, Nature 292: 154-156 (1981); Bradley et al., Nature 309: 255-258 (1 984); Gossler et al., Proc. Natl. Acad. Sci. USA 83: 9065-9069 (1986); Robertson et al. Nature 322, 445-448 (1986)). Transfected gene is DNA transfection , Microinjection, or retrovirus-mediated transduction It can be efficiently introduced into ES cells by standard techniques. Then, the obtained transformation E S cells can be produced with blastocysts from a non-human animal. Transfer the introduced ES cells Of the chimeric animal obtained Become a cell line (R. Jaenisch et al., Science, 240, 1468-1474 (1988)).   Since IL-1β can function as an independent component of a complex mechanism, IL-1β Must be tested separately and the general mechanism of the inflammatory response or IL-1 If its contribution to other mechanisms, including β, should be understood, Must be tested in a run. Determine contribution of individual genes and their expression products One approach to the problem of using isolated genes is to use totipotent ES cells ( (Such as those described herein), can be selectively inactivated and their wild-type genes can be inactivated. After that, a transgenic mouse is produced. Gene targeted transge The use of gene-targeted ES cells in the generation of nick mice is described by Thomas et al., Cell 51: 503-5. 12 (1987), and for a review, Frohman et al., Cell 56: 145-147 (1989); Capecch. i, Trends in Genet. 5: 70-76 (1989); Baribault et al., Mol. Biol. Med. 6: 481-492 (19 89); Wagner, EMBOJ. 9: 3025-3032 (1990); Bradley et al., Bio / technology 10: 534-539. (1992).   Using targeted homologous recombination to insert specific changes in chromosomal alleles Inactivate any gene region or Several techniques are available for altering any desired mutation. But frequently Compared to homologous extrachromosomal recombination that occurs, homologous plasmid-chromosomal recombination^-6 -10^-3(Lin et al., Proc. Natl. Ac ad.Sci. USA 82: 1391-1395 (1985); Smithies et al., Nature 317: 230-234 (1985); T. Homas et al., Cell 44: 419-428 (1986); Song et al., Proc. Natl. Acad. Sci. USA 84: 6820 -6824 (1987)). Heterologous plasmid-chromosome interactions are based on the corresponding homologous insertion. 10^Five(Lin et al., Proc. Natl. Acad. Sci. USA 82: 1391-1395 (12985))-10.^TwoTimes (Th omas et al., Cell 44: 419-428) (1986); Song et al., Proc. Natl. Acad. Sci. USA 84: 68 20-6824 (1987)).   To overcome this low rate of targeted recombination in mouse ES cells, rare homologs Various strategies have been developed to detect or screen for recombination. Detect homologous alterations One approach uses homologous insertion using the polymerase chain reaction (PCR). Elephants are screened for pools of transformed cells and then individual clones are screened. Cleaning (Kim et al., Nucleic Acids Res. 16: 8887-8903 (1988) Kim et al., Gene 103: 227-233 (1991)). Alternatively, if a homologous insertion occurs, simply construct a marker gene that is active Develop a positive gene selection method to select these recombinants directly (Sedivy et al., P roc.Natl.Acad.Sci.USA 86: 227-231 (1989)). Open to select homologous recombinants One of the most common methods issued was the gene without direct selection for modification (IL-1 (eg, β) is a positive-negative selection (PNS) method developed for Nature 336: 348-352 (1988); Capecchi, Science 244: 1288-1292, (1989); Capecc hi, Trends in Genet. 5: 70-76 (1989)). Herpes simplex virus thymidine kinase Gancyclo (HSV-TK) gene and against its heterologous insertion Bill (GANC) or FIAU (1- (2-deoxy-2-fluoro-BD- Selection by herpes drug such as arabinofuranosyl) -5-iodouracil) By doing so, the heterologous recombinants are selected. Surviving by this reverse sorting The number of homologous recombinants in the transformant can be increased.   As used herein, "target gene" or "knockout" (KO) is defined above as Human-modified non-human animal germline, including but not limited to methods Is the DNA sequence to be introduced. The target gene of the present invention is a cognate endogenous allele. Child specific DNA sequences designed to be modified into   Examples will be described below, but it is understood that the examples limit the scope of the present invention. should not do.                               Example 1 Isolation of mouse IL-1β cosmid clone   Mouse cosmid library cESI was used as a probe with mIL-1β cDNA. And used for screening. HB101 cells obtained from ES-J1 cells An ES cell genomic DNA library grown in bacterial cells was used for mouse IL-1βcD Screening was performed using the NA sequence. Bill-neo Plasmi as template And the following oligonucleotide primers: 5'-GCT AGC GTT CCT GAA AAC TTG-3 '(SEQ ID NO: 1) and 5'-CTA GC T TAG GAA GAC ACA GAT TCC ATG GT-3 '( PCR using SEQ ID NO: 2) resulted in a 0.8 kb mouse IL-1β cDNA plasmid. A lobe was made. E. FIG. in situ localization of plasmid DNA in E. coli colonies The conditions for conversion are described in Sambrook et al., “Molecular Cloning: A Laboratory Manual”, Cold  It is described in Spring Harbor Press (1989). 60 μg kanamycin Approximately 85,000 for each of the six 150 mm NZCYM plates containing Colony forming units were plated. Hybridization conditions should be different but different To reduce the chance of isolating homologous genes and related pseudogenes, Maintained at Trinity.   Primary clones were purified by a second and third screening. Positive cosmid DNA was prepared using standard conditions (Sambrook et al., Supra).   Cosmid DNA is digested with different restriction endonucleases and agarose gel And mouse IL-1βc using standard conditions (Sambrook et al., Supra). Hybridized with the DNA sequence.                               Example 2 Characterization of mouse IL-1β clone   The cosmid clone of mouse IL-1β of Example 1 was (ordered) mapped with restriction endonucleases by partial digestion (Evans et al., Gene 79: 9-20 (1989)). IL-1β hybridization of IL-1β gene Location and extent of the mouse IL-1β cDNA and oligonucleotide Fully digested and end-ordered partially digested cosmids using otide probe Positioning was performed by hybridizing with.   Characteristics of the cloned mouse IL-1β gene (designated p12849-146-3) Sex determinations were made to determine the location of restriction endonuclease sites and exons. Mau DNA orientation, restriction endonucleases, By enzyme digestion and Southern hybridization analysis (Sambrook et al., Supra) And a previously cloned mouse IL-1β sequence (Telford et al., Nucl. Acad Res. .V.14.No.24,9955-9963 (1986)) and the predicted restriction endonuclease digestion pattern Determined by comparison. p12849-146-3 is the 5'pro of mouse IL-1β. It was determined to include the motor region and exons. 136b upstream of exon 1 mouse IL-1β gene starting from p and extending to about 1 kb downstream of exon 7 The 7.7 kb EcoRI restriction fragment containing the offspring was cloned into clone p12849-14. The gel was isolated from 6-3. Beginning at approximately 4 kb upstream of exon 1 and exon 6 On a 9.5 kb KpnI restriction fragment extending to a KpnI site between 5 'untranslated IL-1β geno Sequence was isolated. Both the KpnI and EcoRI fragments were converted to pBlues subcloned into a script SK (Stratagene). ue / EcoRI-IL-1β and pBlue / KpnI-IL-1β Was. Using these plasmids, further analysis and IL-1β targeting vector The construction of the ter was done.                               Example 3 Construction of IL-1β gene targeting vector   Knowledge of the genomic structure of the mouse IL-1β gene with respect to restriction sites and exons? (Example 2) Gene targeting vector for inactivating the IL-1β gene Was prepared using standard cloning techniques (Sambrook et al., Supra). Target The carrying vector p12849-316-1 has a long arm of the IL-1β gene. 4.0 kb BamHI fragment and 1.3 kb KpnI-Bgl as short arm  II fragment. Exon 1 part of IL-1β gene to int 5.7 kb sequence between the BamHI to KpnI restriction enzyme sites encoding It was deleted and the selection marker PGKneo was inserted in the cis orientation. PGKtk gene Was inserted at the end of the short arm. HSK-TK remains By FIAU screening for genes, Mansour et al., Nature 336: 348-352 (198 8); Capecchi, Science 244: 1288-1292 (1989); Capecchi, Trends in genet. Five : 70-76 (1989). Gene target By using the vector obtained in the above, the neomer is added to the IL-1β coding region. Part of IL-1β sequence including insertion of car and ATG initiation codon and ICE cleavage site Deletion occurred.   More specifically, plasmid pGEM7 (TK) is a highly efficient mouse phosphoglycoside. Herpes simplex regulated by the serate kinase-1 promoter (PGKp) Contains the viral thymidine kinase gene (TK). Plasmid pGEM7 (TK ) Was digested with EcoRI, which cuts just upstream of the PGKp-TK cassette, and T4 After blunt-ending with DNA polymerase (T4pol), bovine intestinal alkaline phosphatase Dephosphorylated with CIase (CIAP). The short arm of the mouse IL-1β gene was replaced with pBl 1.3 kb KpnI-BglII flag from ue / EcoRI-IL-1β And blunt ended with T4pol. KpnI is a mouse IL- Since the cleavage between exons 6 and 7 of 1β genomic DNA, the short arm F) 208 bp and required 3 ' Includes all 641 bp of exon 7, including untranslated DNA sequences. 1.3 kb short arm Was ligated to the blunt-ended EcoRI site of pGEM7 (TK), and plasmid A was ligated. Formed. Plasmid A contains an EcoRI site immediately upstream of IL-1β exon 7. A PGKp-TK cassette for reproducing the position and the short arm in the same direction. EcoR After digestion with I, plasmid A was blunt-ended with T4pol and removed with CIAP. Oxidation. PGKp-regulated neomycin resistance gene (NEO) As a 1.8 kb EcoRI-SalI fragment from the plasmid pGK-neo Isolated. The PGKp-NEO fragment was blunt-ended with T4pol, and It was ligated to the blunted EcoRI site of Smid A. Plasmid obtained Named Smid B, PGKp-N was located in the same direction as the short arm and PGKp-TK fragment. Includes EO cassette. Plasmid B was placed just upstream of the PGKp-NEO cassette. Digest with XhoI to cut, blunt-end with T4pol, dephosphorylate with CIAP did. The long arm of the mouse IL-1β gene was changed to pBlue / KpnI-IL-1β Isolated as a 4.0 kb BamHI fragment and blunt-ended with T4pol. Was. Long arm fragments are most likely IL-1β5 ′ untranslated DNA sequences. And contains only 54 bp of exon 1 (without ORF sequence). 4.0k b The long arm fragment was ligated to blunt-ended XhoI digested plasmid B. . The obtained gene replacement vector was named p12849-316-1. The vector contains a 4.0 kb IL-1β gene fragment as the 5 ' PGKp-NEO selection marker located between the arm and the 1.3 kb IL-1β gene short arm And the PGKp-TK marker gene linked to the carboxy terminus of the short arm Including. All of the component fragments of p12849-316-1 are oriented in the same direction I have. For electroporation of ES cells, the supplier (5 Prime- 3 Prime, Inc. ), PZ SalI endonuclease that cuts at the junction between the gene and the pGEM7 polylinker It was linearized by digestion with ose.                               Example 4 Target disruption of IL-1β gene in mouse ES cells   The gene targeting vector used in the TL-1β gene disruption experiment was The p12849-316-1 vector of Example 3. IL-1β knockout ( IL-1βKO) Recombining this vector with the wild-type IL-1β allele Exons 1 to 6 of the chromosome region were deleted (FIG. 1). SalI linearization in many experiments The mouse embryonic stem cell line AB2.1 was electroporated using p12849-316-1. Poration was done. All AB2.1 ES cells were obtained from Robertson, Teratocarc as described in inomas and embryonic stem cells, IRL Press, pp. 71-112 (1987) Cultured on SNL feeder cells. 230V, 5 using Bio-Rad Gene Pulser 1 × 10 in 0.8 ml of PBS buffer at 00 μF⁷ES cells and linearized vector 2 Electroporation was performed using 5 μg. The ES cell transformant was 24 hours after clotroporation G418), and several transformants were cloned in 48 to enrich for homologous recombinants. After an hour, the cells were reverse-screened with FIAU (Bristol Myers Squibb; 0.2 μM). Mouse leukemia A disease inhibitory factor (LIF: ESGRO, Gibco BRL, Inc.) was used at 200 U / ml. FIA Sorting by U yielded about 1/8 colonies compared to sorting with G418 alone, Thereby, the isolation of the target transformant was enhanced. G418- and FIAU-resistant ES clones were isolated, grown and -Analyzed by Southern protocol (Ramirez-Solis, R. et al., Anal. Biochem. 201: 331). -335, 1992). A total of three target colonies were analyzed for 350 double resistant colonies. Knee identified. Frequency of targeted recombination versus random integration at the IL-1β locus The degree is 1/930. Target chromatography using 5'-, 3'- and neo probes Detailed analysis of the Southern blots of the IL-1β gene Both showed the expected integration pattern and no other integration events other than targeted recombination. Was shown.                               Example 5 Injection of IL-1β KO clone into donor blastocyst   Southern hybridization for all of the IL-1β target AB2.1 cell lines Characterization by analysis of the IL-1β gene confirmed. The cell line was grown in culture and characterized. Grows normally, A target cell line that does not contain a constant percentage of differentiated cells (Robertson, supra) The vesicle culture is treated with trypsin and the feeder cells are allowed to bind for 30-45 minutes and unbound E S cells were removed and separated from feeder cells. Recipe the ES cells Blastocysts were injected. Three IL-1β target ES clones (# 214, # 31 8, # 334) using a previously described technique (Bradley, A. "Production and analysis o f chimeric mice. In Teratocarcinomas and Embryonic Stem Cells: A Practic al Approach ″, edited by E.J.Robertson, Oxford: IRL Press, (1987), pp113-151). Were injected into C57B1 / 6J recipient blastocysts in separate experiments. Injected The obtained C57B1 / 6J recipient blastocysts were transformed into Tac: SW (fB R) Mice were reimplanted into the uterus and allowed to develop to term. The child is first fur color chimera Screening, but the agouti color (ES cell background strain) 1 shows a cell chimera. From tail samples obtained from these mice By Southern hybridization analysis performed on the isolated genomic DNA , IL-1β targeting was confirmed.   Injection of IL-1β target lines # 214 and # 318 resulted in eleven male chimeras And 6 female chimeras were obtained, the percentage of chimeras being 5-100%. ES cells Line AB2.1 was injected because the agouti (A) fur color gene is homozygous. Invasion of ES cells into C57B1 / 6 blastocysts (black skin color) resulted in chimeric skin Hair-colored mice were obtained.                               Example 6 Breeding chimeric mice   A chimeric skin-colored mouse was used as a wild-type C57B1 / 6 (black fur) female mouse and 1 They were crossed with 29 / J (agout fur) female mice. Chimera x C57B1 / 6 Some of the offspring show that if the chimeric male is If it had a substance, it was expected to be an agooth Dominant). Obtain only agochi mice by crossing chimera x129 / J Will. Transfer ES cell genetic information, including destroyed IL-1β alleles, to offspring These crosses were made to allow them to cross. From both clones # 214 and # 318 Of 3 male chimeras and 1 female chimera with C57B1 / 6J female Agochi's child was obtained.   To determine IL-1β genotype, genomic DNA was isolated from each mouse after weaning. Purified from approximately 1 cm of their tail. Genomic DNA was described in Laird et al. (Supra). Phenol: chloroform extraction and ethanol precipitation. Saza A hybridisation analysis (described in Example 5) was performed and the destroyed IL-1 Offspring containing the beta allele were identified. These transformers The transgenic offspring were heterozygous for IL-1β destruction. Transgenic The genomic DNA of the heterozygous mouse and the non-transgenic mouse (tail) was Digestion with RI and hybridization with 3 'flanking DNA probe Finally, the transgenic IL-1β structure was confirmed. Southern Hybridization The analysis of the modified IL-1β allele revealed that the structure of the IL-1β target E It was the same as expected and characterized previously for the S clone.                               Example 7 Breeding of heterozygous mice and production of homozygous IL-1β-deficient mice   Each containing one copy of a modified IL-1β allele (heterozygous mice), Male and female transgenic mice were bred and both copies of the IL-1β gene were copied. Mice that were targeted and were modified IL-1β genes. mouse Were predicted to be homozygous for the modified IL-1β gene. Surviving offspring were genotyped by Southern hybridization as described above. (FIG. 3). 25 out of 102 offspring (24.5%) were homozygous Type IL-1β- /-. 23 (22.5%) were wild-type IL-1β + / +, 53% Was heterozygous IL-1β − / +. These numbers indicate the number of surviving ILs after weaning. No significant decrease in the number of transgenic mice lacking -1β Indicates that                               Example 8 Characterization of homozygous IL-1β deficient mice   Surviving homozygous IL-1β-deficient mice of Example 9 were replaced with wild-type mice or Were mated with heterozygous mice to determine if they were fertile. test All of the homozygous TL-1β − / − males and females were fertile. I Between the L-1β-deficient mouse and wild-type or heterozygous mouse No significant difference was observed in the tissue structure.                               Example 9 Confirmation of IL-1β inactivation by Northern and ELISA   Both wild-type and IL-1βKO miceP.acnesI. p. Sensitization by injection 6 days later, 10 μg of LPS i. p. Challenge by injection and release IL-1β Induced the present. At 3.3.75 hours after the challenge, the mice are_TwoKilled by suffocation Was. Hepari Untreated blood was obtained by cardiac puncture. The peritoneal cavity was washed. Plasma and cell-free washing Both fluids were assayed for IL-1β by ELISA (Table 1). Table 1 is L It is a result of ELISA analysis of IL-1 (beta) protein after PS induction. Expected Thus, in contrast to wild-type controls, knockout mice have significant IL-1β activity Not shown.   Liver RNA was prepared from the same mouse, and mouse IL-1β cDNA was used as a probe. Northern hybridization was performed according to the standard method used in FIG. ). As expected, liver RNA from knockout mice showed IL-1β expression. Although not shown at all, wild-type control animals showed significant amounts of IL-1β activity.

────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventors Hoffman, Jaslin Jei             New Jersey, United States             07065, Lowway, East Linker             N Avenue 126 (72) Inventor Juan del Proof, Leonardo             Su Her Ta             New Jersey, United States             07065, Lowway, East Linker             N Avenue 126 (72) Inventor Trambauer, Myana E             New Jersey, United States             07065, Lowway, East Linker             N Avenue 126 (72) Inventor Tsuen, Hui             New Jersey, United States             07065, Lowway, East Linker             N Avenue 126

Claims (1)

[Claims] 1. Somatic cells and germ cells containing a modified gene encoding a modified form of IL-1β A transgenic animal or animal at embryonic stage using embryonic stem cells The modified gene to replace the wild-type IL-1β allele with an ancestor of The transgenic animal. 2. The transgenic animal according to claim 1, wherein the animal is a mouse. Animal. 3. The mouse is fertile and capable of transmitting the modified IL-1β gene to its progeny The mouse according to claim 2, wherein: 4. Embryo stage by microinjection of modified embryonic stem cells into mouse blastocysts Characterized in that a modified IL-1β gene has been introduced into a mouse ancestor at the floor. The mouse according to claim 2. 5. Embryo stage by microinjection of modified embryonic stem cells into mouse blastocysts The modified IL-1β gene has been introduced into a mouse at the floor. Item 3. The mouse according to Item 2. 6. The mouse according to claim 2, which is designated as IL-1βKO. 7. The animal is a mouse, and the modified form of IL-1β has a function. The transgenic animal of claim 1, wherein the transgenic animal is absent. 8. Somatic cells and germ cells containing a modified gene encoding a modified form of IL-1β. And using the embryonic stem cells to add wild-type I to the animal or animal ancestor at the embryonic stage The modified gene targeted to replace the L-1β allele. How to create a mouse, (A) Designed to target the IL-1β allele of mouse embryonic stem cells Preparing a gene encoding the modified TL-1β, (B) introducing the modified gene into mouse embryonic stem cells; (C) selecting embryonic stem cells containing the modified gene; (D) injecting embryonic stem cells containing the modified IL-1β gene into mouse blastocysts , (E) transplanting the injected blastocysts into pseudopregnant mice; and (F) Developing embryos to a period to produce the founder transgenic mice , Including. 9. Characterized in that the introduction of the step (b) is by microinjection The method according to claim 8, wherein 10. The following steps: (G) crossing a chimeric transgenic mouse with a wild-type mouse, Obtaining a conjugated (F1) mouse; and (H) Mating heterozygous (F1) mice and homozygous (F2) IL-1 creating a beta transgenic mouse. Item 10. The method according to Item 9. 11. A cell line obtained from the transgenic animal of claim 1.