WO1996012792A1

WO1996012792A1 - INTERLEUKIN-1β DEFICIENT TRANSGENIC ANIMALS

Info

Publication number: WO1996012792A1
Application number: PCT/US1995/013341
Authority: WO
Inventors: Howard Y. Chen; Kathryn J. Hofmann; Leonardus H. T. Van Der Ploeg; Myrna E. Trumbauer; Hui Zheng
Original assignee: Merck & Co., Inc.
Priority date: 1994-10-20
Filing date: 1995-10-16
Publication date: 1996-05-02
Also published as: JPH10507637A; EP0787179A1; EP0787179A4

Abstract

A transgenic animal with alterations in an IL-1β gene is prepared by introduction of a gene encoding an altered IL-1β gene into a host animal.

Description

TITLE OF THE INVENTION

INTERLEUKIN- 1 β DEFICIENT TRANSGENIC ANIMALS

FIELD OF THE INVENTION

The present invention relates to transgenic nonhuman animals wherein an interleukin-1 β gene is mutated.

BACKGROUND OF THE INVENTION

Mammalian interleukin- 1 (IL- 1 ) is an immuno-regulatory protein secreted by certain cell types as part of the general

inflammatory response. The primary cell type responsible for IL- 1 production is the peripheral blood monocyte. Other non-transformed cell types have, however, been described as releasing or containing IL- 1 or IL- 1 -like molecules. These include epithelial cells (Luger et al., J. Immunol. 127: 1493-1498 [ 1981 ]. Le et al., J. Immunol. 138: 2520-2526 [ 1987 ] and Lovett and Larsen, J. Clin. Invest. 82: 1 15- 122 [ 1988]), connective tissue cells (Ollivierre et al., Biochem. Biophys. Res. Comm. 141 : 904-91 1 [ 1986]. Le et al., J. Immunol. 138: 2520-2526 [ 1987 ]). cells of neuronal origin (Giulian et al., J. Exp. Med. 164: 594-604

[ 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 1 1987] and Lindemann et al., J. Immunol. 140: 837-839 [ 1988]). Transformed cell lines have also been shown to produce IL- 1. These include monocytic leukemia lines P388D, J774, THP. l , U-937 (Krakauer and Oppenheimer. Cell. Immunol. 80: 223-229 [ 1983 ] and Matsushima et al., Biochem. 25: 3242-3429 [ 1986], EBV-transformed human B lymphoblastoid lines (Acres, et al.,

J. Immunol. 138: 2132-2136 [ 1987]) and transformed murine

keratinocytes (Luger et al., J. Immunol. 125: 2147-2152 [ 1982]).

Biologically active IL-1 exists in two distinct forms, IL-1 α with an isoelectric point of about 5.2 and IL-1β with an isoelectric point of about 7.0 with both forms having a molecular mass of about 17,500 (Bayne et al., J. Exp. Med. 163: 1267-1280 ] 1986] and Schmidt. J. Exp. Med. 160: 772 [ 1984|). The poly-peptides appear evolutionarily conserved, showing about 27-33% homology at the amino acid level (Clark et al., Nucleic Acids Res. 14: 7897-7914 [ 19861).

Mammalian IL-1 β is synthesized as a cell associated precursor polypeptide of about 31.5 kDa (Limjuco et al., Proc. Natl. Acad. Sci. USA 83: 3972-3976 [ 1986 ]). Precursor IL-1 β is unable to bind to IL- 1 receptors and is biologically inactive (Mosley et al., J. Biol. Chem. 262: 2941 -2944 1 1987 ]). Biological activity appears dependent upon some form of proteolytic processing which results in the

conversion of the precursor 31.5 kDa form to the mature 17.5 kDa form. The complete human nucleotide and predicted amino acid sequence of the pre-IL-1 β translation product is known, and is disclosed by March et al., Nature 315: 641 -647 ( 1985).

Recent studies suggest that the processing and secretion of IL-1 β is specific to monocytes and monocytic cell lines (Matsushima et al., J. Immunol. 135: 1 132 [ 1985 ]). For example, fibroblasts and keratinocytes synthesize the IL-1 β precursor, but have not been shown to actively process the precursor or secrete mature IL-1 β (Young et al., J. Cell Biol. 107: 447 ( 1988) and Corbo et al., Eur. J. Biochem. 169: 669 [ 1987 ]).

Several observations support the hypothesis that the processing and secretion of IL-1 β occurs by a unique pathway distinct from that used by classical secretory proteins. IL-1 β molecules from five different species do not contain hydrophobic signal sequences (Lomedico et al., Nature 312: 458 [1984|, Auron et al., Proc. Natl.

Acad. Sci. USA 8i: 7907 [ 19841, Gray et al., J. Immunol. 137: 3644

[ 1986], Maliszewski et al., Mol. Immunol. 25: 429 [ 1988 ], Mori et al., Biochem. Biophys. Res. Commun. 150: 1237 1 1988 ], and Furutani et al., Nucleic Acid Res. 13: 5869 1 1985|). Furthermore, neither the human nor the murine IL-1 β precursors, when synthesized in vitro, translocate across competent microsomal membranes, and despite the presence of N-linked carbohydrate addition sites, do not contain N-linked

carbohydrate. Finally, light and electron microscopy studies

immunolocalize IL-1 β to the cytoplasm and fail to demonstrate IL-1 β in orcanelles that are involved in classical secretion (Bayne et al., J. Exp. Med. 163: 1267- 1280 [ 1986] and Singer et al.,

J. Exp. Med. 167: 389 [ 1988]).

In activated monocytes, pulse-chase experiments suggest that IL-1 β secretion may be linked to processing. These experiments show that the intracellular pool of unprocessed precursor is chased to extracellular mature IL- l β (Hazuda et al., J. Biol. Chem. 263: 8473 [ 1989|). IL-1 β precursor is occasionally found extracellularly but does not appear to contribute to the formation of 17 kDa IL-1 β unless incubated at high concentrations in the presence of excess trypsin, chvmotrypsin or collagenase in vitro (Hazuda et al., J. Biol. Chem. 264: 1689 [ 1989 ], Black et al., J. Biol. Chem. 263: 9437 [ 1988 ] and Hazuda et al., J. Biol. Chem. 265: 6318 [ 1990]). However, none of these proteinases appear capable of generating mature IL-1 β with the amino terminus at Ala^{1 1 7.}

Proteolytic maturation of precursor IL-1 β to mature, 17 kDa IL- 1 β apparently results from cleavage between Asp^{1 16} and Ala ^{1 17}. An endoproteinase. termed Interleukin-1 β Converting Enzyme (ICE), that is capable of cleaving the IL-1 β precursor at Asp^{1 16}

Asp^{1 17}, as well as at a homologous site at Asp²⁷-Gly²⁸, and generating mature IL-1 β with the appropriate amino terminus at Ala ^{1 1 7} has now been identified. The Asp at position 1 16 has been found to be essential for cleavage, since substitution of Ala (Kostura et al., Proc. Natl. Acad. Sci. 86: 5227-5231 [ 1989]) or other amino acids (Howard et al.,

J. Immunol.. 147, 2964-9, 1991 ) for Asp inhibits this cleavage event.

The precise roles of IL-1 β in normal tissue development and maintenance, as well as in embryonal and fetal development, are not fully understood at this time. Due to the biological importance of IL-1 β in acute and chronic inflammation, it is important to evaluate whether IL- 1 β is a suitable drug target.

The generation of IL-1 β deficient transgenic mice would aid in defining the normal role(s) of IL-1 β, and allow an animal model of IL-1 β deficiency to be used in the design and assessment of various approaches to modulating IL-1 β activity. Such IL-1 β modified transcenic mice can also be used as a source of cells for cell culture. SUMMARY OF THE INVENTION

IL-1 β is a cytokine believed to be the major mediator of chronic and acute inflammation. Transgenic animals having a modified copy of the endogenous native IL-1 β gene are produced. These transgenic animals are useful in the analysis of the in vivo activity of IL-1 β as well as modulators of IL-1 β activity, and are useful as an animal model of IL-1 β-mediated diseases including chronic and acute inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a genomic map of the mouse IL- l β gene and the predicted modification of the mouse chromosomal IL-1 β gene by targeted recombination using the replacement vector pi 2849-316- 1.

Figure 2 is a Southern blot analysis of two targeted embryonic stem (ES) clones having an inactivated IL-1 β (knockout) gene.

Figure 3 is a Southern blot analysis of tail DNA from transgenic mice having an IL-1 β knockout. Southern analysis of genomic DNA from heterozygous x heterozygous crosses yielded the expected number of mice homozygous for the disrupted IL- 1 β allele.

Figure 4 is a Northern hybridization analysis for IL- 1 β RNA in the knockout and wild-type control mice after LPS

induction. DETAILED DESCRIPTION OF THE INVENTION

Transgenic animals are generated which have a partially deleted IL-1 β gene. Among the potential alterations of the naturally occurring gene are nucleotide and amino acid modifications, deletions and substitutions. Modifications and deletions may render the naturally occurring gene nonfunctional, producing a "knockout" animal. These transgenic animals are critical for the creation of animal models of human diseases, and for eventual treatment of disorders or diseases associated with IL-1 β elicited responses. A transgenic animal carrying a "knockout" of IL-1 β is useful for the establishment of a nonhuman model of diseases involving IL-1β, and to distinguish between the activities of the different interleukins in an in vivo system.

The isolation of the mouse genomic IL-1 β gene permits the construction of a targeting vector for the disruption of the mouse IL- 1 β gene. The mouse genomic IL-1 β gene is isolated using the mouse IL-1 β cDNA (Telford, J.L. et al., Nucl. Acids Res. 14: 9955-9963, 1986).

The term "animal" is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. A

"transgenic animal" is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by targeted recombination or microinjection or infection with recombinant virus. The term "transgenic animal" is not intended to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by or receive a recombinant DNA molecule. This molecule may be specifically targeted to a defined genetic locus, be randomly integrated within a chromosome, or it may be extrachromosomally replicating DNA. The term "germ cell line transgenic animal" refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the genetic information to offspring. If such offspring in fact possess some or all of that genetic alteration or genetic information, then they, too, are transgenic animals.

The genetic alteration or genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.

The altered IL-1 β gene generally should not fully encode the same IL-1β as native to the host animal, and its expression product should be altered to a minor or great degree, or absent altogether. However. it is conceivable that a more modestly modified IL-1 β gene will fall within the scope of the present invention.

The genes used for altering a target gene may be obtained by a wide variety of techniques that include, but are not limited to, isolation from genomic sources, preparation of cDNAs from isolated mRNA templates, direct synthesis, or a combination thereof.

A type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells may be obtained from preimplantation embryos cultured in vitro and fused with embryos (M. J. Evans et al., Nature 292: 154-156 ( 1981 ): Bradley et al., Nature 309: 255-258 ( 1984): Gossler et al. Proc. Natl. Acad. Sci. USA 83: 9065-9069 ( 1986): and Robertson et al., Nature 322, 445-448 ( 1986)).

Transgenes can be efficiently introduced into the ES cells by a variety of standard techniques such as DNA transfection, microinjection, or by retrovirus-mediated transduction. The resulting transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (R. Jaenisch, Science 240: 1468- 1474 ( 1988)).

Since IL-1 β can function as an independent component of a complex mechanism, IL-1 β must be examined both individually and in the context of the whole mechanism if its contribution to the

mechanisms of the general inflammatory response or other mechanisms involving IL-1 β are to be understood. One approach to the problem of determining the contributions of individual genes and their expression products is to use isolated genes to selectively inactivate the native wild-type gene in totipotent ES cells (such as those described herein) and then generate transgenic mice. The use of gene-targeted ES cells in the generation of gene-targeted transgenic mice was described 1987

(Thomas et al., Cell 51 : 503-512, ( 1987)) and is reviewed elsewhere

(Frohman et al., Cell 56: 145- 147 ( 1989): Capecchi, Trends in Genet. 5: 70-76 ( 1989): Baribault et al., Mol. Biol. Med. 6: 481 -492, ( 1989):

Wagner. EMBO J. 9: 3025-3032 ( 1990): Bradley et al., Bio/Technology 10: 534-539 ( 1992)). Techniques are available to inactivate or alter any genetic region to any mutation desired by using targeted homologous

recombination to insert specific changes into chromosomal alleles.

However, in comparison with homologous extrachromosomal

recombination, which occurs at a high frequency, homologous plasmidchromosome recombination was originally reported to only be detected at frequencies between 10-6 and 10-3 (Lin et al., Proc. Natl. Acad. Sci. USA 82: 1391 -1395 ( 1985); Smithies et al., Nature 317: 230-234

( 1985); Thomas et al., Cell 44:419-428, ( 1986); Song et al.. Proc. Natl. Acad. Sci. USA 84: 6820-6824 ( 1987)). Nonhomologous plasmidchromosome interactions are more frequent, occurring at levels 105-fold (Lin et al., Proc. Natl. Acad. Sci. USA 82: 1391 - 1395 ( 1985)) to 10²-fold (Thomas et al., Cell 44: 419-428 ( 1986); Song et al., Proc. Natl. Acad. Sci. USA 84: 6820-6824 ( 1987)) greater than comparable homologous insertion.

To overcome this low proportion of targeted

recombination in murine ES cells, various strategies have been

developed to detect or select rare homologous recombinants. One approach for detecting homologous alteration events uses the

polymerase chain reaction (PCR) to screen pools of transformant cells for a homologous insertion event, followed by screening individual clones (Kim et al., Nucleic Acids Res. 16: 8887-8903 ( 1988); Kim et al., Gene 103: 227-233 ( 1991 )). Alternatively, a positive genetic selection approach has been developed in which a marker gene is constructed which will only be active if homologous insertion occurs, allowing these recombinants to be selected directly (Sedivy et al., Proc. Natl. Acad. Sci. USA 86: 227-231 ( 1989)). One of the most general approaches developed for selecting homologous recombinants is the positive-negative selection (PNS) method developed for genes (such as IL-1 β) for which no direct selection of the alteration exists (Mansour et al., Nature 336: 348-352: ( 1988): Capecchi. Science 244: 1288- 1292, ( 1989); Capecchi, Trends in Genet. 5: 70-76 ( 1989)). Nonhomologous recombinants are selected against by using the herpes simplex virus thymidine kinase (HSV-TK) gene and selecting against its nonhomologous insertion with the herpes drugs such as ganciclovir (GANC) or FIAU ( 1 -(2-deoxy 2-fluoro-B-D-arabinofluranosyl)-5-iodouracil). By this counter-selection, the number of homologous recombinants in the surviving transformants can be enriched.

As used herein, a "targeted gene" or "knockout" (KO) is a

DNA sequence introduced into the germline of a non-human animal by way of human intervention, including but not limited to, the above described methods. The targeted genes of the invention include DNA sequences which are designed to specifically alter cognate endogenous alleles.

The following is presented by way of examples and is not to be construed as a limitation on the scope of the invention.

EXAMPLE 1

Isolation of mouse IL-1 β cosmid clones

The mouse cosmid library cESI was screened using mIL- 1 β cDNA as a probe. The ES cell genomic DNA library derived from ES-J 1 cells and propagated in HB 101 bacterial cells was screened using the mouse IL- l β cDNA sequence. The 0.8 kb mouse IL-1 β cDNA probe was generated by PCR using the Bi l l -neo plasmid as a template and the following oligonucleotide primers: 5'-GCT AGC GTT CCT GAA AAC TTG-3' (SEQ ID NO: 1 ) and 5 -CTA GCT TAG GAA GAC ACA GAT TCC ATG GT-3' (SEQ ID NO:2). The conditions for in situ

localization of plasmid DNA in E. coli colonies are described by

Sambrook et al. in "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Press. ( 1989). Approximately 85.000 colony forming units were plated on each of six 150mm NZCYM plates containing 60ug/ml of kanamycin. The hybridization conditions were maintained at high stringency to reduce the chances of isolating different but homologous genes and related pseudogenes.

Primary clones were purified through secondary and tertiary screenings. DNAs for the positive cosmids were prepared using standard conditions ( Sambrook et al., supra). Cosmid DNAs were digested with different restriction endonucleases, electrophoresed on agarose gels, and hybridized with the mouse IL- l β cDNA sequence using standard conditions (Sambrook et al., supra).

EXAMPLE 2

Characterization of the mouse IL-1 β clones

The cosmid clones for mouse IL-1 β of Example 1 were mapped with restriction endonucleases by end-ordered partial digestion (Evans et al., Gene 79: 9-20. ( 1989)). The location and extent of the IL-1 β hybridizing regions of the IL-1 β gene were localized by hybridizing complete and end-ordered partial digestions of the cosmids with the mouse IL-1 β cDNA and oligonucleotide probes.

The cloned mouse IL-1 β gene (designated as p 12849- 146- 3 ) was characterized to determine restriction endonuclease sites and exon locations. Mouse IL-1 β orientation and exon locations were determined by DNA sequencing, digestion with restriction endonuclease and Southern hybridization analysis (Sambrook et al., supra) and by comparison with the predicated restriction endonuclease digestion pattern of the previously cloned murine IL-l β sequence (Telford et al., Nucl. Acad Res. V. 14. No. 24, 9955-9963 ( 1986)). It was determined that pi 2849- 146-3 contains the 5' promoter region and exons of mouse IL-1 β. A 7.7 kb EcoRI restriction fragment containing the mouse IL-1 β gene beginning 136 bps upstream of exon 1 and extending to approximately 1 kb downstream of exon 7 was gel-isolated from clone p i 2849- 146-3. The 5' untranslated IL- 1 β genomic sequence was isolated on a 9.5 kb Kpn I restriction fragment which begins

approximately 4 kb upstream of exon 1 and extends to the Kpn I site between exons 6 and 7. Both the Kpn I and Eco RI fragments were subcloned into pBluescript SK (Stratagene) and designated as

pBlue/EcoRI-IL- 1 β and pBlue/Kpnl-IL-1 β, respectively. These plasmids were used for further analysis and construction of the IL-1 β targetine vector. EXAMPLE 3

Construction of IL-1 β gene targeting vector

From the knowledge of the genomic organization of the mouse IL-1 β gene with regard to restriction sites and exons (Example 2), a gene targeting vector for inactivating the IL-1 β gene was prepared using standard cloning techniques (Sambrook et al., supra). The targeting vector p i 2849-316- 1 contained a 4.0 kb BamH I fragment of the IL- 1 β gene as the long arm and a 1.3kb KpnI-BgIII fragment as the short arm. The 5.7 kb sequence between the BamHI to KpnI restriction enzyme sites encoding part of exon 1 to intron 6 of the IL- 1 β gene was deleted and inserted with the selectable marker PGKneo in cis orientation. The PGKtk gene was inserted at the end of the short arm. Selection against the HSV-TK gene with FIAU allowed for the

enhancement of targeted recombinants as described (Mansour et al., Nature 336: 348-352. ( 1988); Capecchi. Science 244: 1288- 1292, ( 1989): Capecchi. Trends in Genet. 5: 70-76 ( 1989). Use of the resulting vector in gene targeting resulted in the insertion of the neo marker in the IL-1 β coding region and the deletion of a portion of the IL-1 β sequences including the ATG initiation codon and the ICE cleavage site.

More specifically, plasmid pGEM7(TK) contains the herpes simplex virus thymidine kinase gene (TK) driven by the highly efficient mouse phosphoglycerate kinase- 1 promoter (PGKp). Plasmid

pGEM7(TK ) was digested with Eco RI which cuts immediately upstream of the PGKp-TK cassette, made blunt-ended with T4 DNA polymerase (T4 pol) and dephosphorylated with calf intestinal alkaline phosphatase (CIAP). The short arm of the mouse IL- 1 β gene was isolated from pBlue/EcoRI-IL- 1 β as a 1.3 kb, Kpn I-Bgl II fragment and made blunt-ended with T4 pol. Since Kpn I cuts between exons 6 and 7 of the mouse IL-l β genomic DNA, the short arm contains all 641 bps of exon 7 including 208 bps of open reading frame (ORF) in addition to the required 3' untranslated DNA sequence. The 1.3 kb short arm was ligated into the blunt-ended Eco RI site of pGEM7(TK ) to form plasmid A. Plasmid A contains the short arm and the PGKp-TK cassette in the same orientation which regenerates the Eco RI site immediately upstream of the IL-1 β exon 7. Following digestion with Eco RI, plasmid A was made blunt-ended with T4 pol and

dephosphorylated with CIAP. The neomycin resistance gene (NEO) driven by the PGKp was isolated from plasmid pGK-neo as a 1.8 kb Eco RI-Sal I fragment. The PGKp-NEO fragment was made bluntended with T4 pol and ligated into the blunt-ended Eco RI site of plasmid A. The resulting plasmid, designated as plasmid B, contains the PGKp-NEO cassette in the same orientation as the short arm and PGKp-TK fragments. Plasmid B was digested with Xho I which cuts

immediately upstream of the PGKp-NEO cassette, made blunt-ended with T4 pol and dephosphorylated with CIAP. The long arm of the mouse IL- 1 β gene was isolated from pBlue/Kpnl-IL-1 β as a 4.0 kb, Bam HI fragment, and made blunt-ended with T4 pol. The long arm fragment consists mostly of IL-1 β 5' untranslated DNA sequence and contains only 45 bps of exon 1 (no ORF sequences). The 4.0 kb long arm fragment was ligated with the blunt-ended, Xho I-digested plasmid B. The resulting gene replacement vector, designated p 12849-316- 1 , contains a 4.0 kb IL- 1 β gene fragment as the 5'-end long arm, a PGKp-NEO selectable marker located between the long arm and the 1 .3 kb IL-1 β gene short arm and a PGKp-TK marker gene attached to the carboxy-terminal end of the short arm. All of the component fragments in pi 2849-316- 1 are oriented in the same direction. For

electroporation of ES cells, plasmid DNA was prepared using pZ523® columns according to the supplier (5 Prime-3Prime, Inc.) and linearized by digestion with Sal I endonuclease which cuts at the junction between the TK gene and the pGEM7 polylinker. EXAMPLE 4

Targeted disruption of the IL-1 β gene in murine ES cells

The gene targeting vector used in the IL-1 β gene disruption experiments was the pi 2849-316- 1 vector of Example 3. When this vector recombined with the wild-type IL- 1 β allele to generate the IL-1 β knockout (IL-1 β KO), exons 1 to 6 of the coding region were deleted (Fig. 1 ). The mouse embryonic stem cell line AB2.1 was electroporated with SalI-linearized p 12849-316- 1 in multiple experiments. All AB2.1 ES cells were cultured on SNL feeder cells as described (Robertson, in Teratocarcinomas and embryonic stem cells. IRL Press, pp. 7 1 - 1 12 ( 1987)). Electroporations were performed with 1 × 1 07 ES cells and 25 μg linearized vector in 0.8 ml PBS buffer at 230V, 500 μF using a Bio-Rad Gene Pulser. ES cell transformants were selected with the antibiotic GENETICIN® (Gibco G418: 200 μg/ml active G418) 24 hr post electroporation. and some transformants were counter-selected with FIAU (Bristol Myers Squibb; 0.2 μM) 48 hours later for enhancement of homologous recombinants. Murine leukemia inhibitory factor (LIF: ESGRO, Gibco BRL. Inc.) was used at 200 U/ml. Selection with FIAU resulted in about eightfold fewer colonies as compared to G418 selection alone, thereby enhancing the isolation of targeted transformants. G418- and FIAU-resistant ES clones were isolated, grown up and analyzed by a mini-Southern protocol (Ramirez-Solis, R. et al., Anal. Biochem. 201 : 331 -335, 1992). A total of three targeted clones were identified from 350 double resistant colonies analyzed. Therefore, the frequency of targeted recombination vs. random integration at the IL- 1 β locus is 1/930.

Detailed Southern blot analysis of targeted clones using 5'-. 3'- and neo probes showed the expected integration pattern both within and flanking the IL-1 β gene and there is no other integration events in addition to targeted recombination.

EXAMPLE 5 Injection of IL-1 β KO clones into donor blastocysts

All IL-1 β-targeted AB2.1 cell lines were characterized by Southern hybridization analysis to confirm that the IL- 1 β gene was indeed disrupted. The cell lines were grown in culture and

characterized. Targeted cell lines which crew normally and did not contain an abnormal proportion of differentiated cells (Robertson, in supra) were then separated from their feeder cells by treating the cell culture with trypsin. allowing the feeder cell to attach for 30-45 min, and removing the unattached ES cells. The ES cells were injected into recipient blastocysts. Three IL-1 β targeted ES clones (#214, #318 and #334) were injected into C57B1/6J recipient blastocysts in separate experiments using techniques described previously (Bradley, A.

"Production and analysis of chimeric mice. In Teratocarcinomas and Embryonic Stem Cells: A Practical Approach", E.J. Robertson, ed. Oxford:IRL Press, ( 1987), ppl 13- 151 ). The injected C57B1/6J recipient blastocysts were reimplanted into the uteri of day 3

pseudopregnant Tac:SW(fBR ) mice and allowed to develop to term. Progeny were screened initially by coat color chimerism. the agouti color (which is the ES cell background strain) being an indicator of ES cell chimerism. IL- 1 β targeting was confirmed by Southern

hybridization analysis performed on genomic DNA isolated from tail samples obtained from these mice.

Injection of the IL-1 β targeted lines #214 and #318 yielded 1 1 male chimeras and 6 female chimeras, with the chimerism ranging from 5% to 100%. As the ES cell line AB2.1 is homozygous for the agouti (A) coat color gene, penetrance of ES cells into the injected (black coat color) C57BI/6 blastocyst gives rise to chimeric coat color mice. EXAMPLE 6

Breeding chimeric mice

The chimeric coat color mice were bred to wild-type C57BI/6 (black coated) and 129/J (agouti coated) female mice. Some of the progeny from the chimera X C57B1/6 cross were expected to be agouti if the chimeric male had ES cell genetic material incorporated into its germline (agouti is dominant to black coat color). The chimera X 129/J cross would yield only agouti mice. These crosses were performed to transfer ES cell genetic information, including the disrupted IL- 1 β allele. to its offspring. Three male chimeras and one female chimera from both clone #214 and #318 resulted in agouti pups when crossed with C57B1/6J females.

To determine the IL-1 β genotypes, genomic DNA was purified from about 1 cm of tail from each mouse after weaning. The genomic DNA was isolated as described (Laird et al., supr a). followed by phenol: chloroform extractions and ethanol precipitation. Southern hybridization analysis (as described in Example 5) were used to identity offspring which contained the disrupted IL-1 β allele. These transgenic offspring were heterozygous for the IL-1 β disruption. Both transgenic heterozygous and nontransgenic mouse (tail) genomic DNAs weie digested with EcoRI. and were hybridized with a 3' flanking DNA probe to confirm the transgenic IL- 1 β structure. Southern

hybridization analysis confirmed that the structure of the altered IL- 1 β allele was identical to that predicted, and previously characterized in the IL- 1 β targeted ES clones.

EXAMPLE 7 Breeding heterozygous mice and generation of homozygous IL- 1 β deficient mice

Male and female transgenic mice, each of which contained one copy of the altered IL-1 β allele (heterozygous mice), were mated with each other to generate mice in which both copies of the IL- 1 β gene were the targeted, altered transgenic IL-1 β gene. It was predicted that one fourth of the mouse embryos would be homozygous for the altered IL-1 β gene. Surviving offspring were genotyped by Southern hybridization as described above (Fig. 3). It was determined that 25 (24.5% ) of the 102 offspring mice were homozygous IL- 1 β-/-. 23 ( 22.5 %) were wild-type IL-1 β+/+, and 53 % were heterozygous IL-1 β-/+. These numbers indicate that there was no significant decrease in the number of IL- 1 β deficient transgenic mice which survived past weaning. EXAMPLE 8

Characterization of homozygous IL-1β deficient mice

Surviving homozygous IL-1β deficient mice of Example 9 were bred with wild-type or heterozygous mates to determine if they were fertile. All homozygous IL-1β-/- males and females tested were fertile. Significant differences in gross morphology or histology between the IL-1β deficient mice and the wild-type or heterozygous mice were not observed.

EXAMPLE 9

Confirmation of IL- 1 β inactivation by Northern and ELISA

Both the wild-type and IL- 1 β KO mice were sensitized by i.p. injection of P. acnes and challenged by i.p. injection of 10 μg LPS 6 days later to induce the expression of IL- 1 β. 3-3.75 hours after challenge, mice were sacrificed by CO2 asphyxiation. Heparinized blood was obtained by cardiac puncture. The peritoneal cavities were lavaged. Both the plasma and cell-free lavage fluids were assayed for IL- 1 β by ELISA (Table 1 ). Table 1 is the result of ELISA analysis of IL- 1 β protein after LPS induction. As expected, in contrast to the wild- type controls, the knockout mice did not exhibit any significant IL-1β activity.

Liver RNA was prepared from the same mice and

Northern hybridizations were carried out by standard procedures using the mouse IL-1 β cDNA as probe (Fig. 4). As expected, the liver RNA from the knockout mice did not exhibit any detectable IL-1 β

expression, whereas wild-type control animals showed a significant amount of IL- 1 β activity.

Claims

WHAT IS CLAIMED IS:

1 . A transgenic animal whose somatic and germ cells contain an altered gene coding for an altered form of IL-1 β , the altered gene having been targeted to replace a wild-type IL-1 β allele into the animal or an ancestor of the animal at an embryonic stage using embryonic stem cells.

2. The transgenic animal of Claim 1 wherein said animal is a mouse.

3. The mouse of Claim 2, wherein said mouse is fertile and capable of transmitting the altered IL-1 β gene to its offspring.

4. The mouse of Claim 2, wherein the altered IL- 1 β gene has been introduced into an ancestor of the mouse at an embryonic stage by microinjection of altered embryonic stem cells into mouse blastocysts.

5. The mouse of Claim 2, wherein the altered IL- 1 β gene has been introduced into the mouse at an embryonic stage by microinjection of altered embryonic stem cells into mouse blastocysts.

6. The mouse of Claim 2, which is designated IL- 1 β KO.

7. The transgenic animal of Claim 1. wherein said animal is a mouse, and said altered form of a IL-1 β is nonfunctional.

8. A method of producing a mouse whose somatic and germ cells contain a gene coding for an altered form of IL-1 β, the altered gene having been targeted to replace wild-type IL-1 β allele into the animal or an ancestor of the animal at an embryonic stage using embryonic stem cells, which comprises: (a) providing a gene encoding an altered form of IL-1 β designed to target a IL-1 β allele of mouse embryonic stem cells;

(b) introducing the altered gene into mouse

embryonic stem cells;

(c) selecting embryonic stem cells which contain the altered gene:

(d) injecting the embryonic stem cells containing the altered IL-1 β gene into mouse blastocysts: (e) transplanting the injected blastocysts into a pseudopregnant mouse, and

( f) allowing the embryo to develop to term: to produce a founder transgenic mouse.

9. The method of Claim 8 wherein the introducing of step (b) is by microinjection.

10. The method of Claim 9 which further comprises the steps:

(g) breeding the chimeric transgenic mice to wild- type mice to obtain heterozygous (F1 ) mice; and

(h) breeding the heterozygous (F1 ) mice to

generate homozygous (F2) IL- l β transgenic mice.

A cell line derived from a transgenic animal of

Claim 1