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  • A01K67/0276—Knock-out vertebrates
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  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/545—IL-1
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  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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  • A01K2217/00—Genetically modified animals
  • A01K2217/07—Animals genetically altered by homologous recombination
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• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
  • A01K2267/035—Animal model for multifactorial diseases
  • A01K2267/0368—Animal model for inflammation

Definitions

• the present invention relates to transgenic nonhuman animals wherein an interleukin-l ⁇ gene is mutated.
• IL- 1 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 1 1981 1. Le et al.. J. Immunol. 138: 2520- 2526 1 1987
• 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 1 1983
• Biologically active IL-1 exists in two distinct forms, IL-l with an isoelectric point of about 5.2 and IL-l ⁇ 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-l ⁇ 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-l ⁇ 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-l ⁇ translation product is known, and is disclosed by March et aL. Nature 315: 641 -647 ( 1985).
• IL-l ⁇ molecules from five different species do not contain hydrophobic signal sequences (Lomedico et aL, Nature 312: 458 [1984
• IL-l ⁇ secretion may be linked to processing.
• These experiments show that the intracellular pool of unprocessed precursor is chased to extracellular mature IL- l ⁇ (Hazuda et aL, J. Biol. Chem. 263: 8473 [ 1989
• IL-l ⁇ precursor is occasionally found extracellularly but does not appear to contribute to the formation of 17 kDa IL-l ⁇ unless incubated at high concentrations in the presence of excess trypsin, chvmotrypsin or collagenase in vitro (Hazuda et aL, J. Biol. Chem.
• Interleukin-l ⁇ Converting Enzyme that is capable of cleaving the IL-l ⁇ precursor at Asp - 16- Ala l ' 7, as well as at a homologous site at Asp-7-Gly- ⁇ , and generating mature IL-l ⁇ with the appropriate amino terminus at Ala l 17 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 1 1989]) or other amino acids (Howard et aL. J. Immunol.. 147.
• IL-l ⁇ deficient transgenic mice would aid in defining the normal role(s) of IL-l ⁇ , and allow an animal model of IL-l ⁇ deficiency to be used in the design and assessment of various approaches to modulating IL-l ⁇ activity.
• Such IL-l ⁇ modified transcenic mice can also be used as a source of cells for cell culture.
• IL-l ⁇ 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-l ⁇ gene are produced. These transgenic animals are useful in the analysis of the in vivo activity of IL-l ⁇ as well as modulators of IL-l ⁇ activity, and are useful as an animal model of IL-l ⁇ -mediated diseases including chronic and acute inflammation.
• Figure 1 is a genomic map of the mouse IL- l ⁇ gene and the predicted modification of the mouse chromosomal IL-l ⁇ 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-l ⁇ (knockout) gene.
• ES embryonic stem
• Figure 3 is a Southern blot analysis of tail DNA from transgenic mice having an IL-l ⁇ knockout. Southern analysis of genomic DNA from heterozygous x heterozygous crosses yielded the expected number of mice homozygous for the disrupted IL- l ⁇ allele.
• Figure 4 is a Northern hybridization analysis for IL- l ⁇ RNA in the knockout and wild-type control mice after LPS induction.
• Transgenic animals are generated which have a partially deleted IL-l ⁇ gene.
• 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-l ⁇ elicited responses.
• a transgenic animal carrying a "knockout" of IL-l ⁇ is useful for the establishment of a nonhuman model of diseases involving IL-l ⁇ , and to distinguish between the activities of the different interleukins in an in vivo system.
• mouse genomic IL-l ⁇ gene permits the construction of a targeting vector for the disruption of the mouse IL- 1 ⁇ gene.
• the mouse genomic IL-l ⁇ gene is isolated using the mouse IL- l ⁇ 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.
• 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.
• 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-l ⁇ gene generally should not fully encode the same IL-l ⁇ as native to the host animal, and its expression product should be altered to a minor or nreat decree, or absent altogether. However. it is conceivable that a more modestly modified IL-l ⁇ gene will fall within the scope of the present invention.
• 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.
• ES cells may be obtained from pre- implantation 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)).
• IL-l ⁇ can function as an independent component of a complex mechanism, IL-l ⁇ 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-l ⁇ 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. CeU 5J_: 503- 12, ( 1987)) and is reviewed elsewhere
• homologous plasmid- chromosome 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 -1 95 ( 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 plasmid- chromosome 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.
• PPS positive- negative selection
• 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 ( l -(2-deoxy 2-fluoro-B-D-arabinofluranosyl)-5- iodouracil).
• GANC ganciclovir
• FIAU l -(2-deoxy 2-fluoro-B-D-arabinofluranosyl)-5- iodouracil.
• the targeted genes of the invention include DNA sequences which are designed to specifically alter cognate endogenous alleles.
• the mouse cosmid library cESI was screened using mIL- l ⁇ 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 ⁇ cDNA sequence.
• the 0.8 kb mouse IL-l ⁇ cDNA probe was generated by PCR using the Bi 1 1 -neo plasmid as a template and the following oligonucleotide primers: 5'-GCT AGC GTT CCT GAA AAC TTG-3' (SEQ ID NO: l ) 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
• the cosmid clones for mouse IL-l ⁇ 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-l ⁇ hybridizing regions of the IL-l ⁇ gene were localized by hybridizing complete and end-ordered partial digestions of the cosmids with the mouse IL-l ⁇ cDNA and oligonucleotide probes.
• the cloned mouse IL-l ⁇ gene (designated as p 12849- 146-
• Mouse IL-l ⁇ 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 ⁇ 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-l ⁇ .
• a 7.7 kb EcoRI restriction fragment containing the mouse IL- 1 ⁇ 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- l ⁇ 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.
• a gene targeting vector for inactivating the IL-l ⁇ 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 l fragment of the IL- l ⁇ gene as the long arm and a 1.3kb Kpnl-Bglll fragment as the short arm.
• 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 Rl 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).
• T4 pol T4 DNA polymerase
• CIAP calf intestinal alkaline phosphatase
• the short arm of the mouse IL- l ⁇ gene was isolated from pBlue EcoRI-IL- l ⁇ 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 ⁇ 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.
• ORF open reading frame
• the 1.3 kb short arm was ligated into the blunt-ended Eco Rl 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 Rl site immediately upstream of the IL-l ⁇ exon 7. Following digestion with Eco Rl, 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 Rl-Sal I fragment. The PGKp-NEO fragment was made blunt- ended with T4 pol and ligated into the blunt-ended Eco Rl site of plasmid A.
• NEO neomycin resistance gene
• 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- l ⁇ gene was isolated from pBlue/Kpnl-IL-l ⁇ as a 4.0 kb, Bam HI fragment, and made blunt-ended with T4 pol.
• the long arm fragment consists mostly of IL-l ⁇ 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 i 2849-316- 1 , contains a 4.0 kb IL- l ⁇ 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 ⁇ 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.
• 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.
• the gene targeting vector used in the IL-l ⁇ gene disruption experiments was the pi 2849-316- 1 vector of Example 3.
• IL-l ⁇ KO IL-l ⁇ knockout
• exons 1 to 6 of the coding region were deleted (Fig. 1 ).
• the mouse embryonic stem cell line AB2.1 was electroporated with Sall-linearized p i 2849-316- 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 l x l ()7 ES cells and 25 ⁇ g linearized vector in 0.8 ml PBS buffer at 230V, 500 ⁇ F using a Bio-Rad Gene Pulser.
• ES cell transformants were selected with the antibiotic GENETICIN® (Gibco G418: 200 ⁇ g/ l active G41 ) 24 hr post electroporation. and some transformants were counter-selected with FIAU (Bristol Myers Squibb; 0.2 ⁇ M) 48 hours later for enhancement of homologous recombinants.
• FIAU Bacillus Squibb; 0.2 ⁇ M
• Murine leukemia inhibitory factor (LIF: ESGRO, Gibco BRL. Inc.) was used at 200 U/ml.
• 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- l ⁇ 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-l ⁇ gene and there is no other integration events in addition to targeted recombination.
• IL-l ⁇ -targeted AB2.1 cell lines were characterized by Southern hybridization analysis to confirm that the IL- l ⁇ 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.
• IL-l ⁇ targeted ES clones 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", EJ. 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- l ⁇ targeting was confirmed by Southern hybridization analysis performed on genomic DNA isolated from tail samples obtained from these 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- l ⁇ 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.
• 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, a). followed b ⁇ phenol hloroform extractions and ethanol precipitation. Southern hybridization analysis (as described in Example 5) were used to identity offspring which contained the disrupted IL-l ⁇ allele. These transgenic offspring were heterozygous for the IL-l ⁇ disruption. Both transgenic heterozygous and nontransgenic mouse (tail) genomic DNAs weie digested with EcoRI. and were hybridized with a 3' flanking DNA piobe to confirm the transgenic IL- l ⁇ structure. Southern hybridization analysis confirmed that the structure of thereteied IL- l ⁇ allele was identical to that predicted, and previously characterized in the IL- l ⁇ targeted ES clones.
• mice Male and female transgenic mice, each of which contained one copy of the altered IL-l ⁇ allele (heterozygous mice), weie mated with each other to generate mice in which both copies of the IL- l ⁇ gene were the targeted, altered transgenic IL-l ⁇ gene. It was predicted that one fourth of the mouse embryos would be homozygous for the altered IL-l ⁇ 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- l ⁇ -/-.
• mice of Example 9 Surviving homozygous IL-l ⁇ deficient mice of Example 9 were bred with wild-type or heterozygous mates to determine if they were fertile. All homozygous IL-l ⁇ -/- males and females tested were fertile. Significant differences in gross morphology or histology between the IL-l ⁇ deficient mice and the wild-type or heterozygous mice were not observed.
• mice Both the wild-type and IL- l ⁇ KO mice were sensitized by i.p. injection of JP. acnes and challenged by i.p. injection of 10 ⁇ g LPS 6 days later to induce the expression of IL- l ⁇ . 3-3.75 hours after challenge, mice were sacrificed by C02 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- l ⁇ by ELISA (Table 1 ). Table 1 is the result of ELISA analysis of IL- l ⁇ protein after LPS induction. As expected, in contrast to the wild- type controls, the knockout mice did not exhibit any significant IL- l ⁇ activity.
• Liver RNA was prepared from the same mice and Northern hybridizations were carried out by standard procedures using the mouse IL-l ⁇ cDNA as probe (Fig. 4). As expected, the liver RNA from the knockout mice did not exhibit any detectable IL-l ⁇ expression, whereas wild-type control animals showed a significant amount of IL- l ⁇ activity.

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