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(12) Patent Application Publication (10) Pub. No.: US 2009/0113561 A1 Von Melchner Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2009/0113561 A1 Von Melchner Et Al US 2009.0113561A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0113561 A1 Von Melchner et al. (43) Pub. Date: Apr. 30, 2009 (54) GENE TRAP CASSETTES FOR RANDOM (30) Foreign Application Priority Data AND TARGETED CONDITIONAL GENE NACTIVATION Nov. 26, 2004 (EP) .................................. O4O281941 Apr. 18, 2005 (EP) .................................. O5103092.2 (75) Inventors: Harald Von Melchner, Publication Classification Kronberg/Taunus (DE); Frank (51) Int. Cl Schnutgen, Alzenau (DE); AOIK 67/027 (2006.01) Particia Ruiz, Berlin (DE): Silke CI2N 15/87 (2006.01) De-Zolt, Rodenbach (DE); Thomas CI2O I/68 (2006.01) Floss, Oberappersdorf (DE); Jens CI2N 5/06 (2006.01) Hansen, Kirchheim (DE) (52) U.S. Cl. .............. 800/3:536/23.1; 435/325; 800/13; 435/455: 800/25; 435/6:435/463 Correspondence Address: (57) ABSTRACT NORRIS, MCLAUGHILIN & MARCUS, PA 875 THIRDAVENUE, 18TH FLOOR A new type of gene trap cassette, which can induce condi NEW YORK, NY 10022 (US) tional mutations, relies on directional site-specific recombi nation systems, which can repair and re-induce gene trap (73) Assignee: FRANKGEN mutations when activated in Succession. After the gene trap cassettes are inserted into the genome of the target organism, BIOTECHNOLOGIE AG, mutations can be activated at a particular time and place in Kronberg (DE) Somatic cells. The gene trap cassettes also create multipur pose alleles amendable to a wide range of post-insertional (21) Appl. No.: 11/720,231 modifications. Such gene trap cassettes can be used to muta tionally inactivate all cellular genes temporally and/or spa (22) PCT Filed: Nov. 28, 2005 tially. Cells which contain the inventive genetrap cassette can be used for identification and/or isolation of genes and for the (86). PCT No.: PCT/EP2005/056282 creation of transgenic organisms to study gene function at various developmental stages, including the adult, as well as S371 (c)(1), for the creation of animal models of human disease useful for (2), (4) Date: Aug. 3, 2007 in vivo drug target validation. A. Flip Rosageo fit 3 oxPx1 loxPoxs FlipRosaCeo frt-F3 SAT frt f: xless1 loxox51 ruation excision is gain Patent Application Publication Apr. 30, 2009 Sheet 1 of 7 US 2009/0113561 A1 A. Flip Rosabgeo F3 frt F3 SA Bgeo pia OxFOX511 OxPOX51 FlipRosaCeo frt F3 SAT loxP lox51 loxP Ox511 B. nutation step 1 + FLPe i in Versior step 2 SS rig traffi step 4 excision futation Patent Application Publication Apr. 30, 2009 Sheet 2 of 7 US 2009/0113561 A1 &#8 098 Výz 61-I Patent Application Publication Apr. 30, 2009 Sheet 3 of 7 US 2009/0113561 A1 Patent Application Publication US 2009/0113561 A1 3.10 ?EIT Patent Application Publication Apr. 30, 2009 Sheet 5 of 7 US 2009/0113561 A1 A. - 2k - 1 kb -0.65 kb - 0.5 kb s 32 35 Wt t inv re-inv endo fuslendo fus endo fuslendo fus Wit t inv re-inv RNA poll Wt t inv re-inv RBBP7 Patent Application Publication Apr. 30, 2009 Sheet 6 of 7 US 2009/0113561 A1 A. frt F3xx5 - 1 kb s- 0.65 kb - 0.50 kb - FLP iW - GO1 G3 17Bs o Feas Wt t inv re-inv endo fus endo fus endo fus endo fus Wt t inW re-inv GAPDH C Wt t in re-inv G281 fus GAPDH Patent Application Publication Apr. 30, 2009 Sheet 7 of 7 US 2009/0113561 A1 O O t na S. s. 2 S.is S.is S. &S 9 s s N y Suoesus Joaquin US 2009/01 13561 A1 Apr. 30, 2009 GENE TRAP CASSETTES FOR RANDOM spliced in frame to the reporter/selectable marker gene. Since AND TARGETED CONDITIONAL GENE transcription is terminated prematurely at the inserted poly NACTIVATION adenylation site, the processed fusion transcript encodes a truncated and non-functional version of the cellular protein and the reporter/selectable marker (Stanford, W. L. et al., Nat. 0001. The present invention provides for a new type of Rev. Genet. 2, 756-68 (2001)). Thus, gene traps simulta gene trap cassettes, which can induce conditional mutations. neously inactivate and report the expression of the trapped The cassettes rely on directional site-specific recombination gene at the insertion site, and provide a DNA tag (gene trap systems, which can repair and re-induce gene trap mutations sequence tag, GTST) for the rapid identification of the dis when activated in Succession. After the genetrap cassettes are rupted gene. As gene trap vectors insert randomly across the inserted into the genome of the target organism, mutations genome, a large number of mutations can be generated in ES can be activated at a particular time and place in Somatic cells. cells within a limited number of experiments. Gene trap In addition to their conditional features, the gene trap cas approaches have been used successfully in the past by both settes create multipurpose alleles amenable to a wide range of academic and private organizations to create libraries of ES post-insertional modifications. Such gene trap cassettes can cell lines harboring mutations in single genes (Wiles, M.V. et be used to mutationally inactivate all cellular genes. In addi al., Nat. Genet. 24, 13-4 (2000); Hansen, J. et al., Proc. Natl. tion, the invention relates to a cell, preferably a mammalian Acad. Sci. USA 100, 99.18-22 (2003); Stryke, D. et al., cell, which contains the above mentioned gene trap cassette. Nucleic Acids Res. 31, 278-81 (2003); Zambrowicz, B. P. et Moreover, the invention relates to the use of said cell for al., Proc. Natl. Acad. Sci. USA 100, 14109-14 (2003)). Col identification and/or isolation of genes and for the creation of lectively, the existing resources cover about 66% of all protein transgenic organisms to study gene function at various devel coding genes within the mouse genome (Skarnes, W. C. et al., opmental stages, including the adult, as well as for the cre Nat. Genet. 36,543-4 (2004)). However, the genetrap vectors ation of animal models of human disease useful for in vivo which have been used to generate the currently available drug target validation. In conclusion, the present invention resources induce only null mutations and mouse mutants provides a process which enables a temporally and/or spa generated from these libraries can report only the earliest and tially restricted inactivation of all genes that constitute a liv non-redundant developmental function of the trapped gene. ing organism. Therefore, for most of the mutant strains the significance of the trapped gene for human disease remains uncertain, as BACKGROUND OF THE INVENTION most human disorders result from late onset gene dysfunc 0002 With the complete sequencing of the human and tion. In addition, between 20%-30% of the genes targeted in mouse genomes, attention has shifted towards comprehen ES cells are required for development and cause embryonic sive functional annotation of mammalian genes (Austin, C. P. lethal phenotypes when transferred to the germline, which et al., Nat. Genet. 36,921-4 (2004); Auwerx, J. et al., Nat. precludes their functional analysis in the adult (Hansen, J. et Genet. 36,925-7 (2004)). Among the various approaches for al., Proc. Natl. Acad. Sci. USA 100,9918-22 (2003); Mitch addressing gene function, the most relevant for extrapolation ell, K. J. et al., Nat. Genet. 28, 241-9 (2001)). to human genetic disease is mutagenesis in the mouse. 0005 To circumvent the limitations posed by germine Although several model organisms have been used in a vari mutations, conditional gene targeting strategies use site-spe ety of mutagenesis approaches, the mouse offers particular cific recombination to spatially and temporally restrict the advantages because its genome structure and organization is mutation to somatic cells (von Melchner, H. and Stewart, A. closely related to the human genome. Most importantly, F., Handbook of StemCells, ed. Lanza, R., Vol. 1, pp 609-622 mouse embryonic stem (ES) cells, which grow indefinitely in (2004)). The creation of conditional mouse mutants requires tissue culture, allow the generation of mice with defined the generation of two mouse Strains, i.e. the recombinase mutations in single genes for functional analysis and studies recognition strain and the recombinase expressing strain. The of human disease. recombinase recognition strain is generated by homologous 0003. Several mutagenesis strategies have been deployed recombination in ES cells whereby the targeted exon(s) is in mice, ranging from random chemical (ENU) mutagenesis (are) flanked by two recombinase recognition sequences coupled with phenotype driven screens (Cox. R. D. and (hereinafter “RRSs), e.g. loxP or frt. Since the RRSs reside Brown, S. D., Curr. Opin. Genet. Dev. 13, 278-83 (2003); in introns they do not interfere with gene expression. The Brown, S. D. and Balling, R. Curr. Opin. Genet. Dev. 11, recombinase expressing strain contains a recombinase trans 268-73 (2001)) to sequence-based approaches using ES cell gene (e.g. Cre, Flp) whose expression is either restricted to technology, such as gene trapping and gene targeting (Floss, certain cells and tissues or is inducible by external agents. T. and Wurst, W., Methods Mol. Biol. 185, 347-79 (2002); Crossing of the recombinase recognition strain with the Mansouri, A., Methods Mol. Biol. 175, 397-413 (2001)). recombinase expressing strain deletes the RRS-flanked exons 0004 Gene trapping is a high-throughput approach that is from the doubly transgenic offspring in a prespecified tem used to introduce insertional mutations across the mouse porally and/or spatially restricted manner. Thus, the method genome. It is performed with gene trap vectors whose prin allows the temporal analysis of gene function in particular cipal element is a gene trap cassette consisting of a promot cells and tissues of otherwise widely expressed genes.
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