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`Patentamt
`Europfilsches
`European
`Patent Office
`Office européen
`des brevets
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`lllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll
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`EP 1 532 260 B1
`
`(12)
`
`EUROPEAN PATENT SPECIFICATION
`
`(45) Date of publication and mention
`of the grant of the patent:
`10.12.2008 Bulletin 2008/50
`
`(21) Application number: 03790868.8
`
`(22) Date of filing: 06.08.2003
`
`(51) Int CI.:
`C12N 15/90 "””""”
`A01K 67/027‘2""“"’
`
`C12N 15/10(2°”"°”
`
`(86) International application number:
`PCT/EP2003/00871 3
`
`(87) International publication number:
`WO 2004/020645 (11.03.2004 Gazette 2004/11)
`
`(54) CHROMOSOMAL LOCI FOR THE STRINGENT CONTROL OF GENE ACTIVITIES VIA
`TRANSCRIPTION ACTIVATION SYSTEMS
`
`CHROMOSOMSTELLEN ZUR STRINGENTEN KONTROLLE VON GENAKTIVITAT DURCH
`TRANSKRIPTIONSTEUERSYSTEME
`
`LOCI CHROMOSOMIQUES POUR LE CONTROLE STRICT DES ACTIVITES GENIQUES AU MOYEN
`DE SYSTEMES D'ACT|VAT|ON DE TRANSCRIPTION
`
`(84) Designated Contracting States:
`AT BE BG CH CY CZ DE DK EE ES FI FR GB GR
`HU IE IT LI LU MC NL PT RO SE SI SK TR
`
`(56) References cited:
`EP-A- 1 092 771
`
`(30) Priority: 28.08.2002 US 406344P
`
`(43) Date of publication of application:
`25.05.2005 Bulletin 2005/21
`
`(73) Proprietor: TET Systems Holding GmbH & Co. KG
`69120 Heidelberg (DE)
`
`(72) Inventors:
`- BUJARD, Hermann
`69120 Heidelberg (DE)
`- SCHONIG, Kai
`69115 Heidelberg (DE)
`
`(74) Representative: Dick, Alexander et al
`lsenbruck Bésl Hérschler Wichmann Huhn
`Patentanwalte
`Theodor-Heuss Anlage 12
`68165 Mannheim (DE)
`
`o BARON U ET AL: "CO-REGULATION OF TWO
`GENE ACTIVITIES BY TETRACYCLINE VIA A
`BIDIRECTIONAL PROMOTER" NUCLEIC ACIDS
`
`RESEARCH, OXFORD UNIVERSITY PRESS,
`SURREY, GB, vol. 23, no. 17,11 September 1995
`(1995-09-11), pages 3605-3606, XP000775822
`ISSN: 0305-1048
`- ST-ONGE L. ET AL.: "Temporal control of the Cre
`recombinase in transgenic mice by a tetracycline
`responsive promoter" NUCL. ACIDS RES., vol.
`24, no. 19, 1996, pages 3875-3877, XP002270146
`- DATABASE EBI DNA SEOUENCES [Online] 2
`June 2002 (2002-06-02) "Mus musculus BAC
`clone RP23-111G16 from chromosome 6"
`retrieved from EBI Database accession no.
`
`AC122874 XP002270147
`- FESTENSTEIN R. ET AL.: "Locus control region
`function and heterochromatin-induced position
`effect variegation" SCIENCE, vol. 271, 1996,
`pages 1123-1125, XP000876598
`- HENIKOFF S.: "Conspiracy of silence among
`repeated transgenes" BIOESSAYS, vol. 207,
`1998, pages 532-535,
`
`
`
`EP1532260B1
`
`Genentech Exhibit 2005
`
`Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent
`Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the
`Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the op osition fee has been
`Mylan v. Genentech
`paid. (Art. 99(1) European Patent Convention).
`all V. Genentech
`IPR2016-00710
`Genentech Exhibit 2005
`
`Printed by Jouve. 75001 PARIS (FR)
`
`IPR2016-00710
`
`

`
`Description
`
`EP 1 532 260 B1
`
`[0001] The invention relates to a vector for transgenesis by homologous recombination of a MEO and to the use of
`such an vector for transgenesis by homologous recombination of a mammalian cell or organism and to the use of such
`a vector for the preparation of a medicament for somatic gene therapy.
`
`Background of the Invention
`
`[0002] The transgenesis of MEO, that can be e.g. a plant or an animal, comprises the integration of a transcription
`unit into the l\/lEO’s genome that comprises a gene and a regulatory sequence that controls the transcription of said
`gene. To achieve regulation over a maximum amount of orders of magnitude, it is favorable to employ as a regulatory
`sequence one or more transcription control sequence(s) susceptible for binding transactivators fused to an enhancerless
`promoter. The basal activity of a chromosomally integrated minimal (i.e. enhancerless) promotor strongly depends on
`its chromosomal locus. It is well known that the activity of a transcription unit (consisting of at least a promoter, a gene
`to be expressed and a polyadenylation site), which is inserted into a chromosomal locus either by random or targeted
`integration, depends strongly on the context of the surrounding chromatin (Palmiter et al. Annu. Rev. Genet. 29, 465-99).
`When inserted into heterocl‘u'omatin, transcription units are usually silenced.
`in euchromatin, on the other hand, the
`activity of an inserted transcription unit depends on a number of parameters which may affect transcription of the
`integrated gene positively or negatively. When the transcriptional unit is located close to an enhancer, it will show an
`increased activity even in the absence of its cognate activator. This problem can be circumvented, if non»homo|ogous
`recombination is used, by increasing the number of clones (i.e. different integration events) to be screened, or more
`actively by shielding the transcriptional unit via transcriptional silencers which may be susceptible to control by ligands.
`However,
`it is obvious that the first solution of the problem apart from being time consuming leads to the binding of
`personal and financial resources. The latter solution of the problem is complicated and often reduces the maximum level
`of transcription of the gene that can be achieved. Furthermore, also the maximum level of transcription of the gene that
`can be achieved also varies with the chromosomal locus of integration. The locus may also give rise to position effect
`variegation ("PEV“, Palmiter et al. Annu. Rev. Genet. 29, 465-99) and thus, to mosaic expression of the transgene.
`
`Summary of the invention
`
`[0003] The technical problem underlying the present invention is therefore to provide a chromosomal locus for trans—
`genesis of a mammalian cell or organism which does not influence or hamper the regulation of the transcription units
`when introduced into the genome by transgenesis, and that allows the expression of the transgene that forms part of
`the transcription unit at high levels and that does not give rise to any PEV in respect to the transgene.
`[0004] The solution to the above technical problem is achieved by providing the embodiment of the invention as set
`forth in claim 1. Further embodiments of the invention are described in claims 2 to 8.
`
`A vector for transgenes is according to the present invention comprising a chromosomal locus for transgenesis
`[0005]
`of a first multicellular eukaryotic organism (“first MEO") by homologous recombination or random integration of a DNA
`sequence comprising DNA being characteristic for the locus, the locus ensuring both the efficient transcription of the
`gene that is introduced into the genome at said locus (the “transgene"), and the transcriptional control of said transgene
`by a transactivator without disturbing interference with other transcription control elements, comprising the steps of:
`
`(a) providing a transgenic line of a second multicellular eukaryotic organism ("second MEO"), comprising:
`
`(a0) a first and a second transcription unit being integrated into the genome of the second MEO;
`(a l) at least the first transcription unit being stably transmitted to the progeny;
`(a2) the first transcription unit comprising one or more reporter genes and one or more transcription control
`sequence(s), the transcription of the reporter genes being susceptible to control by the binding of a transactivator
`to the transcription control sequence(s) ("first ligand");
`(a3) the second transcription unit comprising a gene encoding the transactivator,
`(a4) the transactivators affinity for the transcription control sequence(s) being susceptible to control by the
`binding of a second ligand; and
`(a5) the transcription of the reporter genes of a cell of the second l\/IEO being susceptible to be stimulated in
`dependence on the concentration of the second ligand, provided that the transactivator is expressed in said cell;
`
`(b) identifying a transgenic line of step (a) further comprising the following teatures:
`
`(b1 ) the ratio of the amount of the reporter gene(s) product present in a cell in the ON state in which transcription
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`of the reporter gene(s) is maximally stimulated to the amount present in the OFF state in which transcription of
`the reporter gene(s) is minimally stimulated, is at least 102;
`(b2) no significant amount of any reporter gene product can be detected during a phase comprising development
`to adulthood if the second MEO is kept in the OFF state during said phase; and
`(b3) no position effect variegation can be observed in respect to the reporter gene(s) in substantially all cells in
`which the transactivator is expressed.
`
`Detailed description of the invention
`
`[0006] The method usually starts with the preparation of a plurality of transgenic MEO by non—homo|ogous recombi-
`nation also known as random integration of the trancription unit into the genome of a second MEO used for identification
`of the locus. The second MEO is preferably a mouse.
`it
`is possible to ensure that all transgenic lines obtained by
`transgenesis exhibit features (a0) to (a5).
`[0007] The feature (a2) refers to the firsttranscription unit that is to be introduced into the genome. The first transcription
`unit comprises one or more reporter genes and one or more transcription control sequence(s), the transcription of the
`reporter genes being susceptible to control by the binding of a transactivator to the transcription control sequence(s)
`(also named 'first ligand’ hereinafter). A transactivator is a protein that binds to cognate DNA sequences within a promotor
`and activates the transcription of this promotor. The affinity of the transactivator for the transcription control sequences
`are dependent on the concentration of a second ligand (e.g. doxycycline). The binding of the transactivator to both
`ligands shows positive or negative cooperativity. This means in the context of the invention that the binding of the first
`ligand positively or negatively influences the binding of the second ligand, and vice versa, depending on the nature of
`the transactivator. The transcription control sequence or the transcription control sequences are preferably built by a
`tetO sequence that may be multimerized to form e.g. a heptamer of H910 sequences. The use of the P)e,bi—1 promotor
`is most preferred (see "Tetracyclines in Biology Chemistry and Medicine" ed. By M. Nelson, W. Hillen and RA. Greenwald,
`Birkhauser Verlag Switzerland (2001), p. 139 et seq. and references therein). The reporter genes are preferably genes
`encoding a Iuciferase and/or a recombinase like cre recombinase.
`[0008] The second transcription unit may be introduced into the genome of the transgenic line of the second MEO by
`breeding with a transgenic MEO line expressing a transactivator in substantially all cells of the MEO or in a subset of
`cells that can be histologically identified. This means that the transgenic line of the second MEO of feature (a) will be
`generally heterozygous in respect to both transcription units. The transactivator encoded by the gene which forms a part
`of the second transcription unit is preferably selected from a group consisting of tTA and rtTA and derivatives thereof
`binding to the fetO sequence as a first ligand and to doxycycline as a second ligand with either negative or positive
`cooperativity. For the transactivators which can be preferably used see "Tetracyclines in Biology Chemistry and Medicine"
`ed. By M. Nelson, W. Hillen and RA. Greenwald, Birkhauser Verlag Switzerland (2001 ), p. 139 ff. and references therein;
`PNAS 895547-5551 (1992); Annu. Rev. Genet. 361153-173 (2002).
`[0009] After providing a plurality of transgenic MEOs each individual is tested for the following features (b1) to (b3):
`
`(b1) the ratio of the amount of the reporter gene(s) product present in a cell in the ON state in which transcription
`of the reporter gene(s) is maximally stimulated to the amount present in the OFF state in which transcription of the
`reporter gene(s) is minimally stimulated is at least 102;
`(b2) no significant amounts of any reporter gene product can be detected during a phase comprising development
`to adulthood, if the second MEO is kept in the OFF state during said phase; and
`(b3) no position effect variegation, PEV can be observed in respect to the reporter gene(s) in all cells in which the
`transactivator is expressed.
`
`In feature (b1) the ability of the reporter genes to be regulated is defined. A ratio of the reporter gene present
`[0010]
`in a cell in the ON state in which transcription of the reporter gene(s) is maximally stimulated to the amount present in
`the OFF state in which transcription of the reporter gene(s) is minimally stimulated of 102 will ensure that the cross talk
`of otherfactors that elevate the transcription background in the OFF state due to enhancers in the vicinity of the integration
`site of the first transcription unit is limited. However, preferably the ratio is at least 104-105, more preferably about 105
`or even higher. It is clear that the ratio can only be determined for the cells of the second MEO expressing the reporter
`genes whose expression level is to be tested. This requires that the transctivator is expressed in these cells, too. The
`expression pattern of the transactivator can be influenced by the choice of the promotor governing the transcription of
`the gene encoding for the transactivator which forms a part of the second transcription unit.
`[0011] The OFF and the ON state can be selected by adjusting the concentration of the second ligand in the cells. If
`the transactivator is tTA or rtTA and the second ligand is a tetracyline, e.g. doxycycline, then the concentration of said
`ligand can be adjusted by offering a liquid nutrient containing the second ligand to the MEOs, preferably being mice. If
`one of the reporter genes is Iuciferase the amount of protein can be detected by conventional testing of the enzymatic
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`If one of the reporter genes is cre recombinase the amount of
`activity of the protein homogenate of the tested cells.
`protein can be detected by using Ft26R transgenic mice as starting material for the production of transgenic mice that
`have a /oxP—flanked DNA insert integrated into their genome that allows simple testing of recombination events as
`described in the examples.
`[0012]
`If the first transcription unit comprises more than one reporter gene all of the reporter genes are preferably
`coregulated.
`[0013]
`In feature (b2) the ability ofthe reporter genes is defined to be kept silent during a phase comprising development
`to adulthood including embryogenesis. The regulation of the reporter genes inserted into the genome at a specific
`chromosomal locus should not vary during development of the MEO. Especially it is important to select for transgenic
`MEO that do not show significant amounts of any reporter gene during a phase comprising the development to adulthood
`of the MEO it held in the OFF state. An amount of a reporter gene product is not significant if the amount is not significantly
`elevated compared to amounts of individuals of the same genotype in the OFF state in any developmental stage. More
`precisely, the amount of a reporter gene product is not significant if
`it
`is 10-fold or less above the detection level of
`individuals with a null genotype. If a reporter gene is a recombinase (e.g. cre recombinase) the amounts of the reporter
`gene in the cells of the second MEO are not significant if no recombination activity in the cells is observed during said
`phase. If the MEO is a mouse the recombination activity is preferably tested using the mouse line R26R. R26R can be
`used as startirg material for the construction of the mouse line trangenic in respect to the first transcription unit or the
`/oxP—f|anked gene can be introduced into the genome by breeding.
`[0014]
`Feature (b3) prescribes that no position effect variegation, PEV can be observed in respect to the reporter
`gene(s) in all cells in which the transactivator is expressed and in which therefore the reporter genes can be transcribed
`and expressed. Generally PEV is presumed to be caused by plasticity of the promoter structure in the surrounding of a
`transcription unit. This plasticity may result in mosaic expression in populations of identical cells.
`[0015]
`In step (c) sequence information of a sequence flanking the first transcription unit is obtained that is sufficient
`to determine the chromosomal locus on the genome of said second MEO. Preferably step (c) further comprises the
`following steps:
`
`(c1) cloning of genome fragments of the second MEO in bacterial artificial chromosomes ("BACs“) or yeast artificial
`chromosomes ("YACs“);
`(c2) testing the clones of step (ct) for the presence of the first transcription unit; and
`(c3) obtaining sequence information of one or both sequence regions that flank the sequence of the firsttranscription
`unit in clones tested positive in step (C2) sufficient to determine the chromosomal locus on the genome of said
`second MEO.
`
`[0016] Cloning of genome fragments in bacterial or yeast artificial chromosomes is known to a person skilled in the
`art (see examples for references). If an artificial chromosome is tested positive for the presence of the first transcription
`unit then the flanking sequences are determined until sufficient information is gained to determine the chromosomal
`locus of the first transcription unit of the respective transgenic line.
`[0017] The invention relates to a vector for transgenesis of a mammalian cell or organism by homologous recombi-
`nation, the vector comprising at least one transcription unit comprising the gene to be introduced into the genome and
`sequences flanking the transcription unit(s), characterized in that the flanking sequences are selected so that homologous
`recombination at a chromosomal LC—l
`locus obtainable by an above method and characterized in mouse by any one
`of SEQ ID Nos: 1
`to 4 is ensured. Such a vector can also be used for somatic gene therapy.
`[0018] The invention further relates to a vector fortransgenesis of a mammalian cell or organism by random integration
`of a DNA sequence comprsing DNA being characteristic for a chromosomal LC’-1 locus, the vector comprising at least
`one transcription unit containing the gene to be introduced into the genome (the "transgene") and sequences flanking
`the transcription unit(s), craracterized in that the flanking sequences comprise a sequence being characteristic for a
`chromosomal LC—1 locus obtainable by an above method and characterized in mouse by any one of SEQ ID Nos: 1
`to
`4. This vector is also usefu for the transgenesis of species for which no ES technology is available. Such a vector can
`also be used for somatic gene therapy.
`[0019] The sequences flanking the transcription unit(s) of this vector will generally be much larger than the flanking
`sequences necessary for ensuring homologous recombination. The length of the flanking sequences is selected so that
`even if the transgenesis is not effected by homologous recombination but by random integration the transcription of the
`transgene is regulated as i would be if transgenesis was effected by homologous recombination. That means that the
`flanking sequences are large enough to emulate the influence of the chromosomal locus obtainable by an above method
`on the transcription of thet ansgene. The flanking sequences will have for example a length of 5 kbp to 150 kbp.
`[0020]
`If a chromosomal locus is identified, sequence information can be obtained that allows the design of the se-
`quences that flank the transcription unit to be introduced into the genome so that homologous recombination at a
`chromosomal locus obtainable by an above method is ensured or that allow random integration as described above.
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`EP 1 532 260 B1
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`The insertion of the transcription unit at the chromosomal locus on the genome of the MEO has the consequence that
`the regulation of said transcription unit is not hampered by enhancers or other sequences in the vicinity of the integration
`site, that the transcription up to high levels is possible provided that the transcription unit contains a suitable gene the
`transcription of which is controlled by a suitable promotor, and that no PEV is observed in respect to the genes of said
`transcription unit.
`[0021]
`A preferred embodiment of such a vector is characterized in that the vector comprises a first and a second
`transcription unit as defined above spaced by a sequence of sufficient length to prevent any influence of transcription
`factors that bind to one of the transcription units and the transcription of the respective other transcription unit. Such a
`vector allows the generation of a transgenic MEO line into which reporter genes encoding e.g. luciferase and/or cre
`recombinase or any other genes of interest and the transactivator gene encoding the transcativator that controls the
`transcription of the reporter gene(s) are introduced by one step. If the vector comprises more than one transcription
`units the transcription units define a cassette that is flanked by sequences being characteristic for a chromosomal locus
`obtainable by an above method so that homologous recombination at a chromosomal locus obtainable by an above
`method is ensured if homologous recombination is chosen.
`[0022]
`A mammalian cell or organism can be obtained by a method of manufacture atransgenic non—human multicellular
`eukaryotic organism by transgenesis via homologous recombination using a vector as described above.
`[0023] The figures show:
`
`Figure 1: Outline of the Tet regulatory principle. Left upper part shows the mode of action of the To controlled
`transactivator (tTA). tTA binds in absence of the effector molecule Dox to the tetO sequence within Pm and activates
`transcription of gene x. Addition of Dox prevents tTA from binding and, thus, the initiation of transcription. Left lower
`part depicts the dose response of Dox on tTA dependent gene expression. Gene activity is maximal in the absence
`of the antibiotic but as effector concentrations increase transcription gradually decreases to background levels at
`Dox concentration > 5 ng/ml. Right upper part illustrates the mechanism of action of the reverse Tc controlled
`transactivator (rtTA). rtTA is identical to tTA with the exception of 4 amino acid substitutions in the TetR moiety. rtTA
`requires Dox for binding to te!O sequences within Ptel in order to activate transcription of gene y. Right lower part
`outlines the dose response of Dox on the rtTA dependent transcription activation. By increasing the effector con-
`centration beyond 20 ng/ml of Dox, rtTA dependent gene expression is gradually stimulated. PM is a minimal
`promoter fused downstream of an array of 7 fetoperators (7). it interacts with tTA as well as with rtTA.
`
`Figure 2: Topography of tTA/rtTA responsive promoters (Ptet). Pte,—1 is composed of a minimal promoter derived
`from the human cytomegalovirus promoter IE of which the sequence between -53 and +75 (+1 being the transcrip-
`tional start site) was fused to an array of 7 equally spaced tet operator sequences (5).
`ln P,e,bi—1. two minimal
`promoters flank either side of the array of tetoperators as described in ref. 9.
`
`Figure 3: Structure of the bidirectional /uc/cretranscription unit and of BAC E1 1 . A. The genes of the firefly luciferase
`and of Cre recombinase, respectively, are coregulated by P[e[bi—1. The /uc gene is flanked by a SV40, the ore gene
`by the human growth hormone (hGH) polyadenylation site (A,,). The expression cassette can be retrieved via unique
`Notl cleavage sites indicated. B. BAC E11 contains three tandemly integrated luc/cre transcription units and at the
`left border a fragment of the hGH polyA site. The insert is flanked at the left side by a 25 kb and at the right side by
`a 50 kb fragment of mouse DNA. Sequence analysis revealed that the two regions stem from mouse chromosome
`6. The size of the insert in BAC E11 is 95 kb, it is flanked by Notl sites as indicated. The cloning vehicle pBe|oBAC—
`HD has been described previously (10).
`
`Figure 4: Localization of the E11 region in C1 of mouse chromosome 6. A. Partial sequences of the E11 insert were
`obtained by probing respective E11 I DNA with primers initiating DNA synthesis from sequences within the vector
`pBe|oBAC (primer 3) or pBluescript (T7 sequencing primer), respectively. B. Blast results ofthe 4 sequences shown
`using the ENSEMBL mouse genomic library (http://www.ensembi.org) C. Position of the E11 region within C1 of
`mouse chromosome 6.
`
`Figure 5: Analysis of E11 transgenic mice. Six mouse lines. E11—1 to E11~6, stably transmitting the intact E11
`fragment were crossed with mice of the TALAP—2 line expressing tTA specifically in hepatocytes. Luciferase activity
`in presence and absence of Dox was determined in extracts of the liver and various other tissues and then compared
`with respective values obtained with the parent LC—1 mouse line. Luciferase activity measured in liver extracts is
`shown. The values given are the means from 4 to 5 animals.
`
`Figure 6: Transcription units incorporated in the LC—1 and rTALAP—1 mouse line, respectively. (A) The bidirectional
`tTA/rtTA responsive promoter Ptetbi—1, present in LC—1 animals, contains an array of seven tet operator sequences
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`flanked by two minimal promoters derived from the human cytomegalovirus promoter IE. The two polyadenylation
`sites are derived from the human growth hormon (ore) and the SV40 early transcription unit (luc), respectively. The
`promoter is activated by rtTA in presence and by tTA in absence of Dox. (B) Animals of the rTAU\P»'| mouse line
`contain a transcription unit consisting ofthe LAP promoter region which extends to -2800, a synthetic gene encoding
`the rtTA2S—S2 transactivator variant, followed by the SV40 polyadenylation site. Transcriptional start sites are denoted
`as +1 .
`
`Figure 7: Luciferase activity in various organs of LC»l animals in the induced and uninduced state. LC—l animals
`were crossed with individuals expressing rtTA or tTA in various cell types/tissues. Luciferase was measured in
`extracts of tissues indicated. (A) rTAClV‘V—3/LC—1, (B) TAlAP—2/LC»1 and (C) rTALAP»t/LC—1 were analyzed. Induced
`and uninduced levels of luciferase are depicted as dark and intermediate grey columns. The light grey columns in
`B and C show luciferase activity in single transgenic LC»1 mice. The instrumental luciferase background around 1
`rlu/ug of protein is indicated. The luciferase values given are the means of 4 to 6 animals.
`
`Figure 8: Examination of LC—1/R26R double transgenic animals for ORE activity. Histological specimen of 14-month»
`old animals are shown. Samples were stained with Xgal overnight and counterstained with nuclear fast red. No b»
`gal activity could be detected in any organ/tissue examined‘ of w'riic'n 6 are exemplified here.
`
`Figure 9: Induction of ORE recombinase in rTALAP—t/LC—t/Fl26R animals. CRE (and luciferase) synthesis was
`induced in triple transgenic animals by ip. injection of Dox. Histological analysis of CRE activity via Xgal staining
`revealed a remarkably specific pattern which reflects the activity of the LAP promoter in the rTALAP—l mouse line.
`Staining as in Fig. 8.
`
`Figure 10: identification of CRE recombinase by immunostaining. LC»1 animals crossed with either rTA'»/V11 or
`‘l'AC3'T"<»t mice were induced by Dox addition or withdrawal, respectively. No sign of CRE protein was detected in
`the uninduced state, whereas intense staining is observed upon induction in hepatocytes and neurons depending
`on the expression pattern of the transactivators in the respective mouse fines.
`
`[0024] The invention is further illustrated by the following examples, which are not to be understood as limiting the
`scope of the claimed subject matter.
`
`Example 1
`
`Introduction
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`[0025] The tetracycline (Tc) controlled gene expression systems (Tet systems) have developed into widely applicable
`tools for the study of gene function in vivo (1 ,2). Particularly in the mouse, presently the prime model of mammalian
`genetics thanks to transgenesis and embryonic stem cell technology, the principle of conditional gene activation as
`provided by the Tet systems allows to dissect gene functions In vivo with unprecedented precision. Indeed, exploiting
`the Tet systems in viva is increasingly yielding new insights into such fundamental processes as development, behaviour
`and disease (3, 4, 5, 6).
`[0026] Thus, the Tet systems have considerable impact not only on basic research in biology, but also on applied
`aspects of life sciences including but not limited to
`
`~ development of cell lines for high throughput screening and fermentation
`= novel animal models for human diseases
`0 gene therapy
`- sterile insect control
`
`- plant breeding, etc.
`
`[0027] The principle of Tc controlled gene expression is depicted in Fig. 1. Two complementary systems have been
`developed (7, 8).
`in both systems, a gene of interest is placed under the control of Pie], a promoter responsive for To
`controlled transcription activators (tTA or rtTA). The binding ofthe transactivators to Pm" I and thus transcription activation
`depends on the presence or absence of doxycycline (Dox), the To derivative most commonly used for Tet regulation in
`vixro and in vivo.
`
`5.7!
`
`A hallmark of a properly set up Tet regulation in any cell or organism is the tightness of control and the wide
`[0028]
`range of regulation which may reach 5 to 6 orders of magnitude.
`[0029]
`importantly, Tet regulation permits not only the repeated activation and inactivation of a gene under study, it
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`EP1 532 260 B1
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`also enables the researcher to tune the expression of a gene to a defined level by administering the appropriate amount
`of the inducer Dox (Fig. 1). This latter feature is of particular interest as it allows to induce graded perturbations into a
`cell/organism.
`[0030] The potential to reverse induced perturbations and the quantitative control of the activity of a gene of interest
`have made the Tet technology the by far most widely applied gene control system (2, 5).
`[0031]
`To achieve optimal Tet regulation, several conditions have to be fulfilled. The invention described herein refers
`to the most crucial one: the properties of the chromosomal loci harbouring Ple, controlled transcription units containing
`the gene(s) of interest. ideally, such loci do not influence Pm, in its inactive state while allowing its high activation by tTA/
`rtTA. Such loci we have termed "silent but activatable" (s/a).
`[0032] Here, the identification, isolation and characterization of a chromosomal region of a transgenic mouse are
`described, which fulfills the conditions of a s/a locus. This locus, designated LC—1 . that harbours both the luciferase and
`the Cre recombinase gene under P,e(bi—1(9; Fig. 2) control, can be transferred via transgenesis to naive mice. In the
`resulting transgenic animals, the PM controlled transcription units can be regulated tightly and over a wide range as in
`the parent animals from which the LC»1 locus was physically retrieved. Thus, the LC—1 locus allows the generation of
`transgenic animals with predictable Tet regulation properties.
`
`Background
`
`It is well known that the activity of a transcription unit (consisting of a promoter, a gene to be expressed and a
`[0033]
`polyadenylation site), which is inserted into a chromosomal locus either by random or targeted integration, depends
`strongly on the context of the surrounding chromatin (11). When inserted into heterochromatin, transcription units are
`usually silenced. ln euchromatin, on the other hand, the activity of an inserted transcription unit depends on a number
`of parameters which may affect transcription of the integrated gene positively or negatively. The locus may also give
`rise to position effect variegation (PEV, 11) and thus to mosaic expression of the transgene.
`[0034]
`In a typical Tc controlled transcription unit, Ptet governs the expression of a gene of interest (Fig. 1). PM consists
`of an array of tefoperators fused upstream of a minimal RNA polymerase II promoter (Fig. 2). Ideally, a minimal promoter
`exhibits no activity on its own when transferred to a cell. Therefore, when fused to tetoperators, its activity will exclusively
`depend on tTA/rtTA an Dox.
`'
`[0035] When a P18, controlled transcription unit is inserted into the genome of a cell, its function will strongly depend
`on the properties of the integration site (7, 11). How a chromatin landscape affects an ectopically inserted transcription
`unit is not understood and, thus, not predictabl