+

WO2008023268A2 - Modèle d'un cancer prolongé exprimant de façon conditionnelle un oncogène chez la souris - Google Patents

Modèle d'un cancer prolongé exprimant de façon conditionnelle un oncogène chez la souris Download PDF

Info

Publication number
WO2008023268A2
WO2008023268A2 PCT/IB2007/003288 IB2007003288W WO2008023268A2 WO 2008023268 A2 WO2008023268 A2 WO 2008023268A2 IB 2007003288 W IB2007003288 W IB 2007003288W WO 2008023268 A2 WO2008023268 A2 WO 2008023268A2
Authority
WO
WIPO (PCT)
Prior art keywords
bcr
cancer
expression
human
human mammal
Prior art date
Application number
PCT/IB2007/003288
Other languages
English (en)
Other versions
WO2008023268A3 (fr
Inventor
Isidro Sanchez-Garcia
Maria Perez-Caro
Original Assignee
Universidad De Salamanca
Consejo Superior De Investigaciones Cientificas
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad De Salamanca, Consejo Superior De Investigaciones Cientificas filed Critical Universidad De Salamanca
Publication of WO2008023268A2 publication Critical patent/WO2008023268A2/fr
Publication of WO2008023268A3 publication Critical patent/WO2008023268A3/fr

Links

Classifications

    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • 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
    • A01K2217/00—Genetically modified animals
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • 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
    • A01K2267/0331—Animal model for proliferative diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/005—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB
    • C12N2830/006—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB tet repressible

Definitions

  • the invention relates, in general, to cancer development; more specifically, leukaemia.
  • the invention relates to transgenic non-human animals that express BCR-ABLp 190 in a controllable fashion.
  • Imatinib mesylate is a moderately potent inhibitor of the kinase BCR-ABL p2l °, the fusion protein product of a chromosomal translocation, named Philadelphia chromosome (Ph), that is involved in the pathogenesis of chronic myeloid leukaemia (CML) (Druker et al., 2001a; Mauro and Druker, 2001; O'Dwyer and Druker, 2001; Chabner and Roberts, 2005) and of the pl90
  • CML chronic myeloid leukaemia
  • BCR-ABL product which is mainly associated with B-cell acute lymphoblastic leukaemia (B-ALL) (Chan et al., 1987; Clark et al., 1987; Hermans et al., 1987; Kurzrock et al., 1987; MeIo, 1996). It has been shown that when Imatinib mesylate is used to treat patients with chronic-phase CML, 90% seem to achieve complete hematological remission and many lose cytogenetic evidence of the malignant clone (Druker et al., 2001a). However, in the acute leukaemic phase of CML or in P190-B-ALL, Imatinib mesylate treatment is not effective (Shah et al., 2002).
  • CML and pl90-B-ALL begins in a hematopoietic stem cell (HSC), a target population that is largely quiescent (Cobaleda et al., 2000; Cox et al., 2004).
  • HSC hematopoietic stem cell
  • Imatinib mesylate treatment Graham et al., 2002; Chu et al., 2005
  • BCR/ ABL kinase activity may be required for abnormal proliferation and expansion of Ph+ cells but may not be essential for preservation of primitive malignant cells and clearly indicates that a complete understanding of the cancer process requires more detailed knowledge of the mechanisms maintaining neoplastic growth.
  • a major obstacle to elucidating the mechanisms that are responsible for cancer maintenance and not just for the initial event that transforms a normal cell into a cancer cell is a lack of an appropriate animal model. Therefore, the generation of mouse models mimicking human cancer pathology is a prerequisite not only for understanding the genesis of human cancer but also for the identification of molecular events responsible for cancer maintenance.
  • the inventors have surprisingly overcome the problems associated with the animal models described in the prior art by developing a conditional transgenic animal model, which reproduces human cancer.
  • transgenic non-human mammal model of a sustained cancer comprising in its genome a human oncogene, wherein the expression of said oncogene can be regulated exogenously by an effector substance.
  • Transgenic non-human mammals of this type are thus useful, among other goals, for studying cancer, in particular hematological malignancies as well as for evaluating potentially useful compounds for treating, diagnosing and/or preventing such pathologies.
  • the animal is particularly useful as a model which faithfully reproduces human hematological malignancies. In these models, and cells derived therefrom, it has been found that inactivation of the oncogene is not able to eliminate the tumour- maintaining cells.
  • the model disclosed herein uses a binary system (i.e. a single plasmid), closely replicates the pathology of the human disease and allows the translation of the target validation/selection/therapeutic response observed to human beings.
  • the model described herein is the first demonstration that it is possible to reproduce human cancer in animals using regulated expression of an oncogene. Furthermore, these animals faithfully predict the human cancer response and can be used to select targets responsible for cancer maintenance and develop human therapeutic and diagnostic strategies.
  • mice harbouring a tetracycline- repressible BCR-ABLp 190 transgene were generated.
  • a single-plasmid system containing the regulating and expression elements of the original binary tetracycline system was used to allow high induction and tight control of human BCR-ABLp 190 gene expression by tetracycline.
  • This system which has the transactivator and the tet- operator minimal promoter driving the expression gene unit on a single plasmid, ensures the integration of the transactivator and reporter gene units in equal copy numbers in a direct c/s-configuration at the same chromosomal locus and prevents genetic segregation of the control elements during breeding.
  • the system is described herein as the CombiTA-pl90 system.
  • the mice produced did not exhibit morphological defects at birth but did develop B-ALL leukaemias similar to those associated with BCR-ABLpl90 expression in humans.
  • BCR-ABLp 190 confers resistance to cell death induced by the withdrawal of survival factors (Sanchez-Garcia and Grutz, 1995).
  • the inventors have successfully confirmed the physiological relevance of CombitTA-pl90 suppression in vitro by assaying survival of Ba/F3 cells expressing CombitTA-pl90 after IL-3 withdrawal.
  • the CombitTA-pl90 mice expressing the BCR-ABLpI 90 chimeric gene product shows consistently the same phenotype as that with which this oncogene is associated in human pathology.
  • BCR-ABLp 190 expression in the model of the invention allows demonstration that the oncogene can initiate leukaemia when activated in both young and adult mice.
  • the survival conferred by BCR-ABLpI 90 while reversible in vitro, can escape such control in vivo.
  • These results are congruent with the poor efficacy of Imatinib mesylate treatment in P190-B-ALL.
  • CombitTA-pl90 leukaemic mice did not show clinical improvement when treated with Imatinib mesylate and transitory expression of the oncogene was enough to initiate leukaemia development.
  • subject refers to members of mammal species, and includes, but is not limited to, domestic animals, rodent, primates and humans; the subject is preferably a human being, male or female, of any age or race.
  • sample can be any biological sample from a subject, such as a liquid sample, for example, blood, serum, etc., or a solid sample, such as a tissue sample, etc.
  • the sample can be obtained by any conventional method, including surgical resection in case of solid samples.
  • the sample can be obtained from a subject previously diagnosed, or not diagnosed, with a hematological malignancy, or also from a subject undergoing treatment, or who has been previously treated, for a hematological malignancy such as leukaemia.
  • the sample is a liquid or solid biological sample from the blood or bone marrow of the subject.
  • cancer refers to epithelial cancer or mesenchymal cancer.
  • epithelial cancer refers to a cancer of which tumour cells are the cells that line the internal and external surfaces of the body.
  • meenchymal cancer refers to a cancer which tumour cells develop into connective tissue, blood vessels and lymphatic tissue, for example sarcomas and hematological malignancies.
  • hematological malignancy refers to one of a type of cancer that affects blood, bone marrow and lymph nodes. As the three are intimately connected through the immune system, a disease affecting one of the three will often affect the others as well.
  • Illustrative, non-limitative examples of said hematological malignancies include lymphomas, leukaemias, such as, for example, chronic myeloid leukaemia, B- cell acute lymphoblastic leukaemia, T-cell acute lymphoblastic leukaemia, acute myeloid leukaemia, chronic myeloid leukaemia and lymphoproliferative syndromes.
  • leukaemia refers to a cancer of the blood or bone marrow characterized by an abnormal proliferation of blood cells, usually white blood cells (leukocytes).
  • leukaemia include acute lymphocytic leukemia (also known as acute lymphoblastic leukemia or ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), B-cell acute lymphoblastic leukaemia and T-cell acute lymphoblastic leukaemia.
  • ALL acute lymphocytic leukemia
  • AML acute myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • B-cell acute lymphoblastic leukaemia B-cell acute lymphoblastic leukaemia and T-cell acute lymphoblastic leukaemia.
  • gene refers to a molecular chain of deoxyribonucleotides encoding a protein.
  • DNA refers to deoxyribonucleic acid.
  • a DNA sequence is a deoxyribonucleotide sequence.
  • cDNA refers to a nucleotide sequence complementary of a mRNA sequence.
  • RNA refers to ribonucleic acid.
  • An RNA sequence is a ribonucleotide sequence.
  • mRNA refers to messenger ribonucleic acid, which is the fraction of total RNA which is translated into proteins.
  • protein refers to a molecular chain of amino acids with biological activity.
  • oncogene refers to a modified gene, or a set of nucleotides that codes for a protein, that increases the potential malignancy of a tumor cell i.e. that increases the chance that a normal cell develops into a tumor cell.
  • the oncogene is a human oncogene, but other mammalian oncogenes may be used.
  • Many oncogenes are known, and are linked to particular types of cancer.
  • BCR-ABLp210 is related to chronic myeloid leukaemia.
  • BCR-ABL pl90 , TEL-AMLl, E2 A-HLF and E2A-Pbxl are related to B-cell acute lymphoblastic leukaemia (B-ALL).
  • BCR-AB Lp210 BCR-ABLpI 90, Slug, Snail, HOXI l, RHOM2/LMO-2, TALI, Maf-B, FGFR, c-maf, MMSET, BCL6, BCLlO, MALTl, cyclin Dl, cyclin D3, SCL, LMOl, LM02, TEL-AMLl, E2A-HLF, E2A-Pbxl, TEL-ABL, AMLl-ETO, FUS-DDIT3, EWS-WTl, EWS FLIl, EWSRl -DDIT3, FUS- ATFl, FUS-BBF2H7, K-RASv 12, Notch 1, etc.
  • any one of these genes that is used may vary.
  • a mammalian oncogene such as a mouse sequence, more preferably a human sequence.
  • the invention includes the use of variant oncogenes so as to include, for example, polymorphic variants, mutants and other gene types not considered wild type.
  • such genes could be used to investigate differences in the oncogenic potency of such genes that might reflect differences in patterns of disease and response to treatment across patient groups and so on.
  • each of the genes referred to above includes within its scope, references to variants of such genes, for example, that share significant homology or sequence identity of 80%, 85%, 90%, 95%, 98%, 99% or more with the sequences described above.
  • the gene may be cDNA, or may comprise genomic sequence. cDNA is preferred.
  • BCR-ABLpl90 gene refers to the product of a chromosomal translocation event called the Philadelphia chromosome (Ph). Parts of two chromosomes, 9 and 22, swap places. The result is that part of the BCR ("breakpoint cluster region") gene from chromosome 22 (region ql l) is fused with part of the ABL gene on chromosome 9 (region q34).
  • BCR Philadelphia chromosome
  • the nucleotide sequence of the human BCR-ABLpJ 90 gene is known (see, for example, NCBI, Accession number AJ 131467) and this is the preferred gene for use in aspects of the invention referred to herein.
  • BCR-ABLp 190 protein refers to the translation product of the BCR-ABLpI 90 gene.
  • the protein is a kinase.
  • the part of the ABL gene encodes a tyrosine kinase domain and, therefore, the BCR-ABLp 190 protein is a tyrosine kinase.
  • the BCR gene encodes a domain which is a serine/threonine kinase.
  • the amino acid sequence of the human BCR-ABLpI 90 protein is known (see, for example, NCBI, Accession number CAA 10377).
  • transcription product of BCR-ABLpl90 gene refers to the mRNA of BCR-ABLpl90 gene.
  • translation product of BCR-ABLpl90 gene refers to BCR-ABLpl90 protein.
  • the human BCR-ABLpl90 protein is a preferred oncogene according to the invention.
  • the invention is based on the discovery that mice harbouring a repressible BCR- ABLp 190 transgene are capable of mimicking human cancer.
  • the invention provides a transgenic non-human mammal model of a sustained hematological malignancy comprising in its genome a human BCR-ABLpI 90 oncogene, wherein the expression of said oncogene can be regulated exogenously by an effector substance.
  • subsequent inactivation of BCR- ABLp 190 is preferably not able to eliminate the tumour maintaining cells.
  • transgenic comprises a transgene in its genome.
  • said transgene may comprise a nucleic acid sequence encoding the BCR-ABLp 190 protein (i.e., said nucleic acid comprises the BCR-ABLpI 90 gene), the expression of said transgene being exogenously regulated by an effector substance.
  • the BCR-ABLp 190 protein is the human BCR-ABLp 190 protein (see, for example, NCBI, Accession number CAA 10377), encoded by the human BCR-ABLp 190 gene (see, for example, NCBI, Accession number AJ 131467), although other forms, such as the murine form, may also be of some utility.
  • a sequence encoding only a portion of the BCR-ABLp 190 protein such as a fragment, or a variant of the BCR-ABLp 190 protein.
  • fragments we mean any portion of the full length BCR-ABLp 190 protein, including, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the full length sequence.
  • a fragment may include a specific domain or combination of domains within the protein structure.
  • variants we mean any variant of the BCR-ABLp 190 protein, such as, for example, a mutant form comprising one or multiple (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 35, 50, 75, 100 or more) insertions, deletions, substitutions and so on.
  • the transgenic non-human mammal provided by this invention possesses a genotype that confers a greater tendency to develop cancer when compared to the non-transgenic mammal.
  • subsequent inactivation of the causative oncogene is not able to eliminate the tumour maintaining cells.
  • cancers that are generated in transgenic models of the type disclosed herein include epithelial cancer, such as lung, prostate, intestine, breast, skin, pancreas and mesenchymal cancers including sarcomas and hematological malignancies.
  • epithelial cancer such as lung, prostate, intestine, breast, skin, pancreas and mesenchymal cancers including sarcomas and hematological malignancies.
  • the transgenic non-human mammal provided by the invention possesses a genotype that confers a greater tendency to develop a hematological malignancy such as lymphomas, lymphoproliferative syndromes and leukaemia, such as, for example, acute lymphocytic leukemia (also known as acute lymphoblastic leukemia or ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML).
  • ALL acute lymphocytic leukemia
  • AML acute myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • the transgenic models of the type disclosed herein develop B-cell acute lymphoblastic leukemia.
  • the introduction of a DNA construct in which BCR-ABLp 190 is expressed in a way which allows regulation by an exogenous factor, into the genome of a model animal causes a genetic anomaly.
  • the genetic anomaly caused by the expression of the BCR-ABLpI 90 chimeric transgene results in a hematological malignancy, in particular leukaemia.
  • the descendants are analysed to evaluate the existence of activated genes and/or genes created by the genetic anomaly associated with the pathology in question.
  • non-human mammal includes any non-human animal belonging to the class of mammals.
  • the non-human mammal is preferably a mouse but may be another mammalian species, for example another rodent, for instance a rat, hamster or a guinea pig, or another species such as a monkey, pig, rabbit, or a canine or feline, or an ungulate species such as ovine, caprine, equine, bovine, or a non- mammalian animal species.
  • the transgenic non-human animal provided by the invention is a murine animal.
  • the term "murine" includes mice, rats, guinea pigs, hamsters and the like.
  • the murine animal is a rat or a mouse; most preferably the non-human mammal of the invention is a mouse.
  • transgenic animals pose questions of an ethical nature, the benefit to man from studies of the types described herein is considered vastly to outweigh any suffering that might be imposed in the creation and testing of transgenic animals.
  • drug therapies require animal testing before clinical trials can commence in humans and under current regulations and with currently available model systems, animal testing cannot be dispensed with. Any new drug must be tested on at least two different species of live mammal, one of which must be a large non-rodent. Experts consider that new classes of drugs now in development that act in very specific ways in the body may lead to more animals being used in future years, and to the use of more primates.
  • transgenic models such as those described herein is not in any limited to mice, or to rodents generally, but encompasses other mammals including primates.
  • the specific way in which these novel drugs will work means that primates may be the only animals suitable for experimentation because their brain architecture is very similar to our own.
  • This aspect of the invention aims to reduce the extent of attrition in drug discovery and development. Whenever a drug fails at a late stage in testing, all of the animal experiments will in a sense have been wasted. Stopping drugs failing therefore saves test animals' lives. Therefore, although the present invention relates to transgenic animals, the use of such animals should reduce the number of animals that must be used in drug testing programmes and decrease attrition rates in clinical assays in humans. Although the Applicant does not wish to be bound by this theory, it is postulated that transitory expression of BCR-ABLpI 90 in the transgenic non-human mammalian model of the invention is enough to initiate leukaemia development and expression of BC R-ABLp 190 is not required to maintain leukaemia development. This suggests that there are other mechanisms responsible for the maintenance of the cancer pathology. The invention, therefore, allows for the investigation of the mechanisms responsible for cancer maintenance.
  • mechanisms responsible for cancer maintenance refers to epigenetic and/or genetic modifications produced in the cells of the transgenic non- human mammalian model of the invention by the initiating oncogenic event.
  • the term “mechanisms responsible for cancer maintenance” encompasses any cellular mechanism which plays a role in maintaining the cancer pathology after the initiating oncogenic event. Such mechanisms may involve the upregulation or downregulation of one or more genes; this includes an increase or decrease in the level of transcription products and translation products of the gene.
  • initiating oncogenic event refers to the molecular event which causes the development of a cancer pathology.
  • the initiating oncogenic event is the expression of the transgenic oncogene.
  • An example of an initiating oncogenic event is the expression of the BCR-ABLp 190 gene.
  • targets responsible for cancer maintenance refers to any gene or protein involved in the mechanisms responsible for cancer maintenance, subsequent to the initiating event.
  • the term also includes any intermediate stage of gene regulation, including any transcription or translation product.
  • effector substance refers to any substance which is capable of regulating the expression of the oncogene in the genome of the transgenic non-human mammal of the invention when said substance is administered.
  • Suitable exogenously regulated expression systems are well known by persons of skill in the art and include the tetracycline system and Cre-Lox technology (see, for example, Maddison K., Clarke AR. 2005. New approaches for modelling cancer mechanisms in the mouse. J. Pathol. 205:181-193).
  • Preferred effector substances include tetracycline and Adeno-Cre.
  • a DNA construct containing the oncogene is made.
  • this gene is introduced into the animal as a DNA construct, preferably comprising regulatory sequences.
  • regulatory sequences may be derived from humans, animals, prokaryotes or other species. In cases where the regulatory genes are not of human origin, the regulatory genes may be derived from the target animal, for example, the mouse.
  • regulatory genes is meant to include any promoter or enhancer sequences, 5' or 3' UTRs, poly-A termination sequences or other DNA sequences, that are necessary for transcription of the gene of interest.
  • Transcripts used for insertion of human sequences are preferably terminated by a poly A motif.
  • the invention may incorporate the endogenous promoter with the coding gene so that the fidelity of wild type expression is retained, developmentally, temporally and in a tissue-specific manner.
  • endogenous promoter is meant the promoter that naturally directs expression of the gene of interest.
  • the endogenous promoter may thus be the endogenous human promoter, or may alternatively be the promoter that is endogenous to that introduced gene in the transgenic animal subject.
  • the expression of the human gene may be directed by the endogenous mouse promoter for that gene.
  • said construct hereinafter referred to as the DNA construct of the invention, thus comprises the oncogene cDNA, for example BCR-ABLpI 90, under the control of an expression system exogenously regulated by an effector substance.
  • an expression system exogenously regulated by an effector substance.
  • the exogenously regulated expression system may be based on the tet-off system (Clontech), i.e. the Combi-tTA
  • the oncogene is preferably under the control of the tet-operator (tetO) minimal promoter.
  • the expression of the oncogene is exogenously regulated by tetracyclin or its derivatives such as doxycyclin, and the effector substance according to the invention is preferably tetracyclin or its derivatives such as doxycyclin.
  • the oncogene of the transgenic non-human mammal according to the invention is preferably silenced in the presence of tetracyclin and activated in the absence of tetracyclin.
  • the original Combi-tTA vector as described by Schultze et al. is preferably modified by the following steps: 1) removal of the tetO-luciferase cassette from said original vector, and 2) introduction of a cassette comprising the tetO minimal promoter and the oncogene.
  • said cassette should be introduced within the ampicillin resistance gene (referred to alternatively as Amp, bla, or the beta-lactamase gene) of the original vector disclosed in Schultze et al.
  • the inventors have improved upon the single-plasmid system of Schultze et al., (1996) containing the regulating and expression elements of the original binary tetracycline system to allow induction and tight control of gene expression by tetracycline in mice.
  • the inventors found that the Schultze system requires some significant modification in order to allow a target gene to be efficiently expressed and appropriately silenced. For example, it has been found that without the modifications described herein, target gene expression is not silenced in the presence of tetracycline or, e.g., tetracyclin derivatives such as doxycyclin, probably because of read-through from the other promoters (e.g. CMV and SV40) that are present on the Schultze plasmid.
  • tetracycline or, e.g., tetracyclin derivatives such as doxycyclin probably because of read-through from the other promoters (e.g. CMV and SV40) that are present on the Schultze
  • one, preferably two or more polyA sequences are introduced in flanking positions around the target gene to ensure that this read-through problem is resolved. Additionally, it has been found useful to introduce a TATA sequence in order to improve expression of the target protein from this system. Preferably, this TATA sequence lies between the tetO sequence and the target gene sequence.
  • said introduced cassette comprises a poly-A sequence, the tetO promotor, a TATA box sequence, the target gene, further two poly-A sequences, an ampicillin resistance gene, and a fourth poly-A sequence.
  • These elements are preferably arranged on said cassette in the aforementioned order.
  • This modified construct, as described above, may be used for expression of any target gene in a manner which is regulated by tetracycline, or its derivatives such as doxycyclin, and forms an independent aspect of the present invention.
  • this aspect of the invention provides a DNA construct adapted for the expression of a target oncogene in a way which allows regulation by an exogenous factor, said construct comprising an origin of replication, at least one promoter, at least one sequence capable of mediating regulation by an exogenous factor, at least one transactivator sequence and a sequence encoding the target gene, wherein the sequence encoding the target gene is flanked on both sides by at least one polyA sequence.
  • the flanking polyA sequences are situated so as to prevent read-through from the promoter sequences, such as in the configuration set out in Figure 1.
  • the construct may contain one, two or more flanking polyA sequences. It is not necessarily essential for the flanking polyA sequences to be directly contiguous with the sequence encoding the target gene. However, in a preferred embodiment, at least one polyA sequence is situated directly 5' and directly 3' to the sequence encoding the target gene.
  • polyA sequence is meant a polyadenylation signal as known from eukaryotic genetics.
  • polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
  • Examples of polyadenylation signals include those derived from SV40, although others will be known to those of skill in the art.
  • Such sequences comprise runs of adenosine nucleotides, preferably between 10 and 500 nucleotides in length, more preferably between 50 and 200 nucleotides.
  • the promoter comprises an SV40 promoter and/or a CMV promoter, more preferably both an SV40 promoter and a CMV promoter.
  • the transactivator comprises the viral VP 16 transactivator domain. More preferably transactivator comprises the viral VP 16 transactivator domain fused to the ⁇ /-repressor protein.
  • transactivator systems will be known to those of skill in the art
  • the construct additionally comprises a promoter sequence, preferably a TATA sequence, preferably situated upstream of the sequence encoding the target gene.
  • the promoter sequence lies between the sequence capable of mediating regulation by the exogenous factor and the sequence encoding the target gene.
  • the sequence capable of mediating regulation by the exogenous factor is tetO, or a functional equivalent thereof, and the exogenous factor is tetracyclin, or a derivative thereof, such as doxycyclin.
  • the sequence encoding the target gene is a gene implicated in predisposition to cancer, including oncogenes, and BCR-ABLp 190. Other useful examples will be clear to those of skill in the art.
  • a preferred embodiment of this aspect of the invention is a construct based on that represented in Figure 1 herein for BCR-ABLpl90.
  • the oncogene is BCR-ABLpl90.
  • the final construct according to the present invention is the Combi-tTA vector resulting from the insertion of said cassette containing the BCR-ABLp 190 gene, and is referred to herein as the Combi-tTA-pl90 vector.
  • the orientation of the poly-A sequence, the tetO promotor, the TATA box sequence, the BCR-ABLpl90 gene, the further poly-A sequences, the ampicillin resistance (beta-lactamase / bla) gene, and fourth poly-A sequence is as shown in Figures IA though, according to some embodiments, the Amp (bla) may also be in the opposite relative orientation.
  • the DNA construct of the invention is next introduced into a non-human mammal, or into a predecessor thereof, in an embryonic state, for example, in the state of a cell, or fertilized oocyte and, generally, not later than the G cell state.
  • a sequence of nucleic acid can be introduced into an embryo of an animal such that it can be incorporated genetically in an active state, all of which can be applied for the generation of transgenic non-human mammals of the invention.
  • One method consists of transfecting the embryo with said sequence of nucleic acid as occurs naturally, and selecting the transgenic animals in which said sequence has been integrated onto the chromosome at a locus that as a result causes the activation of said sequence.
  • Another method implies modification of the nucleic acid sequence, or its control sequences, before introducing it into the embryo.
  • Another method consists of transfecting the embryo using a vector that contains the nucleic acid sequence to be introduced.
  • the introduction of the DNA construct of the invention in the germ line of a non-human mammal is performed by means of microinjection of a linear DNA fragment that comprises the activatable gene into fertilized oocytes of a non- human mammal.
  • the fertilised oocytes can be isolated by conventional methods, for example, provoking the ovulation of the female, either in response to copulation with a male or by induction by treatment with the luteinising hormone. In general, a superovulation is induced in the females by hormonal action and they are crossed with males. After an appropriate period of time, the females are sacrificed to isolate the fertilised oocytes from their oviducts, which are kept in an appropriate culture medium. The fertilised oocytes can be recognised under the microscope by the presence of pronuclei. The microinjection of the linear DNA fragment is performed, advantageously, in the male pronucleus.
  • the linear DNA fragment that comprises the oncogene construct of the invention After the introduction of the linear DNA fragment that comprises the oncogene construct of the invention into fertilised oocytes, they are incubated in vitro for an appropriate period of time or else they are reimplanted in pseudopregnant wet nursing mothers (obtained by making females copulate with sterile males).
  • the implantation can be performed by conventional methods, for example, by anaesthetising the females and surgically inserting a sufficient number of embryos, for example, 10-20 embryos, in the oviducts of the pseudopregnant wet nursing mothers. Once gestation is over, some embryos will conclude the gestation and give rise to transgenic non-human mammals, which theoretically should carry the DNA construct of the invention integrated into their genome and present in all the cells of the organism.
  • This progeny is the GO generation and their individuals are the "transgenic founders".
  • the confirmation that an individual has incorporated the injected nuclear acid and is transgenic is obtained by analysing the individuals of the progeny.
  • the DNA is extracted from each individual and analysed by conventional methods, for example, by PCR using the specific primers or by Southern blot or Northern blot analysis using, for example, a probe that is complementary to, at least, a part of the transgene, or else by Western blot analysis using an antibody to the protein coded by the transgene.
  • antibody refers to a glycoprotein exhibiting specific binding activity to a particular protein, which is called "antigen”.
  • the term “antibody” comprises monoclonal antibodies, polyclonal antibodies, either intact or fragments thereof, recombinant antibodies, etc., and includes human, humanized and non-human origin antibodies.
  • Monoclonal antibodies are homogenous populations of highly specific antibodies directed against a single site or antigenic "determinant”.
  • Polyclonal antibodies include heterogeneous populations of antibodies directed against different antigenic determinants.
  • Other methods for evaluating the presence of the transgene include, without limitation, appropriate biochemical assays, such as enzymatic and/or immunological assays, histological staining for particular markers, enzymatic activities, etc.
  • the preferred transgenic non- human mammal thus generated is preferably obtainable by the procedures mentioned above using the Combi-tTA-pl90 vector.
  • the transgenic non-human mammal of the invention is referred to herein as a Combi-tTA-pl90 mouse.
  • the inserted transgene is transmitted as a Mendelian characteristic and so it is not difficult to establish stable lines for each individual. If the GO individuals are crossed with the parent strain (retrocrossing) and the transgene behaves with Mendelian characteristics, 50% of the progeny will be heterozygotic for the inserted transgene (hemizygotic). These individuals constitute the Gl progeny and a transgenic line that can be maintained indefinitely, crossing hemizygotics of the Gl generation with normal individuals. Alternatively, individuals of the Gl generation can be crossed among themselves to produce 25% homozygotics for the inserted transgene, 50% hemizygotics and 25% without the transgene provided the transgene does not affect the viability of the descendents.
  • the progeny of the transgenic non-human mammal of the invention such as the progeny of a transgenic mouse provided by this invention can be obtained, therefore, by copulation of the transgenic animal with an appropriate individual, or by in vitro fertilization of eggs and/or sperm of the transgenic animals.
  • the term "progeny” or "progeny of a transgenic non-human mammal” relates to all descendents of a previous generation of the transgenic non-human mammals originally transformed. The progeny can be analysed to detect the presence of the transgene by any of the aforementioned methods.
  • the progeny of the transgenic non-human mammal of the invention hereinafter referred to as the progeny of the transgenic non-human mammal of the invention, constitutes a further aspect of the present invention.
  • the invention also relates to a cell line comprising in its genome a human oncogene, wherein the expression of the oncogene can be regulated exogenously by an effector substance.
  • the oncogene is associated with a human cancer pathology and the inactivation of the oncogene does not eliminate the associated cancer pathology.
  • said cell line is a murine cell line.
  • the invention also relates to a cell line of the transgenic non-human mammal of the invention or of the progeny of the transgenic non-human mammal of the invention, to a primary cell of the transgenic non- human mammal of the invention or of the progeny of the transgenic non-human mammal of the invention or to a tissue sample of the transgenic non-human mammal of the invention or of the progeny of the transgenic non-human mammal of the invention.
  • Said cell line, primary cell or tissue sample contains a DNA construct of the invention in its genome, i.e., a DNA construct containing an oncogene such as the BCR-ABLpl90 gene.
  • said cell line, primary cell or tissue sample is a murine cell line, primary cell or tissue sample.
  • the cell lines of the invention can also further comprising a reporter gene useful for spatiotemporal identification of the onset, progression, dissemination and further physiopathological processes, for evaluating the effect of therapies by molecular imaging techniques, for diagnostic assays, drug discovery and development processes, for target identification and for improving the efficacy and reliability of all phases of the clinical development.
  • Reporter genes are nucleic acid sequences encoding directly or indirectly assayable proteins. They are used to replace other coding regions whose protein products are unsuitable or not amenable to the assay envisaged.
  • suitable reporter genes that are known in the art and may be used in the present invention are selected from those genes encoding proteins including but not limited to: chloramphenicol- acetyltransferase, ⁇ -galactosidase, ⁇ -glucuronidase, luciferase, beta-galactosidase, green fluorescent protein, secreted alkaline phosphatase (SEAP), major urinary protein (MUP) or human chorionic gonadotroph (hCG).
  • SEAP secreted alkaline phosphatase
  • MUP major urinary protein
  • hCG human chorionic gonadotroph
  • the cell lines of the invention may be used as biomarkers for detecting the presence of a gene created and/or activated by a genetic anomaly associated with a human cancer pathology in a subject. They may also be used for assessing the risk or predisposition of a subject to develop a human cancer pathology in a subject, or for determining the stage or severity of a human cancer pathology in a subject.
  • the cell lines of the invention can be used for monitoring the effect of the therapy administered to a subject having a human cancer pathology, or for designing an individualized therapy for a subject suffering from a human cancer pathology, or for designing human clinical trials, or for diagnosis of cancer and/or specific processes and effects of cancer development, like cancer dissemination; or for patient selection for personalized therapeutics; or for therapeutic monitoring and evaluation of therapeutic benefits; or for drug discovery and pharmacokinetics guidance.
  • transgenic non-human mammal of the invention, the progeny thereof, the cell lines, primary cell and tissue sample provided by this invention are useful for, among other applications, screening, searching, identifying, discovering, developing and/or evaluating compounds for the prevention and/or treatment of cancer. Furthermore, the transgenic non-human mammal of the invention, the progeny thereof, the cell lines, primary cell and tissue sample provided by this invention, are useful for evaluating potentially useful compounds which target the mechanisms and targets responsible for cancer maintenance, said mechanisms and targets responsible for cancer maintenance being associated with the development of a sustained cancer.
  • the invention refers to the use of the transgenic non-human mammal of the invention, or of the progeny thereof, or the cell lines, primary cell and tissue sample provided by this invention for identifying potentially therapeutic compounds for the treatment of a cancer, or for evaluating the efficacy of therapy administered to a subject suffering from said cancer, or for monitoring the evolution of said cancer, or for affecting, preferably preventing, cancer dissemination.
  • the invention also refers to the use of the transgenic non-human mammal of the invention, its progeny, or of a cell line, a primary cell or a tissue sample from the transgenic non-human mammal of the invention or its progeny in the screening, identification, validation, optimization and/or evaluation of potentially useful compounds (candidate compounds) for the prevention, treatment and/or diagnosis of a cancer, in particular a leukaemia selected from acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia and chronic myelogenous leukemia, preferably for the prevention, treatment and/or diagnosis of B-cell acute lymphoblastic leukemia.
  • a leukaemia selected from acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia and chronic myelogenous leukemia, preferably for the prevention, treatment and/or diagnosis of B-cell acute lymphoblastic leukemia.
  • the invention refers to a method for screening, searching, identifying, validating, optimizing, discovering, developing and/or evaluating compounds for the treatment and/or prevention of a cancer, in particular leukaemia or for repositioning known drugs or combinations of compounds, which comprises administering a candidate compound to a transgenic non-human mammal of the invention, or to its progeny, and monitoring the response.
  • the screening, searching, identifying, discovering, developing and/or evaluating of the candidate compound for the prevention and/or treatment of a cancer, in particular leukaemia can be performed by administering the candidate compound to the transgenic non-human animal of the invention, at different doses, and evaluating the physiological response of the animal over time.
  • the candidate compound can be administered to the transgenic non-human animal of the invention by any conventional and novel method, typically via oral or parenteral, depending, among other factors, on the chemical nature of the candidate compound. In some cases, it may be appropriate to administer the compound in question along with cofactors that enhance the effect of the compound.
  • the invention refers to a method for identifying a target responsible for cancer maintenance, the method comprising comparing the level of one or more potential target(s) responsible for cancer maintenance between two groups, each group comprising a transgenic non-human mammal of the invention or the progeny thereof, or the cell lines, primary cells or tissue samples from the transgenic non-human mammal of the invention or its progeny, wherein the expression of said oncogene is upregulated in the first group; the expression of said oncogene is downregulated or normal in the second group; and comparing the levels of expression of said one or more potential target(s) responsible for cancer maintenance in the first group with the levels the second group.
  • the expression of the oncogene in the second group may have been upregulated for a period of time prior to being downregulated.
  • the above method comprises identifying, validating, optimizing and selecting a compound which inhibits or reduces targets responsible for cancer maintenance and mechanisms responsible for cancer maintenance.
  • the method comprises identifying, validating, optimizing and selecting a compound which inhibits or reduces targets responsible for cancer maintenance and mechanisms responsible for cancer maintenance after downregulation of the expression of the oncogene incorporated into the animal model, for example the BCR-ABLpI 90 gene or its expression products (both transcription products and translation products, i.e., BCR- ABLpl90 mRNA or BCR-ABLpl90 protein).
  • the candidate compound is administered to a transgenic non-human mammal of the invention or to its progeny, wherein the level of the oncogenic expression products in a tissue/cell sample is known, and, subsequently, the level of expression products in said tissue is quantified, and a compound which is able to inhibit or reduce the malignant cells after dowregulation of the expression of the oncogene, is selected.
  • the invention comprises using a compound identified by any of the methods of the invention in the manufacture of a medicament for treating cancer.
  • the above method comprises identifying, validating, optimizing and selecting a compound which inhibits or reduces the level of expression of the oncogene incorporated into the animal model, for example the BCR-ABLpl90 gene or its expression products (both transcription products and translation products, i.e., BCR- ABLpI 90 mRNA or BCR-ABLp 190 protein).
  • the candidate compound is administered to a transgenic non-human mammal of the invention or to its progeny, wherein the level of the oncogenic expression products in a tissue sample is known, and, subsequently, the level of expression products in said tissue is quantified, and a compound which is able to inhibit or reduce the level of the relevant expression product, e.g. 5C/?- ⁇ 5Z,/?/90 mRNA or BCR-ABLpl90 protein, is selected.
  • the invention refers to a method for screening, searching, identifying, validating, optimizing, discovering, developing and/or evaluating compounds for the treatment and/or prevention of a cancer, in particular leukaemia, or for repositioning known drugs or combinations of compounds, which comprises contacting a cell line, or a primary cell, or a tissue sample of the transgenic non-human mammal of the invention, or its progeny, and monitoring the response.
  • the screening, searching, identifying, validating, optimizing, discovering, developing and/or evaluating of the candidate compound for the prevention and/or treatment of a cancer, in particular leukaemia can be performed by adding the candidate compound to a culture medium containing cells from a cell line or from primary cells or from tissue samples provided by the present invention, for an appropriate period of time, at different concentrations, and evaluating the cellular response to the candidate compound over time using appropriate biochemical and/or histological assays.
  • cells may be used that are transfected with a construct that expresses an oncogene in a manner regulated by an exogenous substance.
  • the Combi-TA-pl90 vector is used, as described herein. At times, it may be necessary to add the compound in question to the cellular culture medium along with cofactors that enhance the effect of the compound.
  • the above method comprises identifying and selecting a compound which inhibits or reduces targets and mechanisms responsible for cancer maintenance after dowregulation of the expression of the oncogene.
  • the candidate compound is contacted with a cell line, or with a primary cell, or with a tissue sample of the transgenic non-human mammal of the invention, or its progeny, wherein the level of BCR-ABLpI 90 expression products in said cell line, primary line or tissue sample is known, and, subsequently, the level of BCR-ABLpI 90 expression products in said tissue is quantified, and a compound which is able to inhibit or reduce the malignant cells after dowregulation of the expression of the oncogene, is selected.
  • the above method comprises identifying and selecting a compound which inhibits or reduces the level of expression of BCR-ABLpI 90 gene or its expression products (both transcription products and translation products, i.e., BCR-ABLpl90 mRNA or BCR-ABLp 190 protein).
  • the candidate compound is contacted with a cell line, or with a primary cell, or with a tissue sample of the transgenic non-human mammal of the invention, or its progeny, wherein the level of BCR-ABLp 190 expression products in said cell line, primary line or tissue sample is known, and, subsequently, the level of BCR-ABLp 190 expression products in said tissue is quantified, and a compound which is able to inhibit or reduce the level of BCR-ABLpI 90 expression products is selected.
  • this compound When a compound inhibits or decreases the levels of the BCR-ABLpl90 expression products or reverts the effects of the increased expression of said gene or the activity of BCR-ABLp 190 protein, this compound becomes a candidate for cancer therapy, especially for treating and/or preventing a hematological malignancy, in particular leukaemia.
  • Illustrative, non limitative, examples of compounds which inhibit or decrease the levels of the mechanisms responsible for cancer maintenance and/or BCR-ABLpl90 mRNA include antisense BCR-ABLp 190 mRNA, ribozymes, triple helix molecules, small interference RNA (siRNA), small organic molecules etc.
  • Illustrative, non limitative, examples of compounds which inhibit or decrease the levels of the mechanisms responsible for cancer maintenance and/or BCR-ABLpWO protein include antibodies anti-BCR-ABLpl90, enzymes or proteins which regulate the activity of the mechanisms responsible for cancer maintenance and/or BCR-ABLpl90 protein, small organic molecules such as small natural or synthetic organic molecules of up to 2000Da, preferably 800Da or less, peptidomimetics, inorganic molecules, peptides, antibodies, structural or functional mimetics of the aforementioned etc.
  • the invention refers to the use of a compound which inhibits or decreases the levels of the targets or mechanisms responsible for cancer maintenance and/or BCR-ABLpl90 expression products or reverts the effects of an increased level of BCR-ABLpI 90 expression products in the manufacture of a pharmaceutical composition for prevention and/or treatment of a leukaemia.
  • a compound which inhibits or decreases the levels of the targets or mechanisms responsible for cancer maintenance and/or BCR-ABLpl90 expression products or reverts the effects of an increased level of BCR-ABLpI 90 expression products in the manufacture of a pharmaceutical composition for prevention and/or treatment of a leukaemia include antisense BCR-ABLp 190 mRNA, ribozymes, triple helix molecules, small interference RNA (siRNA), antibodies anti-BCR-ABLpl90, enzymes or proteins which regulate the activity of BCR-ABLp 190 protein, etc.
  • FIG. 1 CombitTA-p 190 construct, expression and its effect on the survival of the Ba/F3 cells deprived of growth factor.
  • C Survival of Ba/F3 cells expressing CombitTA-pl90 in absence of IL-3. Cells growing exponentially in IL-3 supplemented media were adjusted to 5 x 10 5 cells/ml on day 0 and cultured after removal the IL-3.
  • the cell number of viable cells is shown for Ba/F3+BCR-ABLpl90 cells grown in the absence of IL-3 (Sanchez-Garcia and Grutz, 1995), Ba/F3 cells grown in the absence (Ba/F3-IL3) and in the presence of IL-3 (Ba/F3+IL3), CombitTA-pl90- transfected Ba/F3 cells grown in the presence (+tet) or in the absence (-tet) of doxycycline in IL-3 free medium (the time of treatment with doxycycline was 48 hours), and CombitTA-pl90-transfected Ba/F3 cells grown in the absence of IL3 and doxycycline (-tet) and treated with 10 ⁇ g/ml of STI571 for 48h.
  • FIG. 1 CombitTA-p/90 transgene expression.
  • FIG. 1 B-cell acute lymphoblastic leukaemia development in ComitTA-pl90 mice.
  • PB peripheral blood
  • BM bone marrow
  • spleen of CombitTA-pl90 and control mice were analyzed by flow cytometry with combination of the specific myeloid (Macl) and B-cell lymphoid (B220) markers.
  • STI571 does not prolong the survival of CombitTA-pl90 mice. Mice were randomized to treatment with either STI571 or placebo. The survival curve depicts the percentage of animals alive at the indicated time point. The numbers of mice in each arm (N) is also shown.
  • FIG. 5 Phenotypes in CombitTA-pl90 mice after suppression of BCR-ABLpl90 expression by tetracycline treatment.
  • B) Flow cytometry phenotypic characteristics of cells from peripheral blood (pb) and bone marrow (bm) in both lines of CombitTA-pl90 mice after suppression of BCR-ABLpl90 expression by tetracycline treatment (4 mg/ml) for 10 weeks. (ND. Not detected).
  • Figure 6. B-cell differentiation in CombitTA-pl90 mice after downregulation of BCR-ABL expression by doxycycline treatment. Total number of B-cells, ratio of IgM-TIgM+ cells, and the ratio between BCR-ABIf' 90 /abl determined by real time PCR was measured in control, untreated CombitTA-pl90 mice, and CombitTA-pl90 mice under doxycycline treatment. A representative flow cytometry analysis and representative H/E stained sections of liver and spleen are shown per each group of mice. (ND. Not detected).
  • the cDNA for human BCR-ABLP 190 was cloned into the Combi-tTA vector (Schultze et al., 1996; Perez-Mancer a et al., 2005a; Perez-Mancera et al., 2005b).
  • Linear DNA fragments for microinjection were obtained by No/I digestion and injected into CBA x C57BL/6J fertilized eggs.
  • We identified transgenic mice by Southern analysis of tail snip D ⁇ A after EcoRl digestion as described (Garcia-Hernandez et al., 1997).
  • mice included in this study were subjected to standard necropsy. All major organs were closely examined under the dissecting microscope, and samples of each organ were processed into paraffin, sectioned and examined histologically. All tissue samples were taken from homogenous and viable portions of the resected sample by the pathologist and fixed within 2-5 min of excision. Haematoxylin-and eosin-stained sections of each tissue were reviewed by a single pathologist (T.F.). For comparative studies, age- matched mice were used (wild-type or CombitTApl90 mice in the continuous presence of tetracycline). Cell culture.
  • Cell lines used include Ba/F3 (Palacios et al., 1985) and Ba/F3 cells expressing the human proteins BCR-ABL” 190 (Ba/F3+pl90), BCR-ABL” 210 (Ba/F3+p210) (Sanchez- Garcia and Grutz, 1995) and CombitTApl90.
  • Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). When required, 10% WEHI-3B-conditioned medium was added as a source of IL-3.
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • Ba/F3 cells were transfected by electroporation (960 ⁇ F, 220 V) with 20 ⁇ g of either CombitTA-/?/90 or empty vector (CombitTA).
  • the neomycin-resistant pool of cells (Ba/F3+CombitTA-p790) was analysed by RT-PCR for CombitTA-/;/ 90 expression in the presence and in the absence of tetracycline (20 ng/ml). These cells were resistant to IL-3 withdrawal during growth in the absence of tetracycline.
  • the TET OFF repression system was analysed in Ba/F3+ CombitTApl90 cells in presence or absence of tetracycline by Northern-blot, using the total RNA of these cells. Cells were screened for resistance to IL-3 withdrawal and cell viability was determined by trypan blue exclusion.
  • cytoplasmic RNA of different cells was glyoxylated and fractionated in 1.4% agarose gels in 10 mM Na2HPO4 buffer (pH 7.0). After electrophoresis, the gel was blotted onto Hybond-N (Amersham), UV-cross-linked, and hybridised to 32p_i a b e lled ABL cDNA probe. Loading was monitored by reprobing the filter with a ⁇ -actin probe.
  • DNA was prepared from mice tails. DNA was digested with restriction endonucleases as described (Garcia-Hernandez et al., 1997), separated by electrophoresis in 0.8% agarose, transferred to Hybond-N (Amersham). Filters were UV cross-linked and hybridised to a 32 P-radiolabeled full-length ABL probe.
  • RT was performed according to the manufacturer's protocol in a 20- ⁇ l reaction containing 50 ng of random hexamers, 3 ⁇ g of total RNA, and 200 units of Superscript II RNase H' reverse transcriptase (GIBCO/ BRL).
  • Real-time quantitative PCR was carried out for the quantitation of CombitTA-/?/90. Fluorogenic PCRs were set up in a reaction volume of 50 ⁇ l using the TaqMan PCR Core Reagent kit (PE Biosystems). cDNA amplifications were carried out using the same primers in a 96-well reaction plate format in a PE Applied Biosystems 5700 Sequence Detector.
  • Thermal cycling was initiated with a first denaturation step of 10 min at 95 0 C.
  • the subsequent thermal profile was 40 cycles of 95 0 C for 15 s, 56 0 C for 30 s, 72°C for 1 min.
  • Multiple negative water blanks were tested and a calibration curve determined in parallel with each analysis.
  • the AbI endogenous control (PE Biosystem) was included to relate CombitTA-p/90 to total cDNA in each sample.
  • BCR-ABLP 190 sense primer 5'-CCGCAAGACCGGGCAGAT-S', antisense primer 5'- CAGATGCTACTGGCCGCTGA-3' and probe 5 ' -TGGCCCAACGATGGCGAGGG- 3';
  • c-Abl sense primer 5'-CACTCTCAGCATCACTAAAGGTGAA-S', antisense primer 5'-CGTTTGGGCTTCACACCATT-3', and probe 5'-
  • Nucleated cells from total bone marrow (flushing from the long bones), peripheral blood, thymus, liver and spleen cell suspensions were made.
  • contaminating red blood cells were lyzed with 8.3% ammonium chloride and the remaining cells were then washed in PBS with 2% FCS. After staining, all cells were washed once in PBS with 2% FCS containing 2 ⁇ g/mL propidium iodide (PI) to allow dead cells to be excluded from both analyses and sorting procedures.
  • PI propidium iodide
  • Monoclonal antibodies were obtained from Pharmingen and included: CD45R/B220, CD 19, Ly51, CD43, IgM and IgD for B lineage staining; CD4, CD8 and CD3 for T cell lineage; CDl Ib and GrI for myeloid lineage and Seal for stem cell.
  • Single cell suspensions from the different tissue samples obtained by routine techniques were incubated with purified anti-mouse CD32/CD16 (Pharmingen) to block binding via Fc receptors and with an appropriate dilution of the different antibodies at room temperature or 4 0 C, respectively. The samples and the data were analysed in a FACScan using CellQuest software (Becton
  • mice 4- to 6-week-old athymic (nude) male mice were injected subcutaneously on both flanks with 10" cells resuspended in 200 ⁇ l of phosphate-buffered saline (PBS). The animals were examined for tumour formation for up to 3 weeks.
  • PBS phosphate-buffered saline
  • mice stock solutions of 5 mg/mL and 10 mg/ml were prepared fresh in water, sterile filtered and administered to mice in a volume of 250 ⁇ l by gavage twice a day.
  • Mice were started on STI571 or placebo (the same volume of diluent water) beginning on day after leukaemia was confirmed (day 0) by means of an STI571 regimen of 50 mg/kg every morning and 100 mg/kg every evening by gavage.
  • STI571 was administered in a volume of 250 ⁇ l sterile water by means of straight or curved animal feeding needles. Mice tolerated the therapy well and no interruption of therapy was necessary. Mice were followed clinically 3 times a week, and periodic peripheral blood counts were obtained by tail vein blood draw as indicated.
  • the death endpoint was determined either by spontaneous death of the animal or by elective killing of the animal because of signs of pain or suffering according to established criteria (Wolff and Ilaria, 2001) .
  • transgenic mice In order to determine the role of BCRABLpl90 in maintenance of leukaemia, we generated transgenic mice using the Combi-tTA system, in which the expression of BCR-ABLF 190 gene could be exogenously regulated.
  • This system which has the transactivator and the tet-operator minimal promoter driving the expression gene unit on a single plasmid (Schultze et al., 1996), ensures the integration of the transactivator and reporter gene units in equal copy numbers in a direct cw-configuration at the same chromosomal locus and prevents genetic segregation of the control elements during breeding (Schultze et al., 1996; Perez-Mancer a et al., 2005a; Perez-Mancera et al., 2005b).
  • the tet-repressor protein (fused to the viral VP 16 transactivator domain) binds to an engineered tet-operator minimal promoter and activates BCR-ABLf' 90 transcription (CombitTA-p/PO).
  • BCR-ABL expression protects Ba/F3 cells from apoptosis following IL-3 withdrawal (Sanchez- Garcia and Grutz, 1995) and, as we demonstrated, the level of CombitTA-p/90 expression was sufficient in Ba/F3 cells to prevent cell death.
  • the sensitivity to IL-3 removal was restored by the addition of tetracycline (Figure 1C).
  • CombitTA-p/90 mice were generated to analyse the effect of the BCR- ABLF 190 expression in vivo (Table I).
  • a total of 115 transgenic animals 61 mice corresponded to line 1353A and 54 mice to line 1353B) and 37 control animals were used in this study. Both transgenic lines were analysed in detail and similar phenotypic features were seen in both lines.
  • CombitTA-p/90 mice were born alive without overt morphological abnormalities, and were fully fertile with no differences apparent in the progeny.
  • CombitTA-p/90 mice develop acute leukaemias
  • All CombitTA- pl90 mice analyzed became unwell from approximately 5-7 months of age onward with clinical manifestations that included decreased spontaneous movements in the cage, increased respiratory rates, piloerection, shivering, and animals displayed a sustained loss of body weight (Table I). This condition persisted and animals were sacrificed.
  • Macroscopic analysis showed that CombitTA-/?7P0 mice were pale with splenomegalia and hepatomegalia with pronounced white spots. The thymi and lymphoid nodes generally appeared normal. This examination is consistent with hematological disease.
  • CombitTA-/?i90 mice specifically develop B-cell acute lymphoblastic leukaemias.
  • the BCR-ABLPl*® chimeric product is associated with B-cell acute lymphoblastic leukaemia (B-ALL) (Chan et al., 1987; Clark et al., 1987; Hermans et al., 1987; Kurzrock et al., 1987; MeIo, 1996), therefore, we next defined the acute leukaemic disease generated in our CombitTA-p/90 mice.
  • B-ALL B-cell acute lymphoblastic leukaemia
  • BCR-ABV 190 can initiate leukaemia in both young and adult mice
  • the BCRABL pl90 -B-ALL occurs mainly in adults (Seeker- Walker, 1991; Copeland et al., 1995).
  • the above results support that BCR-ABV 190 expression is the responsible of the beginning of the leukaemia, but they do not demonstrate whether the disease can be initiated if the genetic alteration is activated in adult cells.
  • mice were monitored for evidence of leukaemia both clinically and by serial peripheral blood count. Mice treated with placebo developed a B- ALL around 6 months of age, but the group of mice treated with doxycycline were healthy and did not develop leukaemia. During this period, BCR-ABLF 190 expression was not detected by real-time PCR in the group of mice treated with doxycyline. After the tetracycline treatment for 6 months, doxycycline was removed from the drinking water and BCR-ABLF 190 expression was restored (Table II). A similar B-ALL was detected after doxycycline withdrawal (Table II), indicating that BCR-ABLpI 90 can initiate a B- ALL in both young and adult cells.
  • DOX was removed just for 4 weeks myeloid markers (100) withdrawal and ND (100) and then treatment was re-established once treatment was restored
  • BCR-AB Lp 190 is able to generate leukaemia just by transient expression.
  • CombitTA- p 190 mice subjected to doxycycline treatment (4 mg/ml) until 6 months of age (Table II).
  • doxycycline was removed from the drinking water until we could detect expression of BCR-ABLp 190 in peripheral blood cells of CombitTA- p 190 mice (Table II).
  • BCR-ABLp 190 expression was detected, we restored doxycycline treatment.
  • Doxycycline treatment was able to suppress BCR-ABLp 190 expression and these CombitTA-pl90 mice were monitored for evidence of leukaemia development by serial peripheral blood analysis.
  • mice with leukaemia do not demonstrate marked clinical improvement with the tyrosine kinase inhibitor STI571
  • mice of identical age were randomized to receive either STI571 or the same volume of placebo (water) twice a day beginning on day after leukaemia was confirmed by peripheral blood analysis.
  • STI571 treatment regimens were based on previous pharmacokinetics studies of STI in BCR-ABL tumour-bearing mice (Druker et al., 1996; Ie Coutre et al., 1999). Mice were monitored clinically and by serial peripheral blood count for evidence of leukaemia. STI571 did not prolong the survival of these mice ( Figure 4), reminiscent of human P190-B-ALL.
  • BCR-ABL cancer- associated oncogenes
  • BCR-ABLp 190 is sufficient to generate leukaemias and that the growth of these cells becomes independent of BCR-AB Lp 190 -expression, these results do not imply a specific requirement for BCR-ABLpl90 in the maintenance of malignant phenotype.
  • BCR- ABLp 190 in a disease state mimicking human disease.
  • BCR-ABL expression required to reverse B-cell differentiation block in CombitTA-pl90 mice.
  • BCR-ABL expression is tightly regulated according to the concentration of doxycycline, although we have not found a minimum level of oncogene expression necessary to initiate a neoplastic phenotype.
  • the next question we addressed was whether the level of oncogene expression was related to the differentiation level of blast cells.
  • untreated CombitTA-pl90 mice the total number of B cells is increased in the peripheral blood. This increase is due to the presence of undifferentiated B-cells as measured, for example, by the ratio of IgM- verus IgM+ cells in CombitTA-pl90 mice ( Figure 6).
  • BCR-ABL pl90 is mainly associated with B-cell ALL (Chan et al., 1987; Clark et al., 1987; Hermans et al., 1987; Kurzrock et al., 1987; MeIo, 1996).
  • This chimeric molecule represents an ideal therapeutic target because it is unique to the disease state and it exists in the tumour cells but not in the normal cells of the patient (Sanchez-Garcia I , 1997;Cobaleda et al., 1998).
  • Inhibition of chimeric gene expression by antitumour agents specifically kills the cancer cells without affecting the normal cells (Szczylik et al., 1991; Skorski et al., 1994; Choo et al., 1994; Zao et al., 1997; Huettner et al., 2000).
  • the presence of the chimeric molecule is necessary for the persistence of the tumour cells.
  • these systems used are still limited in representing in vivo biology of the disease.
  • BCR-ABL p190 confers resistance to cell death induced by the withdrawal of survival factors (Sanchez-Garcia and Grutz, 1995).
  • the physiological relevance of the CombitTA-pl90 suppression was confirmed in vitro by assaying survival of Ba/F3 cells expressing CombitTA-pl90 after IL-3 withdrawal.
  • the CombitTA-pl90 mice expressing the BCR-AB LpI90 chimeric gene product shows consistently the same phenotype with which this oncogene is associated in human pathology (Tabernero et al., 2001) .
  • the possibility of modulating BCR-ABL p190 expression in our CombitTA-pl90 model allow s us to demonstrate the oncogene can initiate leukaemia when activated in both young and adult mice.
  • the survival conferred by BCR-ABL pl9 ° while reversible in vitro, can escape such control in vivo.
  • BCR-ABL p190 inactivation does not stop tumour growth
  • previous studies showing that BCR-ABL is required for tumour persistence (Szczylik et al., 1991; Skorski et al., 1994; Choo et al., 1994; Zao et al., 1997; Huettner et al., 2000).
  • BCR-ABL pl9 ° inactivation may depend on the mechanism by which BCR-ABL contributes to tumorigenesis, which is likely to vary according to the genetic and cellular context.
  • BCR-ABL pl9 ° causes a B-ALL, as described here and observed in humans (Tabernero et al., 2001), its inactivation does not revert neoplastic growth.
  • BCR-ABL just transforms a cell into tumourigenic, its inactivation would result in tumour regression (Szczylik et al., 1991; Skorski et al., 1994; Choo et al., 1994; Zao et al., 1997; Huettner et al., 2000).
  • BCR-ABLpI 90 inactivation to eliminate cancer cell progenitors suggests that B-ALL patients will require to combine Imatinib mesylate therapy with other therapeutic strategies (Graham et al., 2002; Bathia et al., 2003; Towatari et al., 2004; Chu et al., 2005).
  • this model could have important implications for the development of new cancer drugs targeting essential aspects of tumour maintenance.
  • a primitive hematopoietic cell is the target for the leukemic transformation in human philadelphia-positive acute lymphoblastic leukemia. Blood, 2000. 95(3): 1007-13.
  • Copelan EA McGuire EA. The biology and treatment of acute lymphoblastic leukemia in adults. Blood. 1995 Mar 1;85(5):1151-1168.
  • Druker BJ Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon NB. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med. 1996 ;2(5):561-566. Druker B.J., Talpaz M., Resta D.J., Peng B., Buchdunger E., et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001 (a). 344:1031-7.
  • Etzioni R., U N., Ramsey, S., Mclntosh, M., Schwartz, S., Reid, B., Radich, J., Anderson, G., Hartwell, L. The case for early detection. Nat Rev Cancer. 2003. 3:243- 52.
  • Wendel HG de Stanchina E, Cepero E, Ray S, Emig M, Fridman JS, Veach DR, Bornmann WG, Clarkson B, McCombie WR, Kogan SC, Hochhaus A, Lowe SW. Loss of p53 impedes the antileukemic response to BCR-ABL inhibition. Proc Natl Acad Sci U S A. 2006; 103(19):7444-7449.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Wood Science & Technology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Biophysics (AREA)
  • Animal Husbandry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne le développement du cancer, de façon plus précise, de la leucémie. En particulier, l'invention concerne des animaux transgéniques comprenant, dans leur génome, un oncogène humain qui peut être régulé de façon exogène par une substance effectrice. L'invention concerne en outre des animaux transgéniques comprenant, dans leur génome, le gène BCR-ABLp 190. De plus, l'invention concerne l'utilisation des mammifères non humains transgéniques de l'invention pour la découverte et le développement de composés pour la prévention et/ou le traitement du cancer.
PCT/IB2007/003288 2006-08-24 2007-08-23 Modèle d'un cancer prolongé exprimant de façon conditionnelle un oncogène chez la souris WO2008023268A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP06017630.2 2006-08-24
EP06017630 2006-08-24

Publications (2)

Publication Number Publication Date
WO2008023268A2 true WO2008023268A2 (fr) 2008-02-28
WO2008023268A3 WO2008023268A3 (fr) 2008-07-31

Family

ID=39107179

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2007/003288 WO2008023268A2 (fr) 2006-08-24 2007-08-23 Modèle d'un cancer prolongé exprimant de façon conditionnelle un oncogène chez la souris

Country Status (1)

Country Link
WO (1) WO2008023268A2 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991007489A1 (fr) * 1989-11-22 1991-05-30 Childrens Hospital Of Los Angeles Animaux transgeniques bcr/abl utilises comme modeles de la leucemie myelogene chronique positive et lymphoplastique aigue du chromosome de philadelphie

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991007489A1 (fr) * 1989-11-22 1991-05-30 Childrens Hospital Of Los Angeles Animaux transgeniques bcr/abl utilises comme modeles de la leucemie myelogene chronique positive et lymphoplastique aigue du chromosome de philadelphie
US5849996A (en) * 1989-11-22 1998-12-15 Childrens Hospital Of Los Angeles BCR/ABL transgenic animals as models for Philadelphia chromosome positive chronic myelogenous and acute lymphoblastic leukemia

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BRAIN JULIA M ET AL: "The kinase inhibitor STI571 reverses the Bcr-Abl induced point mutation frequencies observed in pre-leukemic P190Bcr-Abl transgenic mice." LEUKEMIA RESEARCH, vol. 26, no. 11, November 2002 (2002-11), pages 1011-1016, XP002480384 ISSN: 0145-2126 *
CHOO Y ET AL: "IN VIVO REPRESSION BY A SITE-SPECIFIC DNA-BINDING PROTEIN DESIGNED AGAINST AN ONCOGENIC SEQUENCE" NATURE, NATURE PUBLISHING GROUP, LONDON, vol. 372, 15 December 1994 (1994-12-15), pages 642-645, XP000986225 ISSN: 0028-0836 *
COBALEDA C ET AL: "In vivo inhibition by a site-specific catalytic RNA subunit of RNase P designed against the BCR-ABL oncogenic products: A novel approach for cancer treatment" BLOOD, W.B.SAUNDERS COMPANY, ORLANDO, FL, vol. 95, no. 3, 1 February 2000 (2000-02-01), pages 731-737, XP002239466 ISSN: 0006-4971 *
GARCÍA-HERNÁNDEZ B ET AL: "Retroviral vector design for gene therapy of cancer: specific inhibition and tagging of BCR-ABLp190 cells." MOLECULAR MEDICINE (CAMBRIDGE, MASS.) JAN 1996, vol. 2, no. 1, January 1996 (1996-01), pages 125-133, XP001013586 ISSN: 1076-1551 *
PEREZ-CARO M ET AL: "Sustained leukaemic phenotype after inactivation of BCR-ABLp190 in mice" ONCOGENE, vol. 26, no. 12, March 2007 (2007-03), pages 1702-1713, XP002480386 ISSN: 0950-9232 *
VITALE-CROSS LYNN ET AL: "Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis" CANCER RESEARCH, vol. 64, no. 24, 15 December 2004 (2004-12-15), pages 8804-8807, XP002480385 ISSN: 0008-5472 *

Also Published As

Publication number Publication date
WO2008023268A3 (fr) 2008-07-31

Similar Documents

Publication Publication Date Title
Garzia et al. A hematogenous route for medulloblastoma leptomeningeal metastases
Kalamarides et al. Identification of a progenitor cell of origin capable of generating diverse meningioma histological subtypes
Tang et al. Ablation of Smurf2 reveals an inhibition in TGF‐β signalling through multiple mono‐ubiquitination of Smad3
Larsson et al. Abnormal angiogenesis but intact hematopoietic potential in TGF‐β type I receptor‐deficient mice
Wagner et al. Tsg101 is essential for cell growth, proliferation, and cell survival of embryonic and adult tissues
Pérez‐Caro et al. Cancer induction by restriction of oncogene expression to the stem cell compartment
Rahrmann et al. Identification of PDE4D as a proliferation promoting factor in prostate cancer using a Sleeping Beauty transposon-based somatic mutagenesis screen
McPherson et al. Essential role for Bclaf1 in lung development and immune system function
Mélet et al. Generation of a novel Fli-1 protein by gene targeting leads to a defect in thymus development and a delay in Friend virus-induced erythroleukemia
Vaillant et al. A full-length Cbfa1 gene product perturbs T-cell development and promotes lymphomagenesis in synergy with myc
Alam et al. A uterine decidual cell cytokine ensures pregnancy-dependent adaptations to a physiological stressor
JP4733644B2 (ja) 膵臓腺癌の動物モデルおよびその使用
Hu et al. Mesenchymal-specific deletion of C/EBPβ suppresses pulmonary fibrosis
Dahl et al. Spi‐B can functionally replace PU. 1 in myeloid but not lymphoid development
Zhang et al. Acquisition of cellular properties during alveolar formation requires differential activity and distribution of mitochondria
He et al. A knock-in mouse model of thymoma with the GTF2I L424H mutation
de Roo et al. Axin2‐mTurquoise2: A novel reporter mouse model for the detection of canonical Wnt signalling
US20100061973A1 (en) Graded Expression of Snail as Marker of Cancer Development and DNA Damage-Based Diseases
Giuriato et al. Conditional TPM3-ALK and NPM-ALK transgenic mice develop reversible ALK-positive early B-cell lymphoma/leukemia
Lelièvre et al. Deficiency in hereditary hemorrhagic telangiectasia-associated Endoglin elicits hypoxia-driven heart failure in zebrafish
Perez-Caro et al. Sustained leukaemic phenotype after inactivation of BCR-ABLp190 in mice
CN108135150B (zh) 经遗传修饰的非人类动物和涉及补体依赖性细胞毒性的方法
Liu et al. Triplications of human chromosome 21 orthologous regions in mice result in expansion of megakaryocyte-erythroid progenitors and reduction of granulocyte-macrophage progenitors
Cheng et al. Humanization of the mouse Tert gene reset telomeres to human length
JP2008545405A (ja) 齧歯類幹細胞及びその用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07825546

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

NENP Non-entry into the national phase in:

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 07825546

Country of ref document: EP

Kind code of ref document: A2

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载