WO1999036528A2 - Transgenic non-human mammal, a method for the production thereof, and the utilization thereof - Google Patents

Abstract

The invention relates to a method for producing a transgenic non-human mammal, whereby an embryonal stem cell line of a non-human mammal is transfected with a suited recombination vector, stable transfected cellular clones are isolated and implanted in suitably prepared female animals of a non-human mammal. The offspring of said animals are selected based on the existence of the gene exchange. The method is characterized in that the entirety or a part of the p53-gene is functionally replaced by a homologous p53-gene or by a homologous p53-gene segment of another mammal during the recombination event, whereby each channeled sequence is available as a genomic sequence. The invention also relates to the obtained transgenic non-human mammals and to the utilization thereof for testing medicaments, chemicals and therapeutic applications.

Description

Transgenic non-human mammal, process for its production and use

The invention relates to a method for producing a transgenic non-human p53 mammal and its use for testing chemicals, drugs and therapeutic approaches. In particular, the invention relates to the generation of a p53 human knock-in mouse.

The invention further relates to a transgenic non-human mammal in which all or part of the p53 gene is functionally replaced by a homologous p53 gene or a homologous p53 gene segment from another mammal, the respectively introduced sequence being present as a genomic sequence .

Acquired somatic mutations in the tumor suppressor gene p53 very often correlate with the spontaneous onset of cancer in humans and animals, for example colon, lung, liver, breast, bladder, esophageal cancer as well as lymphomas, osteosarcomas, neurofibrosarcomas. Between 30 and 70% of all malignant tumors of almost every organ or tissue type show a point mutation in one of the two p53 gene copies and / or the loss of the other alien. The "normal" p53 protein (wild type) encoded by this gene has a cancer-inhibiting function in that it can stop the cell cycle and induce apoptosis (programmed cell death). With the help of p53-dependent mechanisms, the damaged cell is either repaired again or completely destroyed, whereby the neoplastic process is interrupted in both cases. The p53 protein acts as a negative regulator of cell growth, in which it is induced after DNA damage. Functional loss of p53 can result from point mutation, allele loss, intragenic rearrangement and intragenic deletion. Germline p53 mutations are associated with cancer in early life. The oncogenic potential of various mutations in the p53 gene has been investigated in numerous studies (cf. Harris et al., New Engl. Journ. of Med., 329, 1 31 8-1 327 (1 993)). Certain mutations, so-called hot spol mutations, are more common in some types of tumor. Common mutation sites in the coding sequence of p53 are, for example, codons 273, 248, 1 75, 249 or 245.

Mutated p53 proteins are very often associated with a poor prognosis for the respective cancer and can even inhibit wild-type p53 dominant-negative and perform other additional functions, e.g. stimulate the cell cycle, thus favoring a neoplastic process. An overview of the functional and clinical significance of mutations in the p53 gene can be found in Sidransky et al., Annu. Rev. Med. 47: 285-301, 1 996.

All mutations that have been researched so far and their correlation with the respective cancers are compiled in a database that is maintained by the International Agency for Research on Cancer (IARC) in Lyon, France and is available on CD-ROM (cf. Hollstein et al. , Nucleic Acids Res., 24, 1, 141-146, 1 996).

To identify possible human carcinogens, mice (including transgenic) and other rodents have been exposed to the respective substances and the genes from the tumors formed have been analyzed (cf. Goodrow et al., Molecular Carcinogenesis, 5, 9-1 5, 1 992) . Human cell lines were also exposed in vitro and the mutations in certain genes examined. Such cells were then injected into nude mice and the educated ^¬ th tumors were analyzed for mutations in oncogenes and tumor suppressor genes. More recently, transgenic mice have been used to study carcinogenesis. Transgenic animals are well suited as a model for targeted testing for the effects of a mutation in a tumor suppressor gene such as the p53 gene. For example, a p53 defect mouse was paired with a p53 null allele with a transgenic mouse that contained multiple copies of a mutated mouse p53 gene (Val 135) outside the normal chhromatin context. Animals which are related to the endogenous wild-type p53 gene with the mutated transgenes were hemizygous, developed a tumor more quickly and showed an altered tumor spectrum compared to the non-transgenic control group (Kemp et al., Nature Genetics, 9, 305-31 1 1 995).

However, such carcinogenesis models in animals allow only a few conclusions to be drawn about mutation patterns in humans. On the one hand, the exposures mostly do not correspond to the complex exposures customary in humans, e.g. Tobacco smoke, environmental factors, lifestyle, eating habits etc. Because of the degeneration of the genetic code, according to current knowledge, an animal's mutation spectrum always differs from that of humans (cf. Duflot et al., Carcinogenesis, 1 5, 7, 1 353-1 357, 1 994 and Dumaz et al., Carcinogenesis 1 8, 897-904, 1 997). For example, although the amino acid sequence of the highly conserved DNA binding region of the p53 protein is almost identical in humans, mice and rats, the DNA sequences differ in many nucleotides. It could be shown that the induction of a mutation depends to a large extent on the exact DNA sequence, which is different in the mouse than in the human.

Furthermore, in the transgenic animals described so far, the p53 gene was not in its natural form: in one case the mouse transgene was mutated (not functional) and not in its natural chromosomal location; in the other case the gene sequence was not integrated as a pattern of intron and exon sequences, but rather as cDNA and moreover not under the control of the naturally present regulatory elements and was not expressed in all tissues. These models therefore do not correspond to natural conditions and allow no or only limited information about a possible mutagenesis or carcinogenesis in humans. In a third model, the p53 alleles (mouse) were switched off by "knock-out" and are therefore no longer available for the mutation analysis. There is therefore a need for models which can be used to gain meaningful knowledge about the development and progression of cancer, in particular in humans, with regard to the development of cancer drugs. The present invention is therefore based on the object of providing a model on which carcinogenicity tests with significance for mammals, in particular humans, can be carried out. These carcinogenicity tests are designed to relate to the oncogenic potential of changes in the p53 gene. The invention is also intended to provide a model on which mutation spectra in the human p53 transgene, which are typical for certain classes of human carcinogens or carcinogen exposure, can be represented. The use of carcinogenic substances is said to be able to identify mutation hot spots in the p53 gene which are also important in the natural environment of human p53, ie in humans. This should, for example, enable occupational, nutritional, and lifestyle patterns to be found. A model is to be created on which chemical and pharmaceutical products can be tested with regard to their mutagenic and carcinogenic potential in the human p53 gene in various organs. Furthermore, a model is to be provided with which pharmaceutical products of a new generation can be tested for their effectiveness against human tumors. The model is intended to allow the development of agents which are suitable for diagnostic and / or therapeutic measures in various tumors which carry a mutated p53 gene, and in particular for generating a mutation pattern in the p53 gene which is typical for humans. The interaction of different mutations in the development of tumors should also be investigated.

The present invention therefore relates to a method for producing a transgenic non-human mammal, wherein an embryonic stem cell line of a non-human mammal is transfected with a suitable recombination vector, stably transfected cell clones are isolated and implanted in suitably prepared female animals of a non-human mammal and their progeny are selected for the presence of the gene exchange, the method being characterized in that, in the event of the recombination, all or part of the p53 gene by a homologous p53 gene or a homologous p53 gene segment of another mammal is functionally replaced, the respectively introduced sequence being present as a genomic sequence.

The invention further relates to a transgenic non-human mammal in which part or all of the p53 gene of the mammal is functionally replaced by a homologous p53 gene or a homologous p53 gene segment from another mammal, the respectively introduced sequence being genomic Sequence is present. The respective p53 gene is present in its natural genetic environment and preferably codes for an expressed, non-mutated protein.

The invention further relates to a method in the form of a mammal model for testing new drugs for their effectiveness against human tumors, which is characterized in that the above-mentioned transgenic non-human mammals are treated with the new drug and the tumor regression or remission after method known per se. In addition, the invention relates to a method for testing chemicals for their mutation and cancer-inducing potential, which is characterized in that the above-mentioned transgenic non-human mammal is treated with the chemical and the mutation spectra or tumor induction are determined by methods known per se.

Surprisingly, it was found that the p53 gene of one mammal can be integrated in the form of genomic sequences into the genome of another mammal instead of the p53 gene present there and is then under the control of the regulatory elements naturally present in this genome.

The p53 sequence to be introduced comes from the genome of a mammal other than the transgenic mammal to be generated (eg mouse, rat, rabbit, horse, cow, sheep, goat, monkey, pig, dog, cat). The comes preferably Sequence to be introduced from the human genome. However, it can also come from the genome of another mammal, such as a pet (eg dog, cat etc.) or a farm animal (eg horse, cattle, pig etc.). The p53 sequence can be introduced as a whole or in parts. It can exist as a wild-type sequence or as a mutated sequence, depending on whether a model for testing for carcinogens or drugs is to be obtained. It can also contain one or more mutation (s) that are typical of a specific cancer. The mutation is preferably typical for a type of cancer, for example for colon, lung, liver, breast, bladder, esophageal, as well as lymphomas, osteosarcomas and neurofibrosarcomas.

To generate models for carcinogenicity tests, the exons / introns of the p53 gene of another mammal are preferably introduced, which usually have the greatest number of mutations in tumors. These are preferably human exons 5-9. In models for testing for new medicinal products, those gene parts are preferred which have one or more mutations typical of a specific cancer, e.g. To be able to specifically investigate oncogenesis and therapeutic options for the specific tumor disease.

Additional gene transfer constructs for "gene targeting" were prepared in which e.g. by homologous recombination (e.g. with the constructs 14-1 -3 and 29-38 mentioned below) additional mouse p53 exons were replaced by human p53 exons (e.g. exon 4, exon 10). Finally, constructs were made in which non-coding mouse sequences that control p53 gene transcription were replaced by human control sequences. These control elements include e.g. the 3'-UTR (UTR = Untranslated Region) sequences, the 5'-promoter sequences and intron 4, which contains tissue-specific transcription factor binding sites.

The invention relates in particular to the production of the p53 human knock-in mouse (Phuki mouse). This genetically modified mammal carries the exons and Introns of the human p53 gene as a transgene. The part of the human p53 gene that is most important for tumor development (preferably wild-type p53 or parts thereof) is introduced by homologous recombination instead of the corresponding sequence of the mouse gene. This created a natural situation for the human p53 gene, which is that the p53 gene is present in the form of genomic sequences, ie as intron and exon sequences, but not as cDNA in multiple copies in unknown locations in the genome. Furthermore, the p53 gene is under the control of regulatory elements that are naturally present in the mouse. As a result, the number of copies, the location of the gene and its context are not changed, so that the regulation typical of mammalian cells is retained unchanged for the host organism. With additional plasmids, regulatory elements can be introduced which are intended to bring about gene regulation typical for humans. Such a situation has not previously been described in the known transgenic p53 animals. As a result, the present invention makes it possible, by using carcinogenic substances, to identify mutation hot spots in the p53 gene or p53 protein which are also important in the natural environment of human p53, ie in humans. This enables targeted development of agents that are suitable for diagnostic and / or therapeutic measures for mutant p53 in precancerous diseases and various cancers.

In the method according to the invention, a gene substitution vector or recombination vector is generated in which the p53 gene of a mammal is completely or partially replaced by the corresponding counterpart of another mammal.

This construction is preferably transfected into embryonic stem cells (eg mouse 1 29 / SV) via the mechanism of homologous recombination (cf. RM Torres, R. Kühn, Laboratory Protocols for Conditional Gene Targeting, Oxford University Press, 1 997). The homologous recombination between the DNA sequences present in a chromosome and new, added cloned DNA sequences enables the insertion of a cloned gene into the Genome of a living cell instead of the original gene. With this method, embryonic germ cells can be used to obtain via chimeras animals that are homozygous for the desired gene or the desired gene part or the desired mutation.

Mice with the Phuki constructs can of course be crossed with common mouse strains which are used for carcinogenesis tests and are therefore responsible for the mutagenic / carcinogenic activity of various substances: e.g. SENCAR mice (especially used for skin tumorigenesis studies), B6C3F1 (used for various cancer tests). Other strains of mice that are interesting for crossing with the Phuki mouse are e.g. Strains with genetic defects in DNA repair (e.g. lack of enzyme activity from O6-alkylguanine transferase). These mouse strains are all commercially available and are well known to those skilled in the art.

Furthermore, by crossing the transgenic mammal according to the invention with already established transgenic mammals (e.g. models for general or organ-specific inflammation), double-transgenic mammalian models can be produced, which allow a more realistic simulation of human carcinogenesis than before. For the crossing, for example, the hepatitis B surface antigen (HBsAg) transgenic mouse; the TGF-beta 1 transgenic mouse, which has a transforming growth factor cDNA under the control of regulatory elements from the mouse albumin gene; or the hu-HLA-B27 (a human MHC class I allele associated with human inflammatory diseases) transgenic mouse which develops spontaneous inflammatory responses due to a changed immune system repertoire.

In order to produce a preferred mouse invention, the ge ^¬ be desired exons, including exons 5-9, the endogenous mouse p53 gene by the corresponding exons of the human, including the intron sequences replaced by homologous recombination. This is done in an appropriate manner produced Gensubstitutionsvektor used (for example, the plasmid described below 14-1 -3) ^~ and an embryonic stem cell line (eg ES 129 / SV). The recombinant clones of the mouse embryonic stem cells obtained are implanted in suitably prepared female animals for the production of chimeras. "Appropriately prepared female animals" means that the animals have been prepared for their "carrying out" task by various measures, for example by hormone treatment. In this context, reference is also made to "RM Torres, R. Kühn, see above". Since it is advantageous to have ^homozygous animals, the heterozygous strains (p53 + ^phuk 7 +) thus obtained can then be mated with another animal of the same strain or with a different (heterozygous) mouse strain. For example, this can be a p53 knock-out mouse (p53 + / -) [Donehower et al., Nature 356, 21 5-221, 1 992], in which one of the functional endogenous p53 alleles by interrupting the coding sequences had been rendered inoperative by a foreign DNA. The offspring of this mating (p53 + ^phuki / -) house a ^non- functional mouse p53 allele and a functional hybrid mouse / human p53 allele, which differs from the normal functional mouse allele only with regard to the DNA sequence in the corresponding exons (5 -9) and the cutscenes. These are identical to the sequence of the normal human p53 gene. Thus, the recombinant gene is still controlled by the mouse normal p53 promoter. The chromatic structure, the biological parameters and the gene environment are also unchanged.

The plasmids 14-1 -3 and 29-38 which can be used for gene substitution were obtained on December 1, 1,997 at the DSMZ Braunsschweig (German Collection of Microorganisms and Cell Cultures, Braunschweig) under the accession numbers DSM 1 1 904 (14-1 -3) and DSM 1 1 905 (29-38).

In order to test various cancer risk factors using the transgenic mammal according to the invention, use is made of methods known in this field. These are described, for example, in: Hussain et al., Carcinogenesis 1 8, p. 1 21 -1 25 (1 997) Mace et al., Carcinogenesis 1 8, pp. 1 291-1 297 (1 997) Dümaz et al., Carcinogenesis 1 8, pp. 897-904 (1 997) [Treatment with UV light]

Ruggeri et al., Proc. Natl. Acad. Be. 90, p. 101 3-101 7 (1 993) [treatment with benzopyrenes)

Seil et al., Cancer Res. 51, pp. 1 278-1 285 (1 991) [treatment with aflatoxin B1]

The invention is further described with reference to the figures, which show:

Figure 1 Construction of a p53 mouse-human hybrid gene.

FIG. 2 steps for the construction of a gene transfer vector (“gene targeting”) simple lines: p53 intron sequences black rectangles: p53 exon sequences <<: LoxP sites for excision by means of CRE recombinase

Fragment A: Mouse p53 genomic DNA fragment contains exons 2, 3 and 4 with the intron sequences between them cloned from the mouse 1 29 / SV genome

Fragment B: Drug resistance selection cassette contains the coding genetic information for neomycin resistance and gangcylovir sensitivity. The cassette has its own promoters.

Fragment C: Human p53 genomic DNA fragment contains exons 5, 6, 7, 8, 9 with the intron sequences in between

Fragment D: Mouse p53 genomic fragment contains exon 10 with flanking intronse- quence

Fig. 3 recombination scheme

The model according to the invention can be used for mutagenicity or carcinogenicity studies in order to test chemicals of all kinds for their carcinogenic potential. All products or their metabolites that contribute directly (e.g. through direct DNA adduct formation in this gene) or indirectly (e.g. through favoring endogenous mutagenic adducts in this gene) to mutations in p53 are to be classified as potentially carcinogenic for humans, especially if they cause mutations that are already stored in the p53 database. Known methods are available for the investigation of DNA mutation spectra in the new PHUKI mouse, which already have a few mutations in tumors (Lehman et al., Cancer Res. 51, pp. 4090-4096, 1 991) or adducts or a few Can recognize mutations in p53 mutated cells from non-malignant tissues (cf. Science, 274, p. 430-432, 1 996 and Science 264, p. 1 31 7-1 31 9, 1 994). The model can also be used to test diagnostics for the early detection of mutation patterns in humans. With such a model, both the costs and the time required for animal experiments can be considerably reduced, since the time until the generation of a mutation spectrum in p53 in non-malignant tissues is considerably shorter than that for the generation of mutation spectra in tumors (with p53 mutations). necessary period of time.

To test new drugs for their effectiveness against human tumors, tumors with chemical or other carcinogens with human-typical mutations in the p53 gene are generated in the transgenic animal. These are then treated with the respective test pharmaceuticals. An active ingredient that converts mutated, conformationally modified p53 proteins in tumors to wild-type function makes the tumor cells sensitive to apoptosis and thus triggers the suicide program in the tumor. This should result in remission or regression of the tumor. The following examples illustrate the invention.

example 1

Construction of a recombination vector

To construct a gene substitution vector, a gene bank from DNA of the mouse strain 1 29 / SV in the Lambda Fix II phage (from Stratagene Inc. La Jolla, CA) was screened with a cDNA sample which corresponds to the mouse p53 gene transcript. A phage was isolated with a 14 kb insert of the mouse functional p53 gene (called Strat # 1). A 3.8 kb Kpnl / Xbal fragment and a 2.3 kb BamHI / Xbal fragment were cut out of Strat # 1 by restriction digestion and separately cloned into plasmid pGEM 3Z / A (commercially available from Promega), which the Clones 2QG and 4A results. A Kpnl / Xbal fragment (fragment A, see FIG. 2) was cut out of clone 2QG and then cloned into the pBluescript II KS vector (commercially available from Stratagene). The neomycin resistance / thymidine kinase genes contained in the plasmid pHR1 (obtained from Dr. Kästner, DKFZ Heidelberg) flanked by loxP recognition sites (for the subsequent cre recombinase-controlled excision) were identified as Xbal / HindIII fragment (fragment B; see FIG. 2) cloned to fragment A in the Bluescript II KS vector. Clone 25-34 was obtained. The human fragment C (see FIG. 2) was obtained by PCR amplification of human genomic DNA using the primers (MO9: 5'-TGCAG-G TA CCCGGC ATTTT G AG TG TTA G AC-3 'and MO 5 A: 5 '- CG AT AC - TAGTGTTTCTTTGCTGCCGTGTTC-3'). Fragment C contains the human p53 gene from exon 5 to 9 (and intervening introns). Fragment C and a Kpn / Notl fragment (fragment D, see FIG. 2) from clone 4A were cloned into pBluescript KS II, which results in clone 21 -6. The clones obtained were sequenced using fluorescence-labeled dideoxynucleotides with an ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) for verification. A Spel / Notl fragment from clone 21-6 (contains fragments C + D) has now been cloned into plasmid 25-35. The finished construct now carries A + B + C + D. This construct corresponds to the above-mentioned deposited plasmid 29-3B (Phuki test construct). The other deposited clone 14-1 -3 is identical to 29-38, except that clone 29-38 in fragment C contains a missense mutation which triggers amino acid substitution on codon 1 92 in exon 6, while fragment C in 14-1 -3 corresponds to the wild type and thus represents the "prototype". Construct 29-38, on the other hand, can serve as a test mutant in the experiments, which is particularly interesting since 4% of the point mutations in human tumors take place in the modified codon.

Example 2

Generation of the PHUKI mouse

After linearization by restriction digestion with the restriction enzyme "Notl", the plasmids 14-1-3 and 29-38 obtained in Example 1 were each electroplated with a single pulse of 260 V / 500 μF into pluripotent embryonic stem cell lines of the mouse 1 29 / Sv ( 20 μg / 10 ⁷ cells; E14.1 or MB-1 obtained from Dr. Wang, International Agency for Research on Cancer (IARC), Lyon, France) inserted. Suitable neomycin resistant clones bearing the homologous recombination event were selected. The genomic DNA from these clones was tested by Southern blot to confirm the integration of the plasmid by homologous recombination. The recombination scheme is shown in FIG. 3.

Cell clones which contained the correctly recombined sequence after PCR analysis were isolated and further analyzed by Southern analysis.

Stable transfected cell clones were isolated and the selection cassette (fragment B) was cut out with CRE recombinase. This has the advantage that the mouse p53 intron 4 sequences are no longer interrupted by the 3 Kb of the inserted drug resistance gene, which results in the correct p53 gene transcription or mRNA maturation could interfere if fragment B were not removed. The clones now obtained are then injected into blastocytes and implanted in suitably prepared female mice (pseudo-pregnant). The descendants of this mouse then contain the exchanged gene part. After cross-breeding they are called PHUKI mice (p53 human knock-in mouse) and can be used for the desired experiments or event. be mated again with other mouse strains.

Claims

claims 1) Method for producing a transgenic non-human mammal, wherein an embryonic stem cell line of a non-human mammal is transfected with a suitable recombination vector, stably transfected cell clones are isolated, implanted in suitably prepared female animals of a non-human mammal and the progeny obtained be selected for the presence of the gene exchange, characterized in that during the recombination event, all or part of the p53 gene is functionally replaced by a homologous p53 gene or a homologous p53 gene segment from another mammal, the respectively introduced sequence being genomic Sequence is present. 2) Method according to claim 1, characterized in that the introduced sequence is the p53 wild-type sequence of another mammal. 3) Method according to claim 1, characterized in that the introduced sequence or more of a type of cancer selected from colon, lung, liver, breast, bladder, esophagus cancer, as well Lymphoma ^¬ a homen, osteosarcomas and Neurofibrosarkomen, typical Contains mutation (s) in p53. 4) Method according to claim 1, characterized in that the introduced sequence comprises exons 5 to 9 of the p53 gene of another mammal in unchanged or mutated form. 5) Method according to one of claims 1 to 4, characterized in that the introduced sequence comes from the human p53 gene. 6) Method according to one of claims 1 to 5, characterized in that the non-human mammal is a rodent, in particular a mouse. 7) Transgenic non-human mammal, in which all or part of the p53 gene is functionally replaced by a homologous p53 gene or a homologous p53 gene segment from another mammal, the respectively introduced sequence being present as a genomic sequence. 8) Mammal according to claim 7, characterized in that the introduced sequence is the p53 wild-type sequence of another mammal. 9) Mammal according to claim 7, characterized in that the introduced sequence one or more for a cancer, selected from colon, lung, liver, breast, bladder, esophageal cancer, as well as lymphomas, osteosarcomas and neurofibrosarcomas, typical mutation ( en) in p53. 10) Mammal according to claim 7, characterized in that the introduced sequence comprises exons 5 to 9 of the p53 gene of another mammal. 1 1) Mammal according to one of claims 7 to 10, characterized in that the introduced sequence comes from the human p53 gene. 1 2) Mammal according to one of claims 7 to 1 1, characterized in that it is a rodent, in particular a mouse. 1 3) selectable labeled recombination vector for homologous Rekom ^¬ bination in a method according to any one of claims 1 to. 6 14) recombination vector according to claim 13, characterized in that it stores the plasmid 14-1 -3 or 29-38, deposited on December 1, 6 997 at the DSMZ Braunschweig (German Collection for Microorganisms and Cell cultures) under the deposit numbers DSM 1 1 904 or DSM 1 1 905. 1 5) Stably transfected cell clone, characterized in that all or part of the p53 gene of a non-human mammal is functionally replaced by a homologous p53 gene or a homologous p53 gene segment from another mammal, the respectively introduced sequence as genomic sequence is present. 1 6) Method for testing new drugs for their effectiveness against human tumors, characterized in that a transgenic non-human mammal according to one of claims 7 to 1 2 is treated with the new drug and the tumor regression or remission itself known methods determined. 17) A method for testing of chemicals on their p53-mutagenic and cancer in ^¬ duzierendes potential, characterized in that a trans ^¬ genes non-human mammal treated according to one of claims 7 to 1 2 with the chemical and mutation spectra or Tumorin ^¬ production according to processes known per se determined. 18) Use of a transgenic, non-human mammal according to one of claims 7 to 1 2 for testing drugs, chemicals and therapeutic approaches.