WO2006018316A1 - Methods for the production of improved live stocks and disease models for therapeutic research - Google Patents

Abstract

The invention inter alia relates to a method of providing an embryo capable of producing a mutated non-human animal, the method comprising treating a somatic cell derived from a non-human animal with a mutagen in vitro. The invention also relates to mutated non-human animals obtained by that method.

Description

Methods for the Production of Improved Live Stocks and Disease Models for

Therapeutic Research

Background of the Invention

During the last decades, the mouse became the most popular animal to generate model systems reflecting human diseases. As^' such disease models exhibit disease symptoms, they can be used to further investigate the disease, which leads to a more comprehensive understanding of the disease and opens the field for developing new therapeutic concepts. In addition, such disease models are useful to study the effect of known pharmaceutical compounds as well as to screen for potentially novel compounds. For example, Harvard scientists made a genetically engineered rodent, called OncoMouse^® (see US Patent 4,736,866), carrying a gene that promotes the development of various human cancers. This animal model is a very well recognized tool for developing treatments and cures of human cancer.

Recently, larger domestic animals are more and more proving to be valid research and disease models due to their anatomical and physiological similarities to humans. For example, the pig has proven to be a good model for pancreatic cancer (Kurahashi et al., 2004) and kidney stone disease (Mandel et al., 2004). Furthermore, the pig is a good ischemia model (Hughes GC et al., 2004).

In addition, larger domestic animals may provide transplant organs like heart, liver, and kidney. Due to the problem of donor rejection, those animals need to be modified to overcome immunological problems prior to their use to generate organs. In pigs, it has turned out that the donor rejection is due to sugar-based molecules called alpha- 1,3-galactosylated moieties located at the surface of pig cells. Lai et al. (2002) described a knockout in pig of one allele of the gene GGTAl, which encodes the enzyme alpha-l,3-galactosyl transferase. This transferase is involved in transferring the sugar molecules onto the pig cell surface. Phelps et al. (2003) described a double-knockout in pig, the second allele being silenced by a natural mutation in exon 9. Thus, the direct and reliable provision of pigs with an inactivation of these genes will render the pig an attractive organism to produce transplant organs for humans. Moreover, the modification of a particular gene's normal function in non- human animals, e.g., domestic/farm animals, for economic or medical reasons gains more and more importance. For example, Myostatin, or GDF8 (Growth Differentiation Factor 8), a member of the transforming growth factor-beta superfamily plays a role in the control and maintenance of skeletal muscle mass (Gonzalez-Cadavid, 1998). This information encouraged scientist to develop methods for inhibiting the expression of the myostatin gene or the activity of the protein. A method to inhibit myostatin in order to promote muscle growth and weight gain is disclosed in US Patent Application 2004/0030114. US Patent Application 2002/02127234 discloses an immunoconjugate comprising a full-length myostatin polypeptide linked to a carrier as vaccine. US Patent Application 2003/0140356 discloses myostatin mimetics, binding to the myostatin receptor.

hi addition, mice ("Mighty mouse") and cattle ("Belgium Blue cattle") with inactivating mutations of myostatin have marked muscle hypertrophy. US Patent 5,994,618 discloses transgenic mice carrying a disrupted endogenous myostatin gene. Transgenic mice were significantly larger than wild-type animals and displayed a large and widespread increase in skeletal muscle mass. US Patent 6,103,466 discloses, e.g., cattle myostatin polynucleotide sequences with an 11 bp deletion, resulting in a non-functional myostatin protein. Additional mutations in cattle, nt419(del7-inslθ), Q204X, E226X, and C313Y, resulting in double-muscleing are described in US Patent Application 2003/0129171 and in European Patent Application EP 1 002 068. Thus, the generation of animals with increased body mass is of valuable economic advantages to the farmers. The above examples demonstrate that the increasing knowledge of disease-causing genes and the increasing understanding of gene function require more efficient methods directed to the provision of organisms carrying mutations in various genes.

One possibility to efficiently generate mutants is via physical and chemical mutagenesis. A well-known chemical mutagen in this respect is, e.g., N-ethyl-N- nitrosourea (ENU).

ENU randomly ethylates DNA, which may subsequently result, e.g., into a non-conservative nucleotide exchange (point mutation) during DNA replication in the next cell cycle. Such subtle DNA mutation may further result in a corresponding amino acid exchange. Consequently, ENU mutagenesis does not introduce foreign DNA into the recipient's genome and will not implicate positional effects like those known from random transgene integration.

In mice, the introduction of point mutations in male germ cell DNA is currently performed by intraperitoneal ENU-injection. The mutagenized mouse or its offspring is analyzed for an aberrant phenotype, e.g., mutation identification may be performed with a screen of phenotypic alterations prior to mutation identification in a gene of interest. Alternatively, the injected animal or its offspring is analyzed for a mutation in a gene of interest on a molecular basis without prior observation of any phenotype (see US

Patent 5,994,075). Embryonic stem (ES) cells may also be subjected to ENU mutagenesis, as described in US Patent 6,015,670, WO 97/44485, and WO 99/67361.

ENU mutagenesis has also been described for other species, e.g., Drosophila melanogaster (Vogel and Natarajan, 1995), ascidians (Moody et al., 1999), or zebrafish (Grunewald and Streisinger, 1992). In contrast to mouse, ascidians and zebrafϊsh have been treated with ENU by direct incubation of the organism in ENU (e.g., Moody et al., 1999). Grunewald and Streisinger (1992) reported in vitro ENU mutagenesis of freshly isolated zebrafish sperm and subsequent mixing with freshly collected eggs for fertilization.

Compared to known transgenic methods, e.g., ES-cell mediated gene transfer (Bradley et al., 1984; Capecchi et al., 1989), DNA microinjection (Betsch et al., 1995), nucleus transfer in combination with an initial manipulation of the donor nucleus by a transgene insertion (Schnieke et al., 1998) or Lenti- virus induced gene transfer (Hofman et al., 2004), ENU mutagenesis has the advantage of introducing particular point mutations, i.e., subtle DNA modifications without introducing foreign DNA (for example antibiotic selection marker gene or viral genetic material) into the recipient's genome - like in transgenic animals. The generation of transgenic animals further poses the risk that the transgene integrates within an endogenous gene (undesirable insertional mutation) with a possible loss of host gene function or other positional effects due to random integration, e.g., inappropriate transgene expression. In respect of the aforementioned such transgenic farm animals are classified as genetically modified organisms (GMO) with subsequent restrictions. Over the last three years many countries signed the "Cartagena Protocol on Biosafety" including Germany, France and UK. The Protocol regulates specifically the handling of GMO for the agriculture use. ENU-modified animals are not classified as GMOs.

As mentioned above, ENU mutagenesis is useful for the generation of hypomorphic, hypermorphic and neomorphic alleles of a gene in a model organism, e.g., by single amino acid substitutions. This may be desirable to create a model organism for, e.g., a human trait or disease in which gene function is modified rather than destroyed.

Furthermore, the ENU method also allows for the identification of an allelic series of mutations in a gene of interest. These mutations might be desirable mutations that merely modify gene function (e.g. hypomorphic or hypermorphic alleles that express the gene with reduced or increased efficiency) or that give rise to a new trait in the animal (e.g. by generating dominant neomorphic alleles which result in a gain-of-function or loss-of function). The usefulness of identifying an allelic series of alterations in a gene of interest was illustrated in the human peroxisome proliferator-activated receptor gamma (PP ARγ) gene (Barroso et al., 1999). A dominant-negative V290M mutation and a dominant- negative P467L mutation in the receptor's ligand-binding domain, respectively, is associated with an unusual syndrome of severe insulin resistance, early onset diabetes and hypertension. Therefore, a new subtype of dominantly inherited type 2 diabetes was described, due to defective transcription factor function of PP ARγ. The underlying point mutations provided the first time evidence for the direct involvement of PP ARγ in the control of insulin sensitivity, glucose homeostasis and blood pressure in man (Barroso et al., 1999).

Current methods of ENU mutagenesis, e.g., in mice, are not necessarily appropriate for higher vertebrates, e.g., domestic/farm animals. ENU mutagenesis in mice is performed as intraperitoneal injection of ENU into male mice (see Example 1 of WO 2004/020619), using defined mg/kg dosages of ENU, which are injected once or several times. Due to ENU-induced sterility, the earliest date at which male mice can be mated to females is fifty days after the final ENU injection, when fertility starts to overcome the ENU-induced sterility. The subsequent Gl offspring represents a "living archive", which is subject to phenotypic and nucleic acid analyses, in order to identify a mutation in a gene of interest. Intraperitoneal application of ENU to larger farm/domestic animal, for example male cattle will, however, be associated with relatively high costs and low efficiency. For cattle with a reproduction cycle of 12 month and on average 1 offspring per pregnancy, it would require much time and space to generate the above-mentioned living archive. In addition, dosage testing and optimization may take some time, since intraperitoneal ENU injection may imply lethality of the ENU recipient at certain doses. Furthermore, large amounts of ENU would be required. For a bull with up to 700 kg body weight approximately 63 g of ENU need to be injected in a single dosage. If it survived injection, the period of sterility of a bull may last up to several months.

As an alternative to the maintenance and analysis of the aforementioned

"living archive" of Gl ENU-mutagenized mice, another method for identifying a mutation in a gene of interest comprises the parallel isolation of a tissue sample (for the further isolation of nucleic acid samples) and of sperm cells from all Gl male offspring. The tissue sample and sperm cells are subjected to freezing, representing a "frozen archive". The step of replacing a "living archive" of up to several thousand Gl offspring with a "frozen archive" reduces costs and increases the speed in analysis and generation of mutant animals: nucleic acid samples are used for the analysis of a mutation in a gene of interest by industrial HTS screening of the nucleic acids. Once a desired mutation is identified, the corresponding sperm cells are thawed and subsequently used for in vitro fertilization. The resulting embryos are implanted into a foster mother's uterus to generate offspring. The offspring is carrying the previously identified mutation in a gene of interest, according to Mendelian rules.

However, sperms of certain animal species, especially those that do not fertilize ex vivo, do not always tolerate a freezing procedure well, which may make it difficult to establish an archive that is representative of a large number of mutations.

Campbell et al., 1996, demonstrated the cloning of an organism based on somatic nuclear transfer, dispelling the dogma that it was "biologically impossible" to clone mammals using nuclear transfer with somatic cells (nuclear cloning). In general, somatic cell nuclear transfer (SCNT) is performed by fusion of a somatic donor cell with an enucleated recipient cell. Alternatively, an isolated somatic cell nucleus is transferred into an enucleated recipient cell by microinjection to generate an embryo (Wakayama et al., 1998). A resulting embryo is subject to embryo transfer into the uterus of a pseudopregnant mother animal in order to generate a living animal offspring. Since then nuclear transfer was proven to be a cloning method suitable for many different species, e.g., cattle (Kato et al., 1998), goat (Ohkoshi et al., 2003), pig (Polejaeva et al., 2000), mouse (Ono et al. 2001; Wakayama et al., 1998), rabbit (Chesne et al., 2002), rat (Iannaccone et al., 2001), cat (Shin et al., 2002), and zebrafish (Lee et al., 2002). After the first successful nuclear cloning efforts it became clear that this technique is capable of rendering many species a potential target for genetic modification and subsequently a source for valuable genetic models.

In order to successfully manipulate an organism it is mandatory to manipulate isolated somatic cells from the donor animal in tissue culture first. Of significant importance is the issue that somatic cells keep competence for nuclear transfer after manipulation. Many researchers concentrated on that particular issue when performing somatic cell nuclear transfer techniques at different species, e.g., bovine (Roh et al., 2000, Kubota et al., 1999, and Cibelli et al., 1998), sheep (McGreath et al., 2000 and Schnieke et al., 1997), pig (Bandioli et al., 2001, and Lai et al., 2002), and zebrafish (Lee et al., 2002).

There continues to be a need in the art for methods of producing mutated non-human animals, which avoid one or more of the above-mentioned drawbacks, or provide improvements in this regard, or which represent valuable alternatives to the prior art methods.

Summary of the Invention

The invention is inter alia directed to a method of generating mutated non- human animals and embryos capable of producing such mutated non-human animals. The method of the present invention is applicable to many species. In addition, the method of the present invention of mutagenizing somatic cells provides the possibility to efficiently mutate every gene in an organism. With this method, it is, e.g., possible to efficiently determine the appropriate dosage regime regarding a particular mutagen in vitro, resulting in a substantial saving of time, costs and animals.

More specifically, the present invention provides in a first aspect a method of providing an embryo capable of producing a mutated non-human animal comprising:

(a) treating a somatic cell derived from a non-human animal with a mutagen in vitro;

(b) generating a cell clone or a clonal cell line from the somatic cell treated according to step (a); and

(c) introducing the nucleus of a cell of the cell clone or the clonal cell line of step (b) into an enucleated oocyte to form a 1 cell-stage embryo; or

(d) introducing the nucleus of a cell of the cell clone or the clonal cell line of step (b) into an enucleated oocyte and transferring the subsequently formed pronucleus or nucleus into a second enucleated oocyte to form a 1 cell-stage embryo; or

(e) introducing the nucleus of a cell of the cell clone or the clonal cell line of step (b) into an enucleated oocyte and consecutively transferring the subsequently formed pronucleus or nucleus into a second and further enucleated oocytes, e.g., into a second and subsequently a third enucleated oocyte, to form a 1 cell-stage embryo.

hi another embodiment of the method of the invention, step (b) comprises

(i) isolating the somatic cell as a single cell from a plurality of somatic cells treated with said mutagen; and

(ii) expanding said single somatic cell to provide said cell clone or clonal cell line.

hi another embodiment, the somatic cell treated in step (a) is derived from a non-human embryo. It may, however, also be derived from an adult non-human animal, hi a preferred embodiment, the somatic cell treated in step (a) is a fetal fibroblast cell, or an adult fibroblast cell, including, but not limited to, a skin fibroblast cell. Other suitable and preferred cells are granulosa cells, cumulus cells, or oviduct cells. The method of the invention may additionally comprise the screening of a nucleic acid sample derived from one or more cells of the cell clone or cell line obtained in step (b) for the presence of a mutation in a gene of interest.

The method may further comprise the step of assigning said mutation to a corresponding cell clone or clonal cell line or the corresponding cell clone or clonal cell line generated according to step (b).

In another aspect, the invention provides a method of producing a mutated non-human animal, which method comprises the use of a cell or cells of a cell clone or clonal cell line prepared according to steps (a) and (b) of the method of the invention. In one embodiment, the method comprises the introduction of the nucleus of a cell from said cell clone or clonal cell line into the cytoplasm of an enucleated non-human oocyte, preferably of the same species from which said clonal cell is derived, and subsequently reimplanting the embryo thus formed into a suitable non-human animal pseudopregnant mother.

The invention further provides an archive comprising stored clonal cells and cell lines generated according to steps (a) and (b) or derived from one or more cells of the non-human animals.

The invention also provides a method of producing a non-human animal, wherein said method further comprises breeding of the non-human animal(s) produced by the method as described herein to produce a plurality of offspring.

Brief Description of the Figure

Figure 1 depicts a flow chart, describing in an exemplary manner the method of the invention for generating non-human animals from mutagenized somatic cells.

Briefly, a somatic cell derived from embryonic or adult tissue is subjected to mutagenesis. After mutagenesis single cells are isolated and expanded to clonal cell lines to be kept in a culture repository for short-term storage ("short-term archive"), or in a frozen repository ("frozen archive") for long-term storage. Cells from each clonal cell line are subjected to DNA isolation for subsequent mutation screening in respect of a gene of interest. Once a mutation in a gene of interest is identified and assigned to a particular clonal cell line in the repository, a single cell (or single cells) from this clonal cell line is (are) used for somatic cell nuclear transfer to generate (a) mutant embryo(s). Following embryo transfer pregnant animals will deliver heterozygous Gl offspring. Heterozygous Gl offspring is used for breeding with wild type animals (het x wt) to produce a G2 generation with a plurality of heterozygous offspring. Breeding of selected heterozygotes from G2 (het x het) is performed to obtain homozygous offspring in G3. A plurality of homozygous offspring is obtained by inter-breeding of homozygous G3 offspring (horn x hom) to generate Gn offspring. Gn refers to any offspring generation following G3 offspring and may be used to expand the number of homozygous offspring.

Detailed Description of the Invention

Definitions

The terms "organism" or "organisms" refer to multicellular eukaryotes that undergo development from an embryonic stage to an adult stage. Accordingly, this includes vertebrates and invertebrates, which fall within the term "animal", as well as plants and fungi. The invention is useful with respect to animals, such as insects, nematodes, fish, such as salmon; or mammals, for example ungulates, such as pig, cattle, goat, or sheep; or odd-toed ungulates, such as horse; or rodents, such as mouse or rat.

The term "treating a cell with a mutagen" refers to contacting the cell with, or exposing it to, a mutagen of choice. This is preferably done under in vitro conditions.

The term "phenotype" as used herein refers to one or more morphological, physiological, behavioral and/or biochemical traits possessed by a cell or organism that result from its genotype. Thus, the term "alteration of the phenotype" as used herein refers to a non- human animal of the present invention displaying one or more readily observable abnormalities compared to the wild-type animal. In a preferred embodiment, an animal obtained via the methods of the invention shows at least 1, at least 2, at least 3, or at least 4 abnormal phenotypic features selected from any of the above categories. In another preferred embodiment, the animal shows a loss of function phenotype. In yet another preferred embodiment, the animal shows a gain of function phenotype.

The term "desired phenotype" as used herein refers to phenotypic alterations that are favorable for medical or economic reasons, such as exhibiting human disease symptoms, disease resistance in farm animals, immunological tolerance, or modulation of gene function.

The terms "Gl offspring", "G2 offspring", and "G3 offspring", as used in connection with the present invention, mean the first (Gl, generation 1), second (G2, generation 2), and third generation (G3; generation 3) of offspring generated by somatic cell nuclear transfer techniques. Gl offspring is heterozygous for a mutation in a gene of interest.

The term "nucleic acid" as used herein, refers to DNA, such as genomic DNA, or cDNA, but also RNA.

The term "gene" as used herein refers to a segment of DNA which may be transcribed into RNA, and which may comprise an open reading frame, intronic sequences, and also includes the regulatory elements which control expression of the transcribed region. Therefore, a mutation in a gene may occur within any region of the DNA, which is transcribed into RNA, or outside of the open reading frame and within a region of DNA which regulates expression of the gene (i.e., within a regulatory element). In diploid organisms, a gene is composed of two alleles.

The term "mutation" as used herein refers to a difference in the nucleotide sequence of a given gene or regulatory sequence from the naturally occurring or normal nucleotide sequence, e.g., a single nucleotide alteration (deletion, insertion, substitution), or a deletion, insertion, or substitution of a number of nucleotides. The term "mutation" also includes chromosomal rearrangements. "Introduced mutation" as used herein means a mutation introduced by chemical or physical mutagens.

The term "archive" as used in the present invention refers to a collection of samples from different sources stored under conditions suitable to preserve the integrity of the material. The collection can encompass nucleic acids of tissues, tissue or cell samples of a non-human organism, including somatic cells or clonal somatic cell lines thereof.

The term "non-transgenic" as used herein refers to an organism that does not carry in its genome a heterologous nucleic acid segment that is artificial or derived from (an)other organism(s) in respect of its sequence.

The term "somatic cell nuclear transfer" (SCNT) as used herein refers to a technique which involves the use of enucleated oocytes, typically prepared by removal of the nucleus from an unfertilized oocyte, and the introduction of the nucleus of a somatic cell into the enucleated oocyte to form a 1 cell-stage embryo. Such introduction may be achieved, e.g., via fusion of a somatic donor cell with an enucleated recipient oocyte, or via transfer by microinjection of an isolated somatic cell nucleus into an enucleated recipient oocyte. After the introduction of the nucleus, a stimulus is typically provided to induce embryonic development (e.g., an electric pulse or incubation with strontium). SCNT may further comprise an additional step of transferring the pronucleus, which is generated by the initial step of transferring the nucleus of a somatic cell into the enucleated oocyte, into a second or further enucleated oocytes. In this case, the second oocyte or the further oocytes are preferably oocytes obtained by enucleation of fertilized oocytes. If the transfer of the pronucleus occurs into said further enucleated oocytes, the transfer is performed consecutively, i.e., the pronucleus is transferred from the first to a second enucleated oocyte, then from the second to a third enucleated oocyte, then optionally from the third to a fourth enucleated oocyte, and so on, depending on how many transfer steps are intended. The transfer of the pronucleus into a second or further enucleated oocyte may be useful to improve synchronisation of the donor nuclei with the recipient oocyte cytoplasm. The serial transfer mentioned above in connection with SCNT may also include the transfer of a nucleus into which a pronucleus has developed, into a second oocyte, or into further enucleated oocytes. The term "cell clone" or "cellular clone" as used herein refers to a cell population derived from a single cell isolated, e.g., from a somatic cell population of a non-human organism. This single cell is expanded in tissue culture, typically under low density conditions. The term "cell clone" or "cellular clone" as used herein is intended to encompass clones formed by two cells derived from a single cell via a mitotic cell division. Typically, however, a "cell clone" or "cellular clone" will be formed by more than two cells derived from a single cell via mitotic cell divisions.

The term "clonal cell line" as used herein refers to a cell population, which represents, or is derived from, a cellular clone, e.g., from a somatic cellular clone of a non- human organism. This cellular clone is expanded by one or more cell divisions in tissue culture, thus generating a clonal cell line. A clonal cell line is capable of being further expanded via further cell divisions in culture, or of being stored under appropriate conditions and subsequently further expanded via further cell divisions in culture.

The term "clonal cell" as used herein refers to an individual cell derived from a cell clone or a clonal cell line.

The term "DNA archive" as used herein refers to nucleic acids, isolated, e.g., from one or several cells of a cell clone or clonal cell line, and stored as a frozen repository.

The terms "cell passage", "passage", or "passaging" as interchangeably used herein refer to the process of transferring cells stored or maintained in tissue culture from one storage or culture compartment (e.g., a tissue culture flask, a Petri dish, or a multi-well tissue culture plate) to another. Cell passage or passaging may involve individualization of the cells, e.g., via treatment with trypsin, particularly in the case of cells that adhere to the substrate they are cultured on, followed, e.g., by subsequent pipetting of the cells. In the course of cell passage or passaging, the cells, or an aliquot or several aliquots thereof, are seeded into one or several new compartments such as a fresh flask or a fresh Petri dish, typically at a cell density lower than the cell density displayed by the cell culture in the previous compartment. The term "morula" as used herein refers to a developmental stage of an embryo, where it consists of approx. 4 to 16 cells.

The term "blastocyst" as used herein refers to a developmental stage of an embryo, where it consists of 16 to approx. 300 cells. It is covered in a layer of trophoblast cells, which eventually form the placenta.

The Method of the Invention

The method of the invention involves the use of a somatic cell derived from a non-transgenic or a transgenic non-human animal, which may be either an adult animal or an animal in an embryonic stage. For example, the somatic cell may be derived from a transgenic non-human animal which has a selected phenotype compared to the wild-type animal and is intended to be subjected to further mutagenesis to alter, e.g., improve said phenotype. However, the method of the invention is, of course, also valuable for generating mutated non-human animals from somatic cells derived from wild-type non- human animals. In both scenarios, the method of the invention inter alia offers the possibility to provide disease models useful for developing novel therapeutic approaches, or animals with improved traits that are useful for farming purposes.

In a preferred embodiment of the invention the somatic cell used in the context of the method described and claimed herein is derived from the non-human animal in the sense that it has been isolated directly from the non-human animal. Also suitable and encompassed by this concept is, however, a somatic cell that has been cultured and/or stored for a longer period of time -after its actual isolation from said non-human animal. Furthermore, it may be a cell from a cell line, which is derived, e.g., by cell passaging, optionally including genetic manipulation, from a somatic cell that was initially isolated from any of the non-human animals to be used as a source of somatic cells in accordance with the invention.

The method of the invention comprises the treatment of the somatic cell with a mutagen, e.g., a chemical or physical mutagen, preferably ENU. Additionally, the method comprises the expansion of the mutagenized (e.g. ENU treated) somatic cell into a cell clone of at least 2 cells or a clonal cell line. This is preferably done by isolating the somatic cell as a single cell from a plurality of cells treated with said mutagen and subsequently expanding said single somatic cell to provide said cell clone or clonal cell line.

After generating the cell clone or clonal cell line, a 1 cell-stage or multicell- stage embryo is formed by a method comprising introducing the nucleus of a cell of the cell clone or clonal cell line into an oocyte, which has previously been enucleated, e.g., via micro-manipulation. For the purposes of the methods of the present invention, preferred enucleated oocytes are those obtained by removal of the nucleus of an unfertilized oocyte.

hi one embodiment, the introduction of the nucleus into the enucleated oocyte may be followed by transferring, or consecutively transferring the pronucleus (or nucleus) formed in the course of such initial introduction into a second enucleated oocyte, or a second and yet further enucleated oocytes, and allowing the second or further recipient oocyte to form a 1 cell-stage or multicell-stage embryo.

It will be understood that the 1 cell-stage or multicell-stage embryos formed in the course of the methods of the invention are preferably embryos capable of producing (or maturing into) a mutated non-human animal upon the implementation of suitable measures, e.g., the application of suitable culture conditions and the reimplantation into suitable foster mothers.

The introduction of the nucleus of the somatic cell into the enucleated oocyte may be conveniently performed by fusing said cell with the enucleated oocyte. It is, however, also possible, e.g., to introduce the nucleus into the enucleated oocyte by direct transfer from the somatic cell into the enucleated oocyte via micro-injection.

It will be appreciated by those skilled in the art that the method of the invention may comprise generating a plurality of cell clones or clonal cell lines from a plurality of somatic cells derived from a non-human animal. It will also be appreciated that the method may further comprise introducing the nuclei of a plurality of such cells (either derived from a single cell clone or clonal cell line, or from a plurality of cell clones or clonal cell lines obtained as described herein) into enucleated oocytes, and in case subsequent pronucleus transfer is involved, transferring a plurality of the resulting pronuclei into second or further enucleated embryos.

In one embodiment, the method of the invention comprises the isolation of one or more cells of the cell clone or clonal cell line generated in accordance with the method of the invention after the mutagenesis step. The cells may be isolated prior to, or after the step of introducing the nucleus of a cell of said cell clone or clonal cell line into an enucleated oocyte. In a preferred embodiment, a nucleic acid sample is prepared from said one or more isolated cells, which in turn is then screened for the presence of a mutation in a gene of interest.

After identifying a mutation in a gene of interest in the nucleic acid sample, the mutation may be assigned to the corresponding cell clone or clonal cells line from which the cell or cells that served as the source for the nucleic acid sample were derived. Thus, it is possible to identify cells, cell clones, or clonal cell lines having a mutation in a gene of interest at a very early stage of the process. These cells, which may have meanwhile been stored, e.g., in cell culture or in a frozen archive, may then be used to produce a non-human animal by the method of the invention, e.g., via SCNT. This would comprise, e.g., the introduction or transfer of the nucleus of an individual clonal cell (nucleus donor cell) from a selected cell clone or clonal cell line into an enucleated oocyte (recipient oocyte) and the subsequent reimplantation of the resulting embryo into a non- human animal pseudopregnant mother. The non-human animal generated from the implanted embryo and carrying the mutation in the gene of interest may be further bread to produce a plurality of offspring generations carrying said mutation.

In one embodiment, the resulting non-human animal as described herein is non-transgenic. In a further embodiment, said non-human animal is a vertebrate, e.g., a mammal, a fish, or a bird. Preferably, said mammal is a mammal selected from the group of mouse, rat, hamster, rabbit, cattle, pig, guinea pig, sheep, goat, horse, camel, dog, cat, monkey, e.g., rhesus macaque, baboon, orang-utan, and chimpanzee. Said fish is preferably selected from the group of fish consisting of salmon, trout, tilapia, carp, catfish, medaka, zebrafish, loaches, goldfish, and pikes. Said bird is preferably selected from the group of poultry, most preferably chicken, duck, turkey, and pigeon, and goose and Japanese quail. Another embodiment of the invention is an archive comprising nucleic acid samples isolated from the above-mentioned one or more somatic cells.

A further embodiment of the invention is an archive comprising cells of cell clones or clonal cell lines obtained or obtainable as described herein. The cells in the archive may, for example, be frozen ("frozen archive") or kept under culturing conditions allowing further expansion ("short-term archive").

Mutagenesis

As mentioned above, the methods of the invention encompass mutagenesis of somatic cells, preferably in vitro. Suitable mutations and mutagens are described below.

1. Type of DNA Mutations

Mutations in the DNA may comprise large lesion mutations, e.g., chromosomal breaks, rearrangements, and large insertions or deletions (in the order of kilobases); small lesion mutations, e.g., cytogenetically visible deletions within a chromosome; and/or subtle mutations, e.g., point mutations, such as conservative or non- conservative substitutions, insertions, and small deletions (in the order of several-tens of bases).

The latter category of mutations is preferred in the present invention. Also preferred are substitution mutations, e.g., non-conservative substitutions. Moreover, mutations that do not result in the complete deletion of the gene of interest are preferred, e.g., mutations within the gene or its regulatory sequences.

2. Chemical Mutagenesis and Mutagens

Chemical mutagens may be classified by the chemical modification, which they induce, e.g., alkylation, cross-linking, intercalation, etc.

Useful chemical mutagens according to the invention comprise N-ethyl-N- nitrosourea (ENU), Methylnitrosourea (MNU), Procarbazine hydrochloride (PRC), Triethylene melamine (TEM), Acrylamide monomer (AA), Chlorambucil (CHL), Melphalan (MLP), Cyclophosphamide (CRP), Diethyl sulphate (DES), Ethyl methane sulphonate (EMS), Methyl methane sulphonate (MMS), 6-mercaptopurine (6MP), Mitomycin-C (MMC), Procarbazine (PRC), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), N-nitrosodiethylamine (NDEA)₅ Isopropyl methane sulphonate (iPMS), ³H₂O, Urethane (UR), Bleomycine, Nitrogen Mustard, Vincristine, Dimethyhiitrosamine, 7,12- Dimethylbenz(a)anthracene (DMBA), Ethylene oxide, Hexamethylphosphoramide, Bisulfan, Acridine orange, Ethidium bromide, Proflavin, and ICR-191.

The chemical mutagens mainly cause single nucleotide alterations. For example ENU mainly causes adenosine to thymine or thymine to adenosine base changes, these changes representing roughly 45% of all base changes examined in the mouse germ line upon application of ENU (Noverskoe et al., 2000).

The induction of mutations with chemical mutagens is dependent on several parameters, e.g., the type, dose, and the mode of delivery of the mutagen or the frequency or type of mutations. The skilled person will be readily able to adjust the mutagenesis conditions for a given mutagen to the desired degree of mutation induction.

ENU is a particularly preferred chemical mutagen of the present invention.

As to the frequency/dose, ENU offers the opportunity of obtaining a very large number of mutations in vivo, which gives tremendous power to ENU mutagenesis. For example, in mice it requires 1000 offspring (Gl mice) from a mating of ENU- mutagenized males to wild type females, to obtain one-fold statistical recessive mutation coverage of all mouse genes, which are approximately 30,000 to 35,000 genes (Hitotsumachi et al., 1985). This indicates the presence of 30-35 recessive mutations in each Gl mouse, which equals 1.5 to 1.8 mutations per chromosome.

Thus, in the present invention, a preferred mutation load of the Gl non- human animal is about 0.2 to 5, about 0.5 to 4, about 1 to 3, about 1.5 to 2, and about 1.5 to 1.8 mutations per chromosome.

Another preferred mutation load of the present invention is about one mutation per chromosome. The presence of multiple recessive mutations in each Gl animal frequently led to the concern that a desired phenotype, based on the identified mutation in a gene of interest, may be confounded by the interaction of several mutations. This scenario is rather unlikely, however, based on the following example provided for a Gl mouse. In a mouse with a recombinational genome of 1453 centiMorgan (cM), 30-35 ENU-induced recessive mutations yield an average genetic distance between two functionally relevant mutations of 42-48 cM, indicating that adjacent mutations are almost certain to segregate in the next generation. The average distance between base-pair exchanges of 1.0-2.5 per Megabase (Mb) is large enough so that, for every functional mutation, even the neighboring silent change can be segregated rapidly (Russ et al., 2002). Further breeding for mice homozygous for the mutation will subsequently further reduce the likelihood of such influence.

3. Physical Mutagenesis

Physical mutagens, e.g., radiation mutagenesis via gamma-radiation, X-ray radiation, or neutrons, may also be used in accordance with the invention. Such radiation mutagenesis causes DNA breakage. Due to DNA repair mechanisms, these DNA breaks may lead to regions on the DNA with large lesions, rearrangements, or deletions. In contrast, mutations induced by UV-light, which is likewise a suitable mutagen in connection with the present invention, are largely single nucleotide alterations. UV-light does not penetrate the animal but is generally useful for inducing mutations in cells in culture, e.g., somatic cells as in the present invention.

DNA Amplification

1. Isolation of Cells

In a preferred embodiment of the method of the invention, a cell sample, e.g., one or more cells of a cell clone or a somatic clonal cell line of the invention, optionally previously stored in a "frozen archive" or in a "short-term archive" under conditions allowing (subsequent) cell expansion, is isolated. The one or more cells are then preferably processed for nucleic acid sample preparation, which may then, e.g., be subjected to Primer Extension Preamplification (PEP).

2. Primer Extension Preamplification (PEP)

The cell sample, e.g. the one or more cells isolated from the cell clone or the somatic clonal cell line, is used to prepare nucleic acid samples. Such nucleic acid samples may be DNA or RNA, preferably genomic DNA. Since the amount of nucleic acid in a single cell sample may be very limited, the nucleic acid, e.g., the genomic DNA of the tissue sample will preferably be subjected to amplification in order to allow extensive genetic testing. Sermon et al. (1996) and Cheung and Nelson (1996) describe a method of PCR-based amplification of isolated genomic DNA using partially or fully degenerated oligonucleotides, where the genomic DNA is isolated from cell biopsies. Equivalent methods are variations of the above protocols where oligonucleotides in combination with DNA polymerases are used without thermal cycling for the amplification of whole genome DNA like the method described by Dean et al. (2002).

A variety of suitable methods for whole genome amplification is published

(for review see Lasken and Egholm, 2003; for details see Snabes et al., 1994; Sermon et al., 1996; Chrenek et al., 2000; Bannai et al., 2004). Alternatively or in addition, the cell sample, e.g., the one or more cells isolated from the somatic cell clone or clonal cell line derived from the non-human animal, are cultured in vitro under appropriate conditions in order to allow expansion of such cells, thereby increasing the amount of tissue and nucleic acid derivable therefrom (see, e.g., Example 5).

3. Screening for the Presence of a Mutation

Screening for the presence of a mutation in a gene of interest according to the invention maybe performed, e.g., on a single nucleic acid sample derived from the cell sample as described herein, e.g., derived from one or more cells of a cell clone or somatic clonal cell line from a non-human animal obtained in accordance with the invention.

Alternatively, screening for the presence of a mutation in a gene of interest may be performed on a mixture or pool of nucleic acid samples derived from a plurality of the cell samples as described herein. Another embodiment of the methods of the invention includes screening for the presence of a mutation according to the invention in at least two genes of interest. This may be performed on a single nucleic acid sample or pools of nucleic acid samples as described above.

In yet another embodiment, a mutation in a gene of interest is assigned to a particular phenotype in an individual, e.g., to a disease, after screening for the presence of a mutation in said gene of interest and after generating a non-human animal carrying said mutation and displaying such particular phenotype. In a preferred embodiment, the individual is a human.

As mentioned previously, the somatic cell, cell clone or clonal cell line used in accordance with the invention is preferably derived from a mouse; rat; hamster; rabbit; cattle; pig; guinea pig; sheep; goat; horse; camel; dog; cat; monkey, e.g., rhesus macaque, baboon, orang-utan, chimpanzee; salmon; trout; tilapia; carp; catfish; medaka; zebrafish; loaches; goldfish; pikes; poultry, preferably chicken, duck, turkey, goose; pigeon; or Japanese quail.

A gene of interest is preferably a gene that is already known from an individual, e.g., a disease gene or an economically valuable gene. This information is then used to screen for a suitably mutated somatic cell clone or clonal cell line of a non-human animal according to the methods of the invention. In particular, such somatic cell clone or clonal cell line or a plurality of somatic cell clones or clonal cell lines prepared according to the method of the invention are used as a source for one or more cells, on which screening for the presence of a mutation in said gene of interest is performed, hi a preferred embodiment, said individual is a human. Said somatic cell, cell clone, or clonal cell line of a non-human animal is preferably of mouse; rat; hamster; rabbit; cattle; pig; guinea pig; sheep; goat; horse; camel; dog; cat; monkey, e.g., rhesus macaque, baboon, orang-utan, chimpanzee; salmon; trout; tilapia; carp; catfish; medaka; zebrafish; loaches; goldfish; pikes; poultry, preferably chicken, duck, turkey, goose; pigeon; or Japanese quail, hi another preferred embodiment, said individual is a non-human animal that is from the same species as said somatic cell, cell clone, or clonal cell line produced according to the method of the invention. The screening for the presence of a mutation in a gene of interest according to the invention may be performed by heteroduplex analysis. This analysis is based on detection of a base mismatch or base mismatches in a double-stranded (ds) DNA molecule. Detection can be done either by nondenaturing gel electrophoresis or by using denaturing agents (gradients or constant concentrations) or temperature (gradients or constant temperature) in electrophoretic systems or liquid chromatography. Detection can also be done by chemical cleavage of the mismatch or mismatches using chemical agents as described by Cotton et al. (1988). Detection can further be done by proteins binding to the mismatch with or without subsequent cleavage of the double-stranded (ds) DNA (reviewed in Nollau and Wagener, 1997). Equivalent methods are assays that exploit secondary structures of single stranded DNA or RNA molecules for the electrophoretic separation of nucleic acid strands that exhibit base variations as described by Orita et al. (1989), or assays for allele-specific hybridization to oligonucleotide-coated chips (for a review see Southern, 1996).

A specific example of a heteroduplex analysis is Temperature Gradient

Capillary Electrophoresis (TGCE). The amplified nucleic acid sample, e.g., the genomic DNA₅ is subject to PCR amplification according to standard methods in the art. Genomic DNA fragments of the gene of interest may be obtained by PCR using BioTherm-DNA- Polymerase (GeneCraft, Germany) according to the manufacturer's protocol. Gene- specific oligonucleotide primers may be designed using a publicly available primer design program (Primer3, www, genome, wo .mit. edu) .

For detection of mutations in unknown positions in PCR-amplified DNA fragments of a gene of interest, dsDNA is electrophoresed through a temporal gradient of increasing temperature (Temperature Gradient Capillary Electrophoresis (TGCE); RevealSystem, SCE9610, by SpectruMedix LLC, State College, PA, USA). Because retardation of dsDNA during electrophoresis is greatest at the temperature of partial denaturation, DNA fragments of the same size can be separated according to their thermodynamic stabilities. Base mismatches within dsDNA molecules (heteroduplices) lead to a significant destabilization resulting in significant differences in melting temperatures (T_m) between heteroduplices and perfectly paired dsDNA (homoduplices). Such differences in T_m allow the separation of heteroduplices from homoduplices in a temperature gradient electrophoresis and serve as the basis for mutation detection by TGCE (cf. Example 6).

Alternatively, single strand conformation polymorphism (SSCP) or fluorescent SSCP (fSSCP) may be used as well as, e.g., Denaturing Gradient Gel Electrophoresis; Cleavage of Mismatches; Constant Denaturing Capillary Electrophoresis (CDCE); RNAse cleavage; Mismatch Repair detection; Mismatch Recognition by DNA repair enzyme; sequencing by hybridization; dot-blots; reverse dot blots; allele specific PCR; Primer-Induced Restriction analysis; Oligonucleotide Ligation; Direct DNA sequencing; Mini-sequencing; 5' Nuclease Assay; Representational Difference Analysis; or Microarrays, all described or referenced in WO 97/44485.

Obviously, the screening methods according to the invention are not limited to the methods specifically described herein. Each method that may be useful in the connection with screening a mutation in a gene of interest may be employed.

Storing

Storing according to the invention may comprise any form and any duration of maintaining somatic cells or nucleic acids as described herein. Long-term storage comprises storage for, e.g., a week, several weeks, a month, several months, or even a year or several years up to several decades. Short-term storage comprises storage, e.g., for several minutes, hours or days.

The cells, which are stored in accordance with the invention may, e.g., be one or more cells of a somatic cell clone or clonal cell line, as described herein. For short- term storage, cells are preferably kept in a suitable buffer or culture medium. For example, mouse fetal fibroblasts may be appropriately stored in Dulbecco's modified Eagle's medium (DMEM; 50mg/ml penicillin/ streptomycin) at 37°C under 5% CO₂ and >90% humidity in an incubator for several cell passages. For long-time storage, cells are preferably stored by freezing in a freezing medium, e.g., DMEM containing 20% FCS and 10% DMSO. Certain embodiments of the claimed method also comprise storing of nucleic acids, particularly nucleic acid samples derived from one or more cells of the cell clones or clonal cell lines described herein. In a preferred embodiment such samples are stored prior to being subjected to screening for the presence of a mutation in a gene of interest. Alternatively, they may additionally be stored again for a certain period of time after having been subjected to screening, or both before being and after having been subjected to such screening. Long-term storage of nucleic acids may be conveniently done by freezing the nucleic acids in an appropriate buffer, e.g., TE-buffer (10 mM Tris pH 7.5, 1 mM EDTA), or by lyophilization.

Reimplantation

Reimplantation according to the present invention comprises the reimplantation or transfer of a mutant embryo at its various developmental stages, e.g. zygotes, morulae, blastocysts. For example in mouse, a morula stage embryo can be transferred into the ampulla of an oviduct of a 0.5 days post coitus (dpc) pseudopregnant foster mother, whereas a blastocyst stage embryo is transferred into an uterus horn of a 2.5 dpc pseudopregnant foster mother (see also Example 4).

Example 1 : ENU Treatment of Non-Human Fetal Fibroblasts

1. Preparation of Murine Fetal Fibroblast Cells

Fibroblast cells are prepared from 15.5 days post-coitus (dpc) fetuses derived from murine CD-I x CD-I and CD-I x C57B1/6 breedings. The fetuses are killed by decapitation and sexed by gonadal phentoyping. The internal organs are removed, and the remaining tissue is cut into small pieces (1-2 mm) in preparation for culture. The pieces of tissue are incubated in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen GmbH, Karlsruhe, Germany) containing 0.05% trypsin EDTA (Gibco) for 10 min in a rotator. The cell suspension is filtered after adding the same volume of DMEM medium containing 10% fetal calf serum. The filtered cell suspension is seeded into tissue culture dishes at a concentration of 5xlO⁵/ml. The supernatant is removed after Ih at 37°C culturing in an incubator with an atmosphere of 5% CO₂ and fresh DMEM medium is added. Cells adhering to the culture dish are allowed to grow to confluency.

2. Preparation of ENU Solution

ENU is dissolved in Soerenson Buffer, pH 6.0 for preparation of an ENU stock solution. For preparation of Soerenson buffer 9.078 g of KH₂PO₄ (Merck KgaA, Darmstadt, Germany), (Stock A)_, and 11.976 g Of Na₂HPO₄ (Merck KgaA, Darmstadt, Germany), (Stock B) is dissolved in each 1000 ml of distilled H₂O. 121 ml of Stock B solution is added to 879 ml of Stock A solution, mixed by inversion, and autoclaved. 1 g of ENU (Sigma Aldrich Chemie GmbH, Munich, Germany) is dissolved in 200 ml of Soerenson buffer by vigorous shaking for about 10 min. Final concentration is determined photospectrometrically and the ENU stock solution is continuously kept refrigerated. ENU working solutions for in vitro treatment are prepared by mixing PBS with appropiate amounts of the ENU stock solution.

3. Li Vitro ENU Treatment of Fetal Fibroblasts

After fibroblast cells reach confluency in cell culture, according to Example 1.1, cells are treated with ENU: DMEM medium is aspirated and the cells are washed one time in phosphate-buffered saline (PBS; Invitrogen GmbH, Karlsruhe, Germany). Cells are then incubated with PBS/ENU (0.01-0.6 mg ENU/ml PBS) for 5-60 min at 37⁰C in an incubator with an atmosphere of 5% CO₂. After incubation the ENU/PBS solution is replaced with 1 ml of trypsin. After trypsinization cells are suspended in PBS and harvested by centrifugation. The cell pellet is suspended in 10 ml of DMEM containing 10% fetal calf serum and cells are plated in low density with 2-5 x 10² cells per 10 cm (diameter) petri dish to allow individual cells to grow to individual cellular clones. The incubation is performed at 37°C in an incubator with an atmosphere of 5% CO₂ for at least 4h. Single cellular clones are picked and every individual clone is transferred into one well of a 96 well plate for further expansion. When cells reach confluency each clone is further passaged to each one well of a 24 well plate and incubated at 37°C in an incubator with an atmosphere of 5% CO₂ for further expansion and for generating clonal cell lines.

Example 2: Archiving of ENU-treated Fetal Fibroblast Clonal Cell Lines and corresponding DNAs

When clonal cell lines of Example 1.3 reach confluency, each such clonal cell line is further passaged to one 6 cm (diameter) culture dish each and kept in culture under the conditions described before to further enrich the number of clonal cells of such clonal cell lines. After reaching confluency about half of the clonal cells of each clonal cell line is suspended in medium containing 10% dimethylsulfoxide. These clonal cells are directly frozen in cryotubes to establish a "frozen archive" of mutated fibroblast clonal cell lines. Alternatively, these cells are maintained under culruring conditions to establish a short-term storage archive of mutated fibroblast clonal cell lines (see Example 6).

The remaining clonal cells of each clonal cell line are kept under culruring conditions for further 2 to 6 passages before final trypsinization and collection by centrifugation. The cell pellet is suspended in Proteinase K buffer (0.5 M EDTA, 1 M Tris pH 9.5, 30% Sarkosyl, 20% SDS, Proteinase K 50 mg/ml; Sigma Aldrich Chemie GmbH,

Munich, Germany) and incubated overnight at 55°C for nucleic acid preparation, performed by methods well known in the art. The isolated nucleic acid, e.g., genomic DNA, is stored in TE buffer (10 mM Tris pH 7.5, 1 mM EDTA) at a concentration of 1 mg/ml at minus 70°C. Nucleic acid of each mutagenized fetal fibroblast clonal cell line is subject to mutation detection in a gene of interest, as described in Example 7. Following assignment of a mutation in a gene of interest to (a) particular clonal cell line(s), archived clonal cells of this (these) clonal cell line(s) are subjected to subsequent somatic cell nuclear transfer (SCNT), as described in Example 3 for cells of a "frozen archive", followed by embryo transfer, as described in Example 4.

Example 3: Somatic Cell Nuclear Transfer (SCNT) - Serial Nuclear Transfer The example provided herein mainly follows the protocol for serial nuclear transfer as described in Ono et al., 2001, although methods using a single SCNT event and subsequent embryo transfer are likewise suitable and contemplated, hi the method described herein as an exemplary embodiment, a donor cell arrested at metaphase II is first transferred into an unfertilized enucleated oocyte, and then the resulting pronucleus is again transferred into a fertilized enucleated egg, leading to a 1 cell-stage embryo. This particular SCNT procedure (also called serial nuclear transfer) supports the synchronisation of the donor nuclei, arrested at metaphase II, with the recipient oocyte cytoplasm, which facilitates the further successful development of an embryo.

1. Synchronization of Fibroblast Cells (Donor Cells)

After assigning a mutation in a gene of interest to a particular fetal fibroblast clonal cell line (for method of mutation detection in a gene of interest see Example 7), clonal cells from the corresponding clonal cell line stored in a "frozen archive" are thawed and cultured for 2h under gentle pipetting in DMEM, supplemented with 10% FCS and 0.4 μg/ml nocodazole (Sigma Aldrich Chemie GmbH, Munich, Germany), a microtubule polymerization inhibitor, which induces metaphase arrest. Following this synchronization procedure, cells are collected and an aliquot of these cells is subjected to staining with Hoechst 33258 (10 μg/ml). Metaphase II arrest is observable after staining by the absence of a nuclear membrane and by chromosome condensation. For generating an embryo, several cells arrested in metaphase II are isolated using an Olympus microscope with Hoffmann modulation contrast.

2. Superovulation in Mouse and Isolation of Oocytes (Recipient Cells)

The oocyte donors and zygote donors (see Example 3.4) are female B6CBF1 hybrid mice (C57BL/6 x CBA), superovulated with intraperitoneal injections of 5

LU. of eCG (Peamex; Sankyo Ltd., Tokyo, Japan) and 5 LU. of hCG (Puberogen; Sankyo

Ltd., Tokyo, Japan) given 48 h apart. Oocytes at metaphase II are released from the oviduct 14 h after hCG. Oocyte donor females of the B6CBF1 hybrid strain are sacrificed 14 hours after Puberogen injection. After desinfection with 70% alcohol, each mouse abdomen is opened with surgical scissors from caudal to cranial. The upper end of one uterine horn is grasped with fine forceps and the uterus, oviduct, ovary and the fad pad are removed. A hole is poked with the tip of a fine forceps into the membrane close to the oviduct, for further separation of the whole reproductive tract from the body wall. After stretching the whole reproductive tract and cutting between the oviduct and the ovary, the oviduct is finally removed. The whole procedure is repeated at the other uterine horn. Oviducts and the attached segments of the uterus are transferred into a pre-warmed culture dish, filled with lightweight paraffin oil (embryo tested; Sigma Aldrich Chemie GmbH, Munich, Germany). Oviducts from all the female mice are collected in one culture dish. Collected oviducts are transferred to a culture dish filled with 400 μl of M2 medium, the oviducts still being surrounded with oil. Under oil, the swollen ampullae are opened with the closed tip of fine forceps and the oocyte-cumulus complexes are expelled into the oil. With the closed tip of fine forceps the oocyte-cumulus complexes are pushed into the M2 medium drop. Cumulus cells are removed after transfer and by brief incubation in M2 medium supplemented with 300 U/ml hyaluronidase (see in Quinn et al., 1982).

3. Enucleation of Oocytes

The removal of chromosomes from recipient oocytes, referred to herein as "enucleation", is performed according to a method described by Kono et al., 1993.

Oocytes are placed in a micromanipulator chamber in a small drop of M2 medium (see in Quinn et al., 1982) containing cytochalasin B (5 mg/ml). Using interference microscopy techniques oocytes with properly arrested metaphase II chromosomes are selected. Under microscopic examination, the zona pellucidae of selected oocytes is slit with a glass needle along 10-20% of its circumference at a position close to the metaphase II chromosomes and the spindle located in the cortex of the oocyte. All metaphase II chromosomes are removed using an enucleation pipette, which displays an unsharphened beveled tip. In the rare instance in which enucleation of the oocytes is unsuccessful, the oocytes release a second polar body after fusion with a donor cell as a result of completion of meiosis. Those oocytes are expelled from further procedures. 4. Generation of Fertilized Eggs for Pronucleus Transfer

Fertilized one-cell stage embryos are generated by in vitro fertilization using oocytes and sperm cells of B6CBF1 hybrid mice.

High quality mature sperms, i.e., spermatozoa, are collected from sexually reproducing B6CBF1 males, which have not been mated for at least 10 days. A male mouse is sacrificed by cervical dislocation, followed by immediate dissection of cauda epididymis and vas deferens. (Marschall et al., 1999).

After removal of fat tissue and blood vessels, both testis structures are washed briefly in 0.9% NaCl at room temperature, transferred into 500 μl of HTF fertilization medium (Quinn et al., 1985), and cut into 5 pieces allowing the sperms to flush out. Incubation in HTF medium is for 20 min. at 37°C in an incubator with an atmosphere of 5% CO₂ in air.

Under microscopic examination and using a pipette with an 20 μl E-ART- tip (Molecular Bioproduct, San Diego, California, USA), the oocyte cumulus complexes, as isolated according to Example 3.3, are transferred into fertilization dishes containing capacitated spermatozoa. Oocytes and spermatozoa are incubated for 4 to 6 hours in an incubator (37°C, 5% CO₂ in air), followed by removing from the incubator and subsequent washing of the oocytes of each fertilization dish (with the help of the silicon tube, mouth piece and the glass pipettes) for three times in a separate dish filled with 50 μl drops of KSOM medium (see in Lawitts and Biggers, 1991). Washing in drops of KSOM-medium is for removal of dead sperms and residues of the cumulus complex. After washing, the prepared oocytes are transferred into a fresh culture dish filled with 200 μl of KSOM- medium and are covered with equilibrated lightweight paraffin oil (equilibrated over night with KSOM-medium. Enucleation of the resulting one-cell stage embryos is following, according to the method described in Example 3.3.

5. Embryo Generation

Embryos are generated by serial nuclear transfer using standard micromanipulation procedures described by Kwon and Kono, 1991. All micromanipulations are performed in a micromanipulation chamber in M2 medium (Quinn et al., 1982), containing 5 μg/ml cytochalasin B and 0.4 μg/ml nocozadole. The Sendai virus 2700 used is a virus inactivated with β-propiolactone (McGrath and Solter, 1983).

An assigned metaphase II arrested mutant fibroblast cell is introduced together with a drop of inactivated Sendai virus 2700 (virus concentration: 2000-3000 hemagglutinating activity units/ml) into the perivitelline space of an enucleated oocyte, generated as described in Example 3.3. The virus contacts the oocyte membrane and facilitates the fusion of the donor cell with the recipient cell (McGrath and Solter, 1983). Oocytes that successfully fuse with a donor cell, as detected by microscopical examination, are cultured for 2h in an atmosphere of 5% CO₂, 5% O₂ and 90% N₂ at 37⁰C, and are then artificially activated with 10 mM strontium for 6h, followed by transfer into CZB medium (see in Chatot et al., 1990). After 9 to 12h of incubation the pronucleus of such a constituted egg is again transferred to a previously enucleated fertilized 1-cell embryo obtained by in vitro fertilization, as described in Example 3.4.

An embryo resulting from pronucleus transfer is cultured in CZB medium containing 5.56 mM glucose in an atmosphere of 5% CO₂, 5% O₂ and 90% N₂ at 37°C. A blastocyst obtained is transferred to the uterine horn of a female on day 3 of pseudopregnancy (2.5 dpc), as described in Example 4.

Example 4: Embryo Transfer

1. Production of Mouse Pseudo-Pregnant Females

Pseudo-pregnancy is generated by mating mouse CDl females (8-10 weeks of age, at a body weight of approximately 30 g) to vasectomized or genetically sterile males. It is recommended to mate at least 10 females per one scheduled embryo transfer, with each two females mated over night to one vasectomized male. A vaginal plug is visible the next morning after coitus. A female of 2.5 days post coitus (2.5 dpc), which is day 3 of pseudopregnancy is used for uterus transfer. To archive successful embryo transfer in general, it is necessary to transfer more than one embryo into the uterus of one pseudo-pregnant female mouse. Therefore, in addition to the embryo generated with somatic cell nuclear transfer technique, five to ten 8 cell-stage embryos (blastocyst embryos of the mouse starin C57B1/6J) are co-transferred into the uterus of such a pseudo-pregnant female mouse. These co-transferred embryos are generated by in vitro fertilization of wildtype C56B1/6J oocytes with wildtype C56B1/6J sperm cells.

2. Uterus Transfer

Embryos from Example 3.5 are selected for embryo transfer, washed two times in M2 Medium and subsequently stored in one drop of M2 medium covered with lightweight paraffin oil (embryo tested; Sigma Aldrich Chemie GmbH, Munich, Germany) on a warming plate at 37°C. For the transfer, the pseudo-pregnant female mice are anaesthetized by intra-peritoneal injection of 0.25 ml anesthetic (Rompun 2%/Ketamin

5%). Reflexes of the anaesthetized mice are tested by pricking the tail and foot pads gently with forceps 5 minutes after anaesthetizing. During this time, embryos are prepared for the transfer. Under microscopic examination, visually intact blastocysts are collected with a transfer pipette in approximately 50 μl of M2 medium.

5 minutes after anesthetic injection the mouse is placed onto the lid of a 140 mm (diameter) culture dish, and her back is disinfected with 70% alcohol. A first small transverse incision is made to the skin (approx. 1 cm to the left side of the spinal cord, at the level of the last rib), the peritoneum is opened with fine scissors, the fad pad is picked up, and ovary, oviduct and the uterus horn are pulled out with fine forceps. This tissue complex is fixed on the fad pad with the help of a bullock clamp, located on the back of the mouse. The mouse is placed on the stage of a light microscope (head on the left side, tail to the right side).

Under microscopic examination the top of the uterus is gently lifted with blunt fine forceps and a small hole is made into the uterus, a few millimeters down from the utero-tubal junction, using a 26 gauge needle. The prepared transfer pipette containing the blastocyst embryos, including an embryo carrying a mutation in a gene of interest and several wildtype embryos, is inserted into the hole and the embryos are expelled into the uterus. The bullock clamp is undipped and ovary and oviduct are carefully returned into the abdomen. The body wall is closed with one stitch, the skin is closed with a wound clip. All steps are repeated for the right side located oviduct of the same mouse. After surgery the mouse is left undisturbed on a warming plate for approx. 10 min. until waking it up.

To assess development, recipients are sacrified at 19.5 days of gestation and pubs are recovered from the uterus. Recovering mutant and wildtype mice are visibly distinguishable by their different coat colors.

Example 5: Primer Extension Preamplification

Primer Extension Preamplification (PEP) is used to amplify low amounts of genomic DNA isolated from e.g. a tissue culture sample or from a single cell biopsy sample (see in Sermon et al. (1996) In brief, sample material is resuspended in 5 μl alkaline lysis buffer (200 niM KOH, 50 mM dithiothreitol), heated to 65°C for 10 min. and neutralized by adding 5 μl neutralization buffer (900 mM Tris-HCl, pH 8.3; 300 mM KCl, 200 mM HCl). 5 μl of a 400 μM solution of random 15-base oligonucleotides are added (MWG Biotech AG, Ebersberg, Germany). In a random 15mer oligonucleotide any one of the four bases adenine, cytosine, guanine, and thymine could be present at each position. Following addition of 6 μl of PCR buffer (25 mM MgCl₂/gelatin (1 mg/ml)/100 mM Tris- HCl, pH 8.3), 3 μl dNTP mixture (each 2 mM) and 1 μl Taq polymerase (GeneCraft, Germany; 5 units), the volume is raised to 60 μl with sterile water and 50 primer extension cycles are carried out in a MJ Research (Cambridge, MA) thermocycler. Each cycle consists of 1 min. denaturation at 94°C, 2 min. annealing at 37°C, a ramping step of 10 sec/degree to 55⁰C and a final 4 min. incubation step at 55°C. 3 to 8 μl of the first round PCR products are directly used for a second round of PEP, or for the amplification of myostatin gene sequences, as described in Example 7.

Example 6: In Vitro Expansion of Mutagenized Cells Cells in culture as described in example 2 can be stored for short-term storage as living cells. Every individual cell clone is maintained on a 6 cm dish and the medium (DMEM 10 % FCS) of the cells is changed on a daily basis. Every third day the medium is removed, the cells are washed with PBS (phosphate-buffered saline (PBS; Invitrogen GmbH, Karlsruhe, Germany) and subsequently trypsinized. Trypsinized cells are than resuspended in 4 ml medium (DMEM 10 FCS) and 1 ml medium cell suspension is added to a fresh 6 cm dish containing 4 ml medium.

Example 7: Myostatin Mutation Detection by Heteroduplex Analysis Using Temperature Gradient Capillary Electrophoresis (TGCE)

For detection of heterozygous mutations in nucleic acid from a tissue culture sample, e.g. a single or several cells of a clonal somatic cell line, genomic DNA is used to PCR amplify DNA fragments of the myostatin gene with myostatin-specific PCR primers. The following murine primer pairs Mst-1 and Mst-2, Mst-3 and Mst-4, and Mst-5 and Mst-6 were designed for the amplification of the individual exons in PCR amplification reactions:

Standard PCR reactions (total volume: 20 μl) are carried out. The genomic DNA is either directly used for PCR; or previously enriched by Primer Extension Pre- amplification (see Example 5) or via previous in vitro culturing for sample material enrichment (see Example 6), or both.

Upon initial denaturation at 94°C for 3 min. each cycle consists of a 30 sec denaturation step at 94°C, a 30 sec annealing step at 56°C and a 45 sec synthesis step at 72°C. 40 cycles are carried out in a MJ Research (Cambridge, MA) thermocycler.

Following PCR amplification, hybrids of wild-type and mutant DNA strands are formed in a denaturation/renaturation step, transforming base pair exchanges into heteroduplices with lower thermal stability. A typical temperature profile for denaturation/renaturation is:

heat sample to 95°C and hold for 5 min.

decrease from 95°C to 80°C at 3°C/min.

decrease from 80°C to 55°C at l°C/min.

hold 20 min. at 55⁰C

decrease from 55⁰C to 45⁰C at l°C/min.

decrease from 45⁰C to 25°C at 2°C/min.

Depending on the DNA concentration obtained in the PCR reaction the sample is diluted in a range of 1:10 to 1:50 and subjected to TGCE, as described by Li et al. (2002). Washing, dilution and running buffers are supplied by the manufacturer. Typical operation parameters for TGCE are:

Prerun: 25 min.

Injection voltage: 3-8 kV

Injection time: 3-10 sec.

Running voltage: 10 kV

Electrophoresis time: 60 min. The applied temperature gradient during electrophoresis depends on the base composition (G+C content) of the analyzed fragment and ranges from 55°C to 7O⁰C.

Upon completion the obtained electrophoresis pattern is analyzed for additional bands resulting from decreased mobility of heteroduplices during TGCE, using the manufacturers software program Revelation 2.10. Candidate fragments are further analyzed by DNA sequencing. To this end, PCR products amplified with primers specific for the myostatin gene are purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. PCR products are sequenced using forward/reverse PCR primers and the "Big Dye" thermal cycle sequencing Kit (ABI PRISM, Applied Biosystems, Foster City, CA₅ U.S.A.). The reaction products are analyzed on an ABI 3700 DNA sequencing device.

The sequences are edited manually and different sequence fragments are assembled into one contiguous myostatin sequence using the software Sequencer version 4.0.5. (Gene Codes Corp., Ann Arbor, MI, U.S.A.). The myostatin gene of an ENU-treated heterozygous somatic cell and of a wild-type somatic cell is sequenced. The sequencing results are used to identify mutations by comparing the sequencing results from somatic cells carrying the ENU mutation with wild-type somatic cells.

References

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Claims

Claims

1. A method of providing an embryo capable of producing a mutated non-human animal comprising:

(a) treating a somatic cell derived from a non-human animal with a mutagen in vitro;

(b) generating a cell clone or clonal cell line from the somatic cell treated according to step (a); and

(c) introducing the nucleus of a cell of the cell clone or clonal cell line of step (b) into an enucleated oocyte to form a 1 cell-stage embryo; or

(d) introducing the nucleus of a cell of the cell clone or clonal cell line of step (b) into an enucleated oocyte and transferring the subsequently formed pronucleus or nucleus into a second enucleated oocyte to form a 1 cell-stage embryo; or

(e) introducing the nucleus of a cell of the cell clone or clonal cell line of step (b) into an enucleated oocyte and consecutively transferring the subsequently formed pronucleus or nucleus into a second and further enucleated oocytes, e.g., into a second and subsequently a third enucleated oocyte, to form a 1 cell- stage embryo.

2. The method of claim 1, wherein the enucleated oocyte in step (c), (d), or (e) is an oocyte obtained by enucleating an unfertilized oocyte.

3. The method of claim 1 or 2, wherein the second enucleated oocyte in step (d) or the second and further enucleated oocytes in step (e) are oocytes obtained by enucleating fertilized oocytes.

4. The method of any one of claims 1 to 3, wherein the introduction of said nucleus of the cell of the cell clone or clonal cell line according to steps (c), (d) or (e) is effected by fusing said cell with said enucleated oocyte.

5. The method of any one of claims 1 to 4, wherein the 1 cell-stage embryo of step (c),

(d) or (e) is allowed to develop into a multicell-stage embryo.

6. The method of any one of claims 1 to 5, wherein step (b) comprises

(i) isolating the somatic cell as a single cell from a plurality of somatic cells treated with said mutagen; and

(ii) expanding said single somatic cell to provide said cell clone or clonal cell line.

7. The method of any one of claims 1 to 6, wherein the mutagen is a chemical or a physical mutagen.

8. The method of claim 7, wherein said chemical mutagen is selected from the group consisting of N-ethyl-N-nitrosourea (ENU), Methylnitrosourea (MNU), Procarbazine hydrochloride (PRC), Triethylene melamine (TEM), Acrylamide monomer (AA),

Chlorambucil (CHL), Melphalan (MLP), Cyclophosphamide (CRP), Diethyl sulphate (DES), Ethyl methane sulphonate (EMS), Methyl methane sulphonate (MMS), 6-mercaptoρurine (6MP), Mitomycin-C (MMC), Procarbazine (PRC), N- methyl-N-nitro-N-nitrosoguanidine (MNNG), N-nitrosodiethylamine (NDEA), Isopropyl methane sulphonate (iPMS), ³H₂O, Urethane (UR), Bleomycine, Nitrogen

Mustard, Vincristine, Dimethylnitrosamine, 7,12-Dimethylbenz(a)anthracene (DMBA), Ethylene oxide, Hexamethylphosphoramide, Bisulfan, Acridine orange, Ethidium bromide, Proflavin, and ICR-191.

9. The method of claim 7, wherein said physical mutagen is selected from gamma- radiation, X-ray radiation, UV-light, and neutrons.

10. The method of any one of claims 1 to 9, wherein the somatic cell treated in step (a) is an embryonic somatic cell or an adult somatic cell.

11. The method of claim 10, wherein said embryonic somatic cell is a fetal fibroblast cell.

12. The method of claim 10, wherein said adult somatic cell is an adult fibroblast cell, in particular a skin fibroblast cell; or a granulosa cell, a cumulus cell, or an oviduct cell.

13. The method of any one of claims 1 to 3 and 5 to 12, wherein the introduction of the nucleus into the enucleated oocyte according to step (c), (d) or (e) is effected by nucleus microinjection.

14. The method of any one of claims 1 to 13, wherein said somatic cell is derived from the same non-human animal species as said oocyte and/or said second or further oocyte or oocytes.

15. The method of any one of claims 1 to 13, wherein said somatic cell is derived from a different non-human animal species as said oocyte and/or said second or further oocyte or oocytes.

16. The method of any one of claims 1 to 15, wherein

(i) said somatic cell is derived from a wild-type non-human animal;

(ii) said oocyte and/or said second or further oocyte or oocytes are derived from a wild-type non-human animal;

(iii) said somatic cell and said oocyte are derived from a wild-type non- human animal;

(iv) said somatic cell and said second or further oocyte or oocytes are derived from a wild-type non-human animal;

(v) said somatic cell is derived from a transgenic non-human animal;

(vi) said oocyte and/or said second or further oocyte or oocytes are derived from a transgenic non-human animal;

(vii) said somatic cell and said oocyte are derived from a transgenic non- human animal; or

(viii) said somatic cell and said second or further oocyte or oocytes are derived from a transgenic non-human animal.

17. The method of any one of claims 1 to 16, wherein prior to step (c), (d), or (e) said cell of a cell clone or clonal cell line referred to in step (c), (d), or (e) is stored under freezing conditions, or stored under conditions suitable for maintaining or expanding said cell.

18. The method of any one of claims 1 to 17, wherein the method further comprises the isolation of one or more cells of the cell clone or clonal cell line of step (b).

19. The method of claim 18, wherein prior to isolation said one or more cells are stored under freezing conditions, or stored under conditions suitable for maintaining or expanding said one or more cells.

20. The method of claim 18 or 19, wherein the isolation is effected prior to step (c), (d), or (e).

21. The method of any one of claims 1 to 20, wherein the method further comprises the screening of a nucleic acid sample derived from one or more cells of the cell clone or clonal cell line obtained in step (b) for the presence of a mutation in a gene of interest.

22. The method of claim 21, wherein said one or more cells are one or more cells isolated according to claims 18 to 20.

23. The method of any one of claims 18 to 22, wherein the one or more cells are fetal fibroblast cells; adult fibroblast cells, in particular skin fibroblast cells; granulosa cells; cumulus cells; or oviduct cells.

24. The method of any one of claims 21 to 23, wherein the nucleic acid sample is an RNA or DNA sample.

25. The method of claim 24, wherein the DNA is genomic DNA.

26. The method of any one of claims 21 to 25, wherein the screening method comprises PCR (polymerase chain reaction) specific for the gene of interest; Heteroduplex

Analysis, e.g., Temperature Gradient Capillary Electrophoresis (TGCE); Single Strand Conformation Polymorphism (SSCP); Denaturing High Performance Liquid Chromatography (DHPLC); fluorescent Single Strand Conformation Polymorphism (fSSCP); Denaturing Gradient Gel Electrophoresis (DGGE); Cleavage of Mismatches; Constant Denaturing Capillary Electrophoresis (CDCE); RNAse cleavage; Mismatch Repair detection; Mismatch Recognition by DNA repair enzyme; sequencing by hybridization; dot-blots; reverse dot blots; allele specific

PCR; Primer-Induced Restriction analysis; Oligonucleotide Ligation; Direct DNA sequencing; Mini-sequencing; 5' Nuclease Assay; Representational Difference Analysis; or the use of Microarrays.

27. The method of any one of claims 21 to 26, further comprising the step of amplifying the nucleic acid sample by PCR.

28. The method of any one of claims 21 to 27 further comprising culturing said one or more cells of the cell clone or cell line in vitro under conditions suitable to allow cell expansion prior to screening.

29. The method of any one of claims 1 to 28 wherein

(i) step (b) comprises generating a plurality of cell clones or clonal cell lines from a plurality of somatic cells treated according to step (a), and step (c) comprises introducing the nuclei of a plurality of cells of a cell clone or clonal cell line or of cell clones or clonal cell lines generated in step (b) into enuclated oocytes to form 1-cell stage embryos; or

(ii) wherein step (b) comprises generating a plurality of cell clones or clonal cell lines from a plurality of somatic cells treated according to step (a), and wherein step (d) comprises introducing the nuclei of a plurality of cells of a cell clone or clonal cell line or of cell clones or clonal cell lines generated in step (b) into enucleated oocytes and transferring the subsequently formed pronuclei or nuclei into second enucleated oocytes to form 1 cell-stage embryos; or

(iii) wherein step (b) comprises generating a plurality of cell clones or clonal cell lines from a plurality of somatic cells treated according to step (a), and wherein step (e) comprises introducing the nuclei of a plurality of cells of a cell clone or clonal cell line or of cell clones or clonal cell lines generated in step (b) into enucleated oocytes and consecutively transferring the subsequently formed pronuclei or nuclei into second and further enucleated oocytes to form 1 cell-stage embryos.

30. The method of any one of claims 21 to 29, further comprising the step of assigning said mutation to a cell clone or clonal cell line or the cell clone or clonal cell line generated in step (b).

31. The method of claim 30, wherein the nucleus or the nuclei used in step (c), (d), or (e), or the subsequently formed pronucleus or pronuclei, or the subsequently formed nucleus or nuclei used in steps (d) or (e) are derived from cells from the cell clone or clonal cell line to which said mutation has been assigned.

32. A method of producing a mutated non-human animal comprising allowing a 1 cell- stage or a multicell-stage embryo obtained according to a method of any one of claims 1 to 31 to develop into a non-human animal capable of sexually reproducing.

33. An archive comprising cells of cell clones or clonal cell lines obtained or obtainable by the method as defined in steps (a) and (b) of any one of claims 1 to 31.

34. The archive of claim 33, wherein said cells are stored by freezing or by storing in a culture medium suitable for maintaining or expanding cells.

35. The archive of claims 33 or 34, wherein the cells are derived from embryonic somatic cells or adult somatic cells of a non-human animal.

36. The archive of claim 35, wherein the embryonic somatic cells are fetal fibroblast cells.

37. The archive of claim 35, wherein the adult somatic cells are adult fibroblast cells, in particular skin fibroblast cells; or granulosa cells, cumulus cells, or oviduct cells.

38. Use of a somatic cell of a cell clone or clonal cell line obtained or obtainable by the method as defined in steps (a) and (b) of any one of claims 1 to 31, or of the archive of any one of claims 33 to 37, for producing a non-human animal.

39. A method of producing a non-human animal comprising introducing the nucleus of a somatic cell of a cell clone or clonal cell line obtained or obtainable by the method as defined in steps (a) and (b) of any one of claims 1 to 31, or a cell of the archive of any one of claims 33 to 37, into an enucleated oocyte to produce an embryo capable of developing into a non-human animal, the method optionally further comprising allowing said embryo to develop into a non-human animal capable of sexually reproducing.

40. The use or the method of claim 38 or 39, wherein the non-human animal is non- transgenic.

41. The use or the method of any one of claims 1 to 40, wherein the non-human animal is a vertebrate.

42. The use or the method of claim 41, wherein said vertebrate is a mammal, a fish, or a bird.

43. The use or the method of claim 42, wherein said mammal is selected from the group consisting of mouse, rat, hamster, rabbit, cattle, pig, guinea pig, sheep, goat, horse, camel, dog, cat, and monkey, e.g., rhesus macaque, baboon, orang-utan, and chimpanzee.

44. The use or the method of claim 42, wherein said bird is selected from the group consisting of poultry, preferably chicken, duck, turkey, and goose; and pigeon, and Japanese quail.

45. The use or the method of claim 42, wherein said fish is selected from the group consisting of salmon, trout, tilapia, carp, catfish, medaka, zebrafish, loaches, goldfish and pikes.

46. The method or the use of any one of claims 32, and 38 to 45, wherein the method comprises reimplantation of the embryo, preferably a blastocyst stage, into a non- human animal pseudo-pregnant mother.

47. The method of claim 32, wherein a plurality of non-human animals is produced from the embryos formed in step (c), (d) or (e).

48. The method or the use according to any one of claims 32, and 38 to 46, further comprising breeding of the non-human animal or the plurality of non-human animals to produce a plurality of offspring generations.

49. The method of any one of claims 1 to 32, 39 and 46 to 48, the archive of any one of claims 33 to 37, or the use of any one of claims 38, 40 to 45, and 48, wherein the cell clone or the clonal cell line or the cell clones or the clonal cell lines is/are formed by more than 2 cells derived by clonal expansion from the somatic cell treated in accordance with step (a) of claim 1.