WO2005107447A2 - 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 or a germ cell capable of producing a mutated non-human animal comprising treating sperm cells with a mutagen in vitro and contacting an oocyte with the sperm cells thus obtained. The invention also relates to an archive comprising embryos or germ cells obtained by the method of the invention, and the use of the embryo(s) or the germ cell(s) obtained or obtainable by the method of the invention for producing a non-human animal.

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-l,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.

In 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-insl0), 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 development of larger transgenic non-human animals, e.g., domestic/farm animals, is, however, currently very expensive. Up to date, the major problems are long time periods to produce such animals, low success rate, and high costs due to the lack of reliable methods in higher species.

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 zebrafish 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), and nucleus transfer (Schnieke et al., 1998), ENU mutagenesis has the advantage of introducing particular point mutations, i.e., subtle DNA modifications without introducing foreign DNA (for example antibiotic selection marker genes) into the recipient's genome. 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. 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 (PPARγ) gene (Barroso et al., 1999). A dominant-negative N290M 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 PPARγ. The underlying point mutations provided the first time evidence for the direct involvement of PPARγ in the control of insulin sensitivity, glucose homeostasis and blood pressure in man (Barroso et al, 1999). Current methods of EΝU mutagenesis, e.g., in mice, are not necessarily appropriate for higher vertebrates, e.g., domestic/farm animals. EΝU mutagenesis in mice is performed as intraperitoneal injection of EΝU into male mice (see Example 1 of WO 2004/020619), using defined mg/kg dosages of EΝU, which are injected once or several times. Due to EΝU-induced sterility, the earliest date at which male mice can be mated to females is fifty days after the final EΝU injection, when fertility starts to overcome the EΝU-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 EΝU 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. Cryopreservation is established for many species, e.g., for mouse (Nagakata et al., 1993), cattle (Foote and Kaprotht, 2002), carp (Magyary et al., 1996) and camel (Deen et al, 2003). 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 large number of mutations.

There is 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 animals using sperm mutagenesis in combination with subsequent oocyte fertilization, e.g., INF or Al. 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. The methods of the present invention are applicable to many species. In addition, the mutagenesis on sperm cells provides the possibility to efficiently mutate every gene in a given sexually reproducing organism.

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

(a) treating sperm cells with a mutagen in vitro;

(b) contacting an oocyte with the sperm cells obtained in step (a) for fertilization and allowing the resulting zygote to develop into: (i) an at least 4-cell stage embryo; or

(ii) a non-human animal capable of sexually reproducing; and

(c) storing the embryo or one or more germ cells, e.g., sperm cells or oocytes, of the non-human animal obtained in step (b); or

(d) maintaining the non-human animal obtained in step (b). The method may further comprise the isolation of one or more cells, preferably at least two cells, of the embryo or the non-human animal, preferably isolation prior to step (c).

The method may additionally further comprise the screening of a nucleic acid sample derived from one or more cells, preferably at least two cells, of the embryo or the non-human animal produced 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 the corresponding embryo or the one or more germ cells of the non-human animals stored according to step (c), or to the non-human animal maintained according to step (d).

In another aspect, the invention provides a method of producing a non- human animal from the stored embryo or the stored germ cells of the non-human animal or the use of the stored embryo or the stored germ cells of the non-human animal for the production of a non-human animal. The method of producing a non-human animal may comprise the reimplantation of such an embryo, preferably a morula or a blastocyst stage, into a non-human animal foster mother. In case the stored germ cells are sperm cells, the method of producing a non-human animal further comprises the in vitro fertilization of an oocyte with said one or more sperm cells of the non-human animal and the subsequent reimplantation of the resulting embryo into a non-human animal foster mother or artificial insemination with said one or more sperm cells of the non-human animal.

The invention further provides a method, wherein step (b) comprises contacting and fertilizing a plurality of oocytes with the sperm cells obtained in step (a) and allowing the resulting zygotes to develop into at least 4-cell stage embryos or non- human animals capable of sexually reproducing, and wherein step (c) comprises storing a plurality of the embryos or the germ cells of non-human animals obtained in step (b), or wherein step (d) comprises maintaining the plurality of the non-human animals obtained in step (b).

The invention further provides an archive comprising the stored embryos or one or more germ cells of the non-human animals.

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

Brief Description of the Figures

Figure 1 depicts a flow chart, describing two alternative embodiments

A and B of the method of the invention for generating non-human animals from mutagenized sperm cells. A. Mutagenized sperm cells are subject to In Vitro Fertilization (INF). The resulting zygotes are allowed to develop into the morula or blastocyst stage of embryonic development (morulas/blastocysts) before blastomer sample dissection from these embryos. Blastomeres' nucleic acid is used for mutation identification in a gene of interest. Dissected embryos are either cryopreserved (Embryo "frozen archive") prior to mutation identification on nucleic acid isolated from the blastomer samples (A-l) or directly transferred into foster mothers (Embryo transfer) after the mutation identification on nucleic acid isolated from the blastomer samples (A-2). Gl offspring is delivered. In A-l, mutation identification will select embryos carrying mutations in a gene of interest. The selected embryos, which were stored in the embryo "frozen archive", are subjected to embryo transfer to generate Gl offspring. Subsequent offspring G2 and Gn (where "n" means any offspring following G2 offspring) is generated. In A-2, the Gl embryo is selected based on the results of the mutation identification on nucleic acid isolated from the blastomeres (bypassing the "frozen archive" and the DΝA "archive") to produce Gl offspring. Offspring carrying a mutation in a gene of interest, the mutation having been verified on nucleic acid from an animal's tissue sample, is selected for further breeding and generating G2 and Gn offspring.

B. Mutagenized sperm cells are subject to either Artificial Insemination (Al) or In Vitro Fertilization (INF). Pregnant animals will deliver Gl offspring (Gl Offspring "living archive"), which is subject to tissue sample dissection (for DΝA collection or "archive") and optionally isolation of germ cells. Germ cells are collected to establish a germ cell "frozen archive". The DΝA of the tissue samples is analyzed for mutations in a gene of interest (mutation identification). Once a mutation in a gene of interest is identified, corresponding animals from the Gl offspring are selected for further breeding of G2 and Gn offspring (B-1). Alternatively, once a mutation in a gene of interest is identified, corresponding germ cells are selected for generating G2 and Gn offspring (B- 2).

Tissue samples, nucleic acids derived therefrom, and germ cells from offspring delivered by methods A and B are collected for establishing frozen archives (tissue sample "frozen archive", germ cell "frozen archive", "DΝA archive"). 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 sperm cell with a mutagen" as used herein refers to the contact or exposure of a population of sperms in vitro with a mutagen of choice.

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 term "capable of sexually reproducing" as used herein refers to a non- human or non-human animals that is capable of sexually reproducing via gametes fusion, that is fusion of sperm an egg cell. The term "mature sperm cells" or "spermatozoa" means sperm cells isolated from cauda epidydimis or vas deferens.

The terms "Gl offspring", "G2 offspring", and "Gn offspring", as used in the present invention, mean the first (Gl, generation 1), second (G2, generation 2), and any subsequent offspring generations (Gn, n=any offspring generation following G2 offspring), generated from initial fertilization of an oocyte with an in vitro mutagen-treated sperm cell. Due to the kind of mutagenesis contemplated herein, the Gl offsprings are chimeric animals with respect to the induced mutations.

The terms "chimeric" or "chimera" as used herein mean that the developing embryo starting from the two cell embryo will be a 50% chimera with approximately 50% of its cells being genetically different from the other 50% of its cells.

The term "embryo capable of producing a mutated non-human animal" as used herein refers to an at least 2-cell stage embryo that is capable of developing into a sexually reproducing animal as described herein, e.g., upon (re)imρlantation into a foster mother for development. Alternatively, such an embryo may be an embryo resulting from fertilization, e.g., by Al or normal mating, of an oocyte carried by a non-human animal, which embryo is capable of developing within said non-human animal to an offspring animal.

The term "germ cells capable of producing a mutated non-human animal" as used herein inter alia refers to sperm cells, e.g., spermatogonia, spermatides, or mature sperm cells, which are capable of fertilizing, e.g., via INF, an oocyte in vitro, or which are capable of developing into such mature sperm cells. The term also refers to oocytes, which are capable of being fertilized by sperm cells as described herein in vitro, e.g., via INF. The term, however, also encompassed sperm cells, e.g., spermatogonia, spermatides, or mature sperm cells, which are capable of fertilizing, e.g., via Al or in the course of mating, an oocyte in vivo, or are capable of developing into such mature sperm cells. The term also includes oocytes capable of being fertilized in vivo, e.g., via Al or in the course of mating.

The term "nucleic acid" as used herein, refers to DΝA, such as genomic DΝA, or cDΝA, and also RΝA. 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 occuring 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 terms "tissue sample" or "cell biopsy" as used herein, mean a multicellular sample of an organism's tissue or organ or a single cell sample, the sample having been isolated by biopsy techniques, where the organism is in any stage of development from embryonic developmental stages to adult stage. A tissue sample may comprise a single or one or more, preferably two, blastomer(es) dissected from a morula or blastocyst embryonic developmental stage; or a piece of an organ, e.g., liver, isolated from a non-human animal, e.g., from birds, fish or mammals; or a piece of tissue, e.g., tail, isolated by tail clipping from a non-human animal, e.g., from rats, mice, cattle or pig.

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 or an embryonic developmental stage as well as embryos or germ cells or the non-human animal itself.

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 "blastomer" as used herein refers to any cell resulting from dissection of an embryo.

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.

Methods of the Invention We inter alia describe a method of providing an embryo or a germ cell capable of developing into an at least 4 cell stage embryo, or a mutated non-human animal. This method comprises the treatment of sperm cells with a mutagen, e.g., a chemical or physical mutagen, preferably ENU (see Example 1). The method further comprises contacting an oocyte with said sperm cells for fertilization. After fertilization, the replication of the sperm DNA treated, e.g., with ENU prior to fertilization, produces two different DNA molecules in the diploid zygote. One molecule will carry a mutation as the replication mechanism will introduce with a certain probability a different base at the position of the altered, e.g., ethylated and thereby modified base. Hence, a mutation is introduced in one of the two sperm DNA molecules after the first mitotic duplication. The co-duplicated egg DNA will not carry a mutation. With the division of the zygote a two- cell embryo is produced. One cell carrying the copy of the sperm (paternal) DNA and one copy of the egg (maternal) DNA. Due to the introduced point mutation, one cell differs genetically from the other cell. As a consequence, the developing embryo will be a 50% chimera with eventually approximately 50% of its cells being genetically different from the other 50% of its cells. The method further allows the resulting zygote to develop into an at least 4-cell stage embryo or into a non-human animal capable of sexually reproducing; and storing the embryo or the one or more germ cells of the non-human animal (see Example 7).

The sperm cells treated with a mutagen may be derived from the same or a different non-human animal species compared to the non-human animal species from which the oocyte is derived. They may further derive from a wild-type or a transgenic animal. The sperms and the oocyte used for the contacting step with the sperms may be derived from a wild-type non-human animal. They may, however, also 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.

In one embodiment, said fertilization comprises in vitro fertilization (INF) and said resulting zygote is allowed to develop into an at least 4-cell stage embryo (see Example 2). This embryo is dissected and one or more cells, preferably at least two cells, e.g. blastomeres or trophoectodermal cells of the embryo, are used to isolate a nucleic acid sample, which may be screened for the presence of a mutation in a gene of interest (see Examples 3 and 6). The remaining embryo, e.g. a morula or a blastocyst, preferably an at least 2-cell embryo, is stored, preferably frozen or stored in an appropriate culture medium. Upon screening of the nucleic acid sample for a mutation in a gene of interest, the corresponding stored embryo is assigned to the identified mutation. The assigned embryo is then further used to produce a non-human animal from the embryo according to the methods described herein (see, e.g., Example 8). This step may comprise the reimplantation of the embryo, preferably a morula or a blastocyst stage, more preferably an at least 2-cell embryo, into a non-human animal foster mother. In yet another embodiment, one or more cells, preferably at least two cells, of the embryo are isolated and nucleic acid samples thereof are analyzed for a mutation in a gene of interest. The corresponding embryo is directly used for further breeding without freezing (see, e.g., Example 8). The resulting non-human animal carrying the mutation in the gene of interest may be further bread to produce a plurality of offspring generations carrying said mutation.

In another embodiment, said fertilization step comprises artificial insemination in vivo or INF, respectively, and the subsequent development of a non-human animal capable of sexually reproducing, as mentioned herein (see Example 9). One or more cells are isolated from the non-human animal capable of sexually reproducing. The cells are isolated from an organ, e.g., liver or a tissue, e.g., ear or tail. Also isolated are one or more germ cells, e.g., spermatogonia, spermatides, mature sperm cells, or oocytes. The one or more germ cells are stored, e.g., frozen, or stored in an appropriate culture medium. The one or more tissue or organ cells may be isolated prior to or after the isolation and freezing of said germ cells.

A nucleic acid sample is isolated from said one or more tissue or organ cells, which is screened for a mutation in a gene of interest (see Example 6). Upon screening of the nucleic acid sample, the corresponding stored one or more germ cells are assigned to the identified mutation. The assigned one or more germ cells, e.g., spermatogonia, spermatides, mature sperm cells, or oocytes, are further used to produce a non-human animal. This step comprises, e.g., the INF of an oocyte, e.g., a wild type oocyte, with stored mature sperm cells, and the subsequent reimplantation of the resulting embryo into a non-human animal foster mother. Alternatively, this step comprises the INF of a stored oocyte with mature sperm cells, e.g., wild type sperm cells, and the subsequent reimplantation of the resulting embryo into a non-human animal foster mother according to the methods described herein (see, e.g., Examples 2 and 8). A further alternative is the artificial insemination using said stored sperm cells (see Example 9). In yet another embodiment of the above method, one or more tissue or organ cells are isolated from the non-human animal and their nucleic acid samples are analyzed for a mutation in a gene of interest. The non-human animal is maintained for further breeding without storing, e.g., freezing, of one or more germ cells. The resulting non-human animal 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 may be 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 chicken, duck, turkey, pigeon, goose and Japanese quail.

In another embodiment of the invention, the method comprises contacting and fertilizing a plurality of oocytes and allowing the resulting zygotes to develop into at least 4-cell stage embryos or non-human animals capable of sexually reproducing. The method may furthermore comprise storing a plurality of the resulting embryos or germ cells of the resulting non-human animals, e.g., by freezing or storing in an appropriate culture medium or maintaining the non-human animals obtained from the developing zygote, for, e.g., further breeding. Yet another embodiment of the invention is an archive comprising the stored, e.g., frozen or stored in appropriate culture medium, embryos or one or more germ cells of the non-human animals.

Another embodiment of the invention is an archive comprising one or more cells, preferably at least two cells, from each of a plurality of embryos prepared by the method of the invention, e.g., from at least 4-cell stage embryos, or comprising one or more cells from the non-human animals capable of sexually reproducing prepared by the method of the invention, e.g., cells from their tissue or organs.

Another embodiment of the invention is an archive comprising nucleic acid samples isolated .from the above-mentioned one or more cells, preferably at least two cells, from the embryos, e.g., from the at least 4-cell stage embryos, or from the above- mentioned one or more cells from the non-human animals capable of sexually reproducing, e.g., cells from their tissue or organs.

Yet another embodiment of the invention is an archive comprising the maintained non-human animals that developed from the zygotes resulting from the fertilization of the sperms treated with a mutagen in vitro.

A further embodiment is a plurality of non-human animals, wherein said animals are produced by the plurality of stored embryos or the plurality of stored germ cells of the invention.

Mutagenesis The invention encompasses mutagenesis of sperm cells 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), Isopropyl methane sulphonate (iPMS), ³H₂O, Urethane (UR), Bleomycine, Nitrogen Mustard, Nincristine, Dimethylnitrosamine, 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 EΝU 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 EΝU (Νoverskoe 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 a onefold 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 UN-light, which is likewise a suitable mutagen in connection with the present invention, are largely single nucleotide alterations. UN-light does not penetrate the animal but is generally useful for inducing mutations in cells in culture, e.g., ES cells or sperm cells as in the present invention.

Pre Implantation Genetic Diagnosis (PGD

1. General

Embryo biopsy and subsequent single-cell genetic analysis of the embryonic cells allow screening of embryos at the preimplantation stage of development. In humans, PGD techniques are being used to screen embryos for diagnosing genetic defects, which may lead to inherited diseases and other genetic conditions like postzygotic chromosomal abnormalities. Such abnormalities are likely to contribute to early pregnancy loss (Handyside and Delhanty, 1997). PGD routinely uses a single-cell biopsy for analysis.

In non-human animals, e.g., domestic/farm animals, PGD on single embryonic cells is widely used to determine the sex. The transfer of embryos of specific gender is economically beneficial and has herd management advantages. In addition, with the increasing effort of generating genetically altered non-human animals, e.g., domestic/farm animals, PGD gained importance for screening transgenic embryos in order to confirm their transgenic status for screening embryos to detect undesired genetic alterations (see in Νowshari and Brem, 2000a). Additionally, PGD may also be used to screen embryos for a desired genetic alteration.

2. Isolation of Cells

A tissue sample, e.g., one or more cells, preferably at least two cells, of the embryo of the invention, may be isolated at different embryonic developmental stages, e.g., from an at least 4-cell stage embryo (morula) or a blastocyst for PGD. The dissection of 1 or 2 blastomeres from an 8- to 16-cell stage embryo (morula) for PGD is described for many organisms, for example in mouse (Liu et al., 1993, Takeuchi et al., 1992) and in cattle (Bondioli et al., 1989, Bredbacka, 1994). A blastomer is removed and the embryo is subjected to cryopreservation (see Examples 3 and 7). Alternatively, a biopsy may be taken from the blastocyst stage, where the embryo contains up to 300 cells. With this method, a single cell or several cells may be removed without apparent detrimental effect, because blastocyst biopsy typically involves the preferential removal of the more accessible trophoectoderm cells, whereas the inner cell mass, that is destined to become the fetus, is not damaged (Gentry and Critser, 1995). Tissue sample for PGD may also be isolated from one or more other cells of a non-human animal, e.g. tissues or organs of the adult organisms. A preferred tissue for rodents is, e.g., tissue from the ear or the tail of such rodent. A preferred tissue for birds is, e.g., the liver.

3. Primer Extension Preamplification (PEP) The tissue sample, e.g. the one or more cells, preferably at least two cells, isolated from the embryo or the non-human animal, 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 biopsy 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 tissue sample, e.g., the one or more, preferably two, cells isolated from the embryo or the non- human animal, are cultured in vitro under appropriate conditions in order to allow proliferation of such cells and thereby increasing the amount of tissue and nucleic acid derivable therefrom (see, e.g., Example 5).

4. Screening for the Presence of a Mutation

Screening for the presence of a mutation in a gene of interest may be performed on a single nucleic acid sample derived from the tissue sample as described herein, e.g., derived from one or more cells, preferably at least two cells, of an embryo or one or more cells from a non-human animal obtained in accordance with the invention. If using more than one cell of the non-human animal, these cells may derive from the same or different tissues or organs from said animal. 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 tissue samples as described herein. The plurality of said tissue samples may be, e.g., derived from a plurality of at least 4-cell stage embryos or non-human animals capable of sexually reproducing as described herein or from the offspring produced from the plurality of embryos and non-human animals obtained by the method of the invention.

Another embodiment 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. The nucleic acid sample may be derived from a tissue sample as described herein or a tissue sample derived from the offspring generations of the embryos or the non-human animals obtained by the method of the invention. Alternatively, screening for the presence of a mutation according to the invention in at least two genes of interest may also be performed on a mixture of nucleic acid samples. The mixture of nucleic acid samples may be derived from a plurality of tissue samples as described herein, e.g., from a plurality of at least 4-cell stage embryos or a plurality of non-human animals capable of sexually reproducing or the offspring generations of such embryos or non-human animals.

In yet another embodiment, a mutation in a gene of interest can be 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 in an at least 4-cell stage embryo or a non- human animal capable of sexually reproducing produced according to the methods of the invention. In a preferred embodiment, the individual is a human. Said embryo or non- human animal is preferably a mouse, rat, cattle, or pig.

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 produce an embryo or a non-human animal capable of sexually reproducing according to the methods of the invention. Such embryo or non-human animal or a plurality of embryos or non-human animals capable of sexually reproducing are used to isolate one or more cells, on which screening for the presence of a mutation in said gene of interest is performed. In a preferred embodiment, the individual is a human. Said embryo or non-human animal is preferably a mouse, rat, cattle, or pig. In another preferred embodiment, said individual is a non-human animal that is from the same species as said embryo or non-human animal 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 dsDNA 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-standed (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 DNA, is subject to PCR amplification according to standard methods in the art. Genomic DNA fragments of the gene of interest are obtained by PCR using BioTherm-DNA- Polymerase (GeneCraft, Germany) according to the manufacturer's protocol. Gene- specific oligonucleotide primers are 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 an embryo, cells of an embryo or of a non-human animal, or germ 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. In one embodiment, embryos are stored, e.g., for reimplantation. Long-term storage may comprise freezing of the embryos. Protocols for freezing, such as cryopreservation, are applied to embryos at the pronucleate, cleavage or blastocyst stage of development and depend on the slow diffusion of the cryoprotectant through the zona pellucida (see Eaxmple 7). Protocols for the successful cryopreservation of biopsied embryos are established (see Lui et al., 1993; Nowshari and Brem, 2000a; Tominaga and Hamada, 2004, and Tominaga, 2004). Short-term storage may comprise the storing under appropriate culture conditions, e.g., in Ml 6 medium at 37°C and 5% CO₂, 5%O₂, and 90% N₂ (cf. Example 3). Alternatively the embryos can be stored in KSOM -Medium (Lawitts and Biggers.; 1991) at 37°C and 5%C0₂, 5% O₂ and 90% N₂.

The cells, which are stored in accordance with the invention may, e.g. comprise one or more cells of an embryo as described herein. Alternatively, such cells may comprise, e.g., one or more cells of a non-human animal, e.g., cells from its tissues or organs. For short-term storage, cells are kept in culture medium, e.g., for mouse blastomer cells in DMEM (Dulbecco's modified Eagle's medium; Invitrogen GmbH, Karlsruhe Germany), containing 15% FCS, 2 mM Glutamine, 0,1 mM β-mercaptoethanol, 50 mg/ml penicillin/streptomycin at 37°C, under 5% CO₂ and >90 % humidity in an incubator. For long-time storage, cells are kept frozen in a freezing medium, e.g., DMEM, containing 20% FCS, 10% DMSO . The cells stored in accordance with the invention may also comprise germ cells, e.g., oocytes, spermatogonia, spermatides, or mature sperm cells as described herein. Long-term storage of such germ cells comprises, e.g., freezing. For freezing, e.g., mouse sperm cells, the males are aged between 3-5 month because the spermatozoa of younger males sometimes are less viable. Ideally, males are separated from each other two weeks before freezing, have proven fertility, and have not mated for more than one week. The male mice are sacrifized by cervical dislocation and the two caudae epididymides are dissected, removing as much fat as possible. The tissue is placed into 0.9% NaCl in the appropriate well of a 4-well dish (on ice) and washed in NaCl, i.e., all remaining fat and big blood vessels are removed with a watchmaker forceps and a spring scissors. Both caudae epididymides are placed into the cryoprotecting solution in the appropriate well of the 4-well dish (on ice). As few NaCl as possible is transferred. In the cryoprotecting solution each cauda epididymis and vas deferens is cut several times with a spring scissors. The 4-well dish is left on ice for approximatley 1-2 minutes. In this time, the spermatozoa should disperse from the tissue (grey clouds). To get a homogeneous suspension, the dish is shaked carefully. The desired number of samples (e.g., 10 aliquots per 15 μl) is pipetted on to the lid of the 4 well dish. A 1 ml syringe is connected with the French straw and approx. 100 μl HTF medium are aspirated alternately with one air bubble. The HTF medium just acts as a weight so that the straws will not float when they have to be plunged in liquid nitrogen. Both ends of the straw are welded. The samples are placed in a freezing canister and the freezing canister is put in the liquid vapor phase (-120°C) for 10 minutes (descending cooling rate of -20 to -40°C per minute). After this time, the freezing canister is directly plunged in liquid nitrogen (-196°C) (Nakagata, 1993).

Another method of long-term storage of sperm cells is airdrying of sperm and subsequent fertilisation by ICSI (intracytoplasmatic sperm injection; see, e.g., Annual Meeting of the European Society of Human Reproduction and Embryology, Madrid, Spain, July 1, 2003). In another embodiment, nucleic acids are stored. Long-term storage of nucleic acids may be performed by freezing the nucleic acids in an appropriate buffer, e.g., TE-buffer (10 mM Tris ph 7.5, 1 mM EDTA).

Fertilization

Fertilization according to the present invention comprise all types of fertilization, e.g., fertilization ex vivo, such as the fertilization of zebrafish eggs and sperms, fertilization in vivo, such as fertilization via copulation or via artificial insemination, e.g., any method of surgical and non-surgical insemination, where harvested sperm is manually applied into the vagina of a female recipient (cf, e.g., Example 9), or in vitro fertilization (INF), i.e., incubation of sperms with oocytes outside of a non-human organism (cf, e.g., Example 2).

Reimplantation

Reimplantation according to the present invention comprise the reimplantation or transfer of an embryo at its various developmental stages, e.g. zygotes, morulae, blastocysts. For example in mouse, embryos from the one cell to the morula stage (e.g., day 0.5 - 2.5 days post coitus (dpc)) can be transferred into the ampullae from the oviduct into a 0.5 dpc pseudopregnant foster mother, whereas 3.5 dpc blastocysts are transferred into the uterus horns of 2.5 dpc pseudopregnant foster mother (see also Example 8 and Manipulating the Mouse Embryo; 2003 A Laboratory Manual).

Examples

EXAMPLE 1 : ENU Treatment of Non-Human Sperm cells

Preparation of the ENU-Solution.

ENU was dissolved in Soerenson Buffer, pH 6,0 for preparation of an ENU stock solution. For preparation of Soerenson buffer 9.078 g of KH₂PO (Merck KgaA, Darmstadt, Germany), (Stock A)_, and 11.976 g of Na₂HPO (Merck KgaA, Darmstadt, Germany), (Stock B) were dissolved in each 1000 ml of distilled H₂O. 121 ml of Stock B solution was added to 879 ml of Stock A solution, mixed by inversion, and autoclaved. 1 g of ENU (Sigma Aldrich Chemie GmbH, Munich, Germany) was dissolved in 200 ml of Soerenson buffer by vigorous shaking for about 10 min. Final concentration was determined photospectrometrically and the ENU stock solution was continuously kept refrigerated.

Sperm Isolation from Mice and Preparation.

High quality mature sperms, i.e., spermatozoa, were collected from sexually reproducing male C3H mice, which had not been mated for at least 10 days. A male mouse was 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 were 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 was for 20 min. at 37°C in an incubator with an atmosphere of 5% CO in air.

ENU Treatment of Isolated Mouse Sperm cells.

In a 1.5 ml tube, 20 to 40 μl of the HTF/sperm suspension was combined with an appropriate amount of ENU solution in a maximum volume of 500 μl. Final ENU concentrations were in the range of 0.2 mg/ml to 2.5 mg/ml. The ENU/sperm suspension was incubated at 37°C in an incubator with an atmosphere of 5% CO₂ in air for 20 min., followed by centrifugation at 3800 rpm for 4 min. The supernatant was discarded and the sperm cell pellet was resuspended in 500 μl of HTF medium. The centrifugation step was repeated to ensure proper removal of ENU. The sperm cell pellet was resuspended in 200 μl of HTF medium and the suspension was transferred into a 40 mm culture dish covered with lightweight paraffin oil (embryo tested; Sigma Aldrich Chemie GmbH, Munich, Germany) for further incubation at 37°C in an incubator with an atmosphere of 5% CO₂ in air for 20 min. Capacitated sperms are subject to in vitro fertilization, as described in Example 2.

Alternatively, capacitated sperms are resuspended in 200 μl of PBS pH

7.2/0.3% BSA after the repeated centrifugation step to properly remove ENU. Sperms are further diluted 1:50 in 10% formol saline, and the sperm concentration is determined using a haemocytometer counting chamber. The sperm concentration is adjusted to 2xl0⁶ to

5x10⁶ sperms per 50 μl volume for artificial insemination, as described in Example 9.

EXAMPLE 2: Method of In Vitro Fertilization (IVF)

Superovulation in Mouse.

Three days before in vitro fertilization (day minus 3) Intergonan (Intervet Deutschland GmbH; Unterschleissheim, Germany) was injected intraperitoneally (5

I.E./mouse; 0.1 ml/mouse) into oocyte donor females of mouse strain C57BL/6J. At day minus 1 Ovogest (Intervet Deutschland GmbH; Unterschleissheim, Germany) was injected intraperitoneally (5 I.E./mouse; 0.1 ml/mouse) into the same oocyte donor females.

Preparation of Oocyte Donor Mice. The oocyte donor females were sacrificed 14 hours after Ovogest injection.

After disinfection with 70% alcohol, each mouse abdomen was opened with surgical scissors from caudal to cranial. The upper end of one uterine horn was grasped with fine forceps and the uterus, oviduct, ovary and the fad pad were removed. A hole was 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 was finally removed. The whole procedure was repeated at the other uterine horn. Oviducts and the attached segments of the uterus were 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 were collected in one culture dish. Collected oviducts were transferred to a culture dish filled with 400 μl of HTF medium (see in Quinn et al., 1985), the oviducts still being surrounded with oil. Under oil, the swollen ampullae were opened with the closed tip of fine forceps and the oocyte-cumulus complex expelled into the oil. With the closed tip of fine forceps the oocyte-cumulus complexes was pushed into the medium drop. Culture dish with oocyte-cumulus complexes was incubated at 37°C in an incubator with an atmosphere of 5% CO₂ in air until in vitro fertilization.

In Vitro Fertilization.

Under microscopic examination and using a pipette with an 20 μl E-ART- tip (Molecular Bioproduct, San Diego, California, USA), the oocyte cumulus complexes were transferred into fertilization dishes containing capacitated spermatozoa of Example 1. Oocytes and spermatozoa were 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 was for removal of dead sperms and residues of the cumulus complex. After washing, the prepared oocytes were transferred into a fresh culture dish filled with 200 μl of KSOM-medium and were covered with equilibrated lightweight paraffin oil (equilibrated over night with KSOM-medium). For overnight incubation the culture dish was placed into an incubator adjusted to 37°C and an atmosphere of 5% CO in air. The following day, the number of 2-cell embryos was examined microscopically. Only embryos displaying two symmetrical blastomeres were used for further culturing to an 8-cell embryo in 15 μl droplets of Ml 6 medium containing 5 mg/ml BSA and covered with lightweight paraffin oil (embryo tested; Sigma Aldrich Chemie GmbH; Munich, Germany). After culturing of the zygotes for approximately 48 h in an incubator at 37°C in an atmosphere containing 5% CO₂, 5% O₂, and 90% N₂, 8-cell embryos were observed.

EXAMPLE 3: Pre-Implantation Genetic Diagnosis (PGD) - Method of

Blastomer Dissection

Preparation of Materials. Blastomer dissection of a mouse 8-cell embryo may be performed according to a method described in Liu et al., 1993. In brief, zygotes are prepared as described in Example 2 and than cultured in 15 μl droplets of M16 medium containing 5 mg/ml BSA and covered with lightweight paraffin oil (embryo tested; Sigma Aldrich Chemie GmbH; Munich, Germany). After culturing of the zygotes for approximately 48 h in an incubator at 37°C in an atmosphere containing 5% CO₂, 5% O₂, and 90% N₂, 8-cell embryos are observed. The removal of 2 blastomeres from an 8-cell embryo is performed under microscopic examination using an inverted microscope (Nikon, Tokyo, Japan). Holding and aspiration pipettes are made from 30 ml Drummond microcaps (Drummond Scientific Co., Broomall, PA, USA) on a model 753 Campden pipette puller (Campden Instruments Ltd., London, UK). The holding pipettes have an outer diameter of 60-80 μm and in inner diameter of 30-50 μm and are polished on a de Fonbrune microforge, pulled on the Campden pipette puller and then cut with the de Fonbrune microforge at the place where the outer diameter is 18-20 μm. A bevel angle of 45° is made with a Narishige 6-4 Microgrider. The inner diameter of the aspiration pipette is about 13-15 μm. The holding and aspiration pipettes are connected to 800 μl Narishige micrometer syringes by plastic tubing filled with lightweight paraffin oil.

Blastomer Dissection from Mouse Embryos.

An 8-cell embryo is transferred into 10 μl of HTF collection medium droplets, kept under lightweight paraffin oil in 35 x 10 mm culture dishes at 37°C on a heated stage. The embryo is maintained in a stationary position by gentle suction through a holding pipette. The aspiration of the 2 blastomeres is done by puncturing the zona pellucida and by aspirating 2 blastomeres gently into the aspiration pipette. After the biopsy procedure, the manipulated embryos are transferred back to fresh Ml 6 medium and cultured in an incubator adjusted to 37°C and an atmosphere containing 5% CO₂, 5% O₂, and 90% N₂. Survival of the biopsy is assessed 1 h later under the inverted microscope at magnification of x 100 or x200. An embryo is considered to have survived the procedure if by inspection under the light microscope, all remaining blastomeres are intact. Such embryos are cryopreserved as described in Example 7. Dissected blastomer nucleic acid is subjected either to direct pre-implantation genetic diagnosis (see Example 4 and Example

6) or to in vitro culture (see Example 5) followed by pre-implantation genetic diagnosis as described in Example 4 and/or Example 6. EXAMPLE 4: Pre-Implantation Genetic Diagnosis (PGD) - Amplification of Biopsy Genetic Material

Primer Extension Preamplification (PEP) is used to amplify genomic DNA from single cell biopsies (see in Sermon et al. (1996). In brief, biopsy material is resupended in 5 μl alkaline lysis buffer (200 mM 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 KC1, 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 55°C 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 6.

EXAMPLE 5: In Vitro Culture of Blastomeres Dissected From 8-Cell Embryos

As an alternative approach to generate a nucleic acid sample for PGD, both dissected blastomeres are directly transferred to one well of a gelatin-coated (0.2% gelatin

(Invitrogen GmbH, Karlsruhe Germany) in PBS (Invitrogen GmbH, Karlsruhe Germany))

96-well plate, each well filled with 100 μl of ES cell medium (DMEM (Dulbecco's modified Eagle's medium (Invitrogen GmbH, Karlsruhe Germany), containing 15% FCS,

2 mM Glutamine, 0.1 mM β-mercaptoethanol, 50 mg/ml penicillin/streptomycin). The cells are incubated at 37°C, under 5% CO₂ and >90% humidity in an incubator. Medium is changed daily until day 5, where the cells are washed with PBS and trypsinized with 30 μl of trypsin (Invitrogen GmbH, Karlsruhe Germany) for 5 min 37°C. Cells are resuspended in 150 μl ES cell medium before re-plating on one well of gelatin-coated 96-well plate.

After four passages 1:1, the cells are plated on one well of a 24-well plate and cells are kept growing, ideally until confluency. The cells are trypsinized, resuspended in PBS buffer, collected by centrifugation (5 min. lOOOx g, Sigma clinical centrifuge), and resuspended 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)) for nucleic acid preparation. Nucleic acid is subjected to mutation detection (see Example 6).

EXAMPLE 6: Pre-Implantation Genetic Diagnosis (PGD) - Myostatin Mutation Detection by Heteroduplex Analysis Using Temperature Gradient Capillary Electrophoresis (TGCE)

For detection of heterozygous mutations in nucleic acid from tissue samples, genomic DNA is used to PCR amplify DNA fragments of the myostatin gene with myostatin-specific PCR primers. The following 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 using amplified biopsy genomic DNA of M. musculus strain C57/B16 as template. The genomic

DNA derives from a tissue sample and is either directly used for PCR; or previously enriched by Primer Extension Pre-amplification (see Example 4); or previously in vitro cultured for sample material enrichment (see Example 5), or both.

Upon initial denaturation at 94°C for 3 min. each cycle consisted 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 55°C decrease from 55°C to 45°C at l°C/min. decrease from 45°C 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 70°C.

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, CA, 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 a heterozygous ENU embryo and of a wild type embryo is sequenced. The sequencing results are used to identify mutations by comparing the sequencing results from embryos carrying the ENU mutation with wild-type embryos.

EXAMPLE 7: Cryopreservation of Non-Human Embryos (Frozen Embryo

Archive)

Rapid-freezing of biopsied mouse embryos (6-cell dissected embryo) is performed by a method described in Liu et al., 1993. In brief, 6-cell dissected embryos, grown from in vitro fertilized eggs as described in Example 2 and dissected as described in Example 3, are cryopreserved by a slow freezing procedure with 1.5 M 1,2-ρropanediol (PROH) adapted from the procedure described by Lasalle et al., 1985. The cryoprotectant solution is prepared by adding 1.5 M PROH (Fluka AG, Nel, Leuven, Belgium) and 0.1 M sucrose (British Drug House) to M2 medium. The cryoprotectant mixture is cooled on crushed ice. A single biopsied embryo is transferred into 30 μl of the cold cryoprotectant mixture and then transferred into 0.25 ml plastic straws (Type -2A 175, Industrie de la Medecine Neterinaire, L'Aigle, France). The straws are carefully labeled and put into the freezing chamber of a programmable biological freezer (Planer R294, HNL, Brussels, Belgium) that has been pre-cooled to 0°C. The straws are kept for 15 min. at 0°C and then cooled to -6°C at a cooling rate of-2°C/min. The induction of the extracellular ice crystal formation is done at -6°C by touching the straw with forceps cooled in liquid nitrogen. The embryo is further cooled at -30°C at a cooling rate of- 0.3°C/min. The embryo straw is then plunged into liquid nitrogen. For thawing of the embryo the plastic straw is taken out of the storage bank and transferred to a water bath at 37°C. The thawing rate is approximately 600°C/min. The thawed cryoprotectant mixture with the embryo is transferred into a culture dish containing 100 μl droplets of collection medium with 1.0 M sucrose. The cryoprotectant is removed by incubating the embryo for 10 min. in collection medium with 1.0 M sucrose and for 10 min. in collection medium without sucrose. Thereafter the embryo is rinsed several times in collection medium and transferred into Ml 6 culture medium droplets under lightweight paraffin oil. Droplets, containing the embryos, are then kept in an incubator at 37°C in an atmosphere containing 5% CO₂, 5% O₂, and 90% N₂. The survival after freezing and thawing is assessed after about 1 hour of in vitro culture under an inverted microscope at x200 or x400 magnification. Embryos are considered to have survived the procedure if they contain the same number of blastomeres with the zona pellucida intact, as they do before the cryopreservation.

EXAMPLE 8: Embryo Transfer

Production of Mouse Pseudo-Pregnant Females.

Pseudo-pregnancy is generated by mating mouse CD1 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.

Uterus Transfer.

Embryos for embryo transfer are selected depending on the results of pre- implantation genetic diagnosis. Before the transfer, thawed embryos of Example 7 are 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 2 cell embryos 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 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 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. Foster mothers are kept until offspring is born. Offspring is subject to further phenotypic and molecular genetic analysis.

EXAMPLE 9: Artificial Insemination in Mouse

A successful artificial insemination occurs only when the inseminated female mouse is in the late proestrus/early estrus stage of the estrus cycle. Females of the musculus strain C57BL6/J are used and appropriately staged females are obtained through superovulation (see in Example 2).

Artificial Insemination is performed as described in West et al., 1977. In brief, ENU mutagenized sperms of Example 1 are concentrated to 2xl0⁶ to 5x10⁶ sperms in a 50 μl volume for artificial insemination. The 50 μl sperm suspension is drawn into a 1 ml disposable tuberculin syringe fitted with a 4 cm 22-gauge blunted needle, bent 2 cm from its tip to an angle of about 110°.

A superovulated mouse is prepared for insemination by inserting a vibrating brass rod into the vagina for about 20 sec. The mouse is then etherized and taped by the base of the tail to the stage of a dissecting microscope. The mouse vagina is dilated with curved forceps, and the cervix is penetrated with the blunted needle filled with sperms. After insemination with 50 μl sperm suspension, the vagina is plugged by one or two cotton wool balls soaked in isotonic saline. The vibrating brass rod and cotton wool plugs serve to stretch the vagina, and this stimulation induces the formation of functional corpora lutea necessary for the maintenance of pregnancy.

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Claims

Claims

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

(a) treating sperm cells with a mutagen in vitro; (b) contacting an oocyte with the sperm cells obtained in step (a) for fertilization and allowing the resulting zygote to develop into: (i) an at least 4-cell stage embryo; or (ii) a non-human animal capable of sexually reproducing; and (c) storing the embryo or one or more germ cells of the non-human animal obtained in step (b); or (d) maintaining the non-human animal obtained in step (b).

2. The method of claim 1 , wherein the mutagen is a chemical or physical mutagen.

3. The method of claim 2, 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-mercaρtopurine (6MP), Mitomycin-C (MMC), Procarbazine (PRC), N- methyl-N-nitro-N-nitrosoguanidine (MNNG), N-nitrosodiethylamine (NDEA), Isopropyl methane sulphonate (iPMS), H₂O, Urethane (UR), Bleomycine, Nitrogen Mustard, Nincristine, Dimethylnitrosamine, 7,12-Dimethylbenz(a)anthracene (DMBA), Ethylene oxide, Hexamethylphosphoramide, Bisulfan, Acridine orange, Ethidium bromide, Proflavin, and ICR-191).

4. The method of claim 2, wherein said physical mutagen is selected from gamma- radiation, X-ray radiation, UV-light, and neutrons.

5. The method according to any one of claims 1 to 4, wherein said oocyte and said sperm cells derive from the same or from different non-human animal species.

6. The method according to any one of claims 1 to 5, wherein said sperm cells derive from a wild-type or a transgenic non-human animal.

7. The method according to any one of claims 1 to 6, wherein said oocyte derives from a wild-type non-human animal.

8. The method according to any one of claims 1 to 7, wherein said fertilization comprises in vitro fertilization or artificial insemination.

9. The method according to any one of claims 1 to 8, wherein the embryo stored in step (c) is a morula or a blastocyst.

10. The method according to any one of claims 1 to 8, wherein the one or more germ cells of step (c) are selected from ooyctes, spermatogonia, spermatides, and mature sperm cells.

11. The method of any one of claims 1 to 10, wherein the method further comprises the isolation of one or more, preferably at least two, cells of the embryo or the non- human animal.

12. The method of claim 11, wherein said one or more cells or said at least two cells of the embryo or the non-human animal are isolated prior to step (c).

13. The method of any one of claims 1 to 12, wherein the method further comprises the screening of a nucleic acid sample derived from one or more, preferably at least two, cells of the embryo or the non-human animal produced in step (b) for the presence of a mutation in a gene of interest.

14. The method according to any one of claims 11 to 13, wherein the one or more or the at least two cells of the embryo are blastomeres or trophoectodermal cells.

15. The method according to any one of claims 11 to 13, wherein the one or more cells of the non-human animal derive from an organ or a tissue.

16. The method of claim 15, wherein said tissue is ear or tail tissue or said organ is liver.

17. The method according to any one of claims 13 to 16, wherein the nucleic acid sample is RNA or DNA.

18. The method of claim 17, wherein the DNA is genomic DNA.

19. The method according to any one of claims 13 to 18, wherein the screening method comprises a PCR specific for the gene of interest; Heteroduplex Analysis, e.g., Temperature Gradient Capillary Electrophoresis (TGCE); Single Strand Conformation Polymorphism (SSCP); 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 Microarrays.

20. The method according to any one of claims 13 to 19 further comprising the step of amplifying the nucleic acid sample by PCR.

21. The method according to any one of claims 13 to 20 further comprising culturing said one or more or said at least two cells of the embryo or the non-human animal in vitro under appropriate conditions to allow cell proliferation prior to screening.

22. The method according to any one of claims 13 to 21 further comprising the step of assigning said mutation to the corresponding stored embryo, the one or more germ cells of the non-human animal stored according to step (c), or to the non-human animal maintained according to step (d).

23. The method according to any one of claims 1 to 22, wherein said storing is performed by freezing or by storing in an appropriate culture medium or, in case the stored germ cells are sperm cells, by freeze drying.

24. The method according to any one of claims 1 to 23 wherein step (b) comprises contacting and fertilizing a plurality of oocytes with the sperm cells obtained in step (a) and allowing the resulting zygotes to develop into at least 4-cell stage embryos or non-human animals capable of sexually reproducing, and wherein step (c) comprises storing a plurality of the embryos or the germ cells of the non-human animals obtained in step (b) or wherein step (d) comprises maintaining a plurality of the non- human animals obtained in step (b).

25. An archive comprising the stored embryos or germ cells of the non-human animals obtained by the method of claim 24.

26. The archive of claim 25, wherein said storing is performed by freezing or by storing in an appropriate culture medium or, in case the stored germ cells are sperm cells, by freeze drying.

27. The archive of claims 25 or 26, wherein said embryos are morulas.

28. The archive of claims 25 or 26, wherein said embryos are blastocysts.

29. The archive of claims 25 or 26, wherein said germ cells are selected from oocytes, spermatogonia, spermatides, and mature sperm cells.

30. An archive comprising the non-human animals maintained according to step (d) of claim 24.

31. Use of the embryo(s) or the germ cell(s) obtained or obtainable by the method of any one of claims 1 to 24 or of the archive of any one of claims 25 to 29 for producing a non-human animal.

32. A method of producing a non-human animal comprising allowing the embryo obtained or obtainable by the method of any one of claims 1 to 23 or an embryo of the archive of any one of claims 25 to 29 to develop into a non-human animal.

33. A method of producing a non-human animal comprising allowing the germ cells of the non-human animal obtained or obtainable by the method of any one of claims 1 to 23 or the germ cells of the archive of any one of claims 25 to 29 to develop into a non-human animal.

34. The use or the method according to any one of claims 31 to 33, wherein the non- human animal is non-transgenic.

35. The use or the method according to any one of claims 1 to 34, wherein the non- human animal is a vertebrate.

36. The use or the method of claim 35, wherein said vertebrate is a mammal, a fish, or a bird.

37. The use or the method of claim 36, 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.

38. The use or the method of claim 36, wherein said bird is selected from the group consisting of chicken, duck, turkey, goose, pigeon, and Japanese quail.

39. The use or the method of claim 36, wherein said fish is selected from the group consisting of salmon, trout, tilapia, carp, catfish, medaka, zebrafish, loaches, goldfish and pikes.

40. The use or the method according to any one of claims 31 , 32, or 34 to 39 comprising the reimplantation of the embryo, preferably a morula or a blastocyst stage, into a non-human animal foster mother.

41. The use or the method according to any one of claims 31, or 33 to 39, wherein the one or more germ cells are sperm cells, and wherein said method comprises:

(a) in vitro fertilization of an oocyte with said one or more sperm cells of the non-human animal and the subsequent reimplantation of the resulting embryo into a non-human animal foster mother; or (b) artificial insemination with said one or more sperms of a non-human animal.

42. The use or the method according to any one of claims 31 to 41, wherein a plurality of non-human animals are produced from the embryos or germ cells stored in step (c) or maintained in step (d) of the method of claim 24.

3. The use or the method according to any one of claims 1 to 24 or 30 to 42, further comprising breeding of the (maintained) non-human animal(s) to produce a plurality of offspring generations.