WO2006002902A2 - Procedes de production de betail et de modeles pathologiques ameliores pour la recherche therapeutique - Google Patents

Procedes de production de betail et de modeles pathologiques ameliores pour la recherche therapeutique Download PDF

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WO2006002902A2
WO2006002902A2 PCT/EP2005/007015 EP2005007015W WO2006002902A2 WO 2006002902 A2 WO2006002902 A2 WO 2006002902A2 EP 2005007015 W EP2005007015 W EP 2005007015W WO 2006002902 A2 WO2006002902 A2 WO 2006002902A2
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embryo
human animal
cells
cell
embryos
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WO2006002902A3 (fr
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Michael Christian Nehls
Sigrid Wattler
Ulrike Huffstadt
Reinhard Sedlmeier
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Ingenium Pharmaceuticals GmbH
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Ingenium Pharmaceuticals GmbH
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2517/00—Cells related to new breeds of animals

Definitions

  • 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).
  • 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.
  • 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- 1,3 -galactosyl transferase. This transferase is involved in transferring the sugar molecules onto the pig cell surface. Phelps et al.
  • 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.
  • 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.
  • point mutation non-conservative nucleotide exchange
  • 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.
  • mice 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 AR ⁇ ) 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 PPAR ⁇ .
  • the underlying point mutations provided the first time evidence for the direct involvement of PPAR ⁇ in the control of insulin sensitivity, glucose homeostasis and blood pressure in man (Barroso et al., 1999).
  • 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.
  • 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.
  • 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.
  • sperms of certain animal species especially those that do not fertilize ex vi ⁇ o, 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.
  • the invention is inter alia directed to a method of generating mutated animals using embryo mutagenesis in combination with subsequent embryo transfer. 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.
  • the mutagenesis on embryos provides the possibility to efficiently mutate every gene in a given sexually reproducing organism.
  • 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:
  • step (b) storing the embryo treated in step (a);
  • step (c) allowing the embryo treated in step (a) to develop into a non-human animal capable of sexually reproducing
  • the embryo treated in step (a) is a 1 or 2 cell-stage embryo, or a 4 cell-stage embryo, and step (a) further comprises
  • step (i) allowing said 1 or 2 cell-stage embryo to develop into an at least 4-cell stage embryo following treatment with said mutagen and prior to storing according to step (b); or
  • the method may further comprises the isolation of one or more cells, preferably at least two, cells of the embryo obtained in step (a), or the non-human animal of step (c), preferably isolation prior to step (b) or steps (c)i) or (c)ii), respectively.
  • the method may additionally comprise the screening of a nucleic acid sample derived from one or more cells, preferably at least two cells, of the embryo obtained in step (a) or the non-human animal of step (c), 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 stored according to step (b), to the non-human animal maintained according to step (c), or to the one or more germ cells of the non-human animal stored according to step (c).
  • 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, hi 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.
  • step (b) comprises storing a plurality of embryos treated according to step (a); and wherein step (c) comprises allowing a plurality of embryos treated according to step (a) to develop into a non-human animal capable of sexually reproducing, and maintaining a plurality of said non-human animals, or storing one or more germ cells of a plurality of said non-human animals.
  • 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 breeding of the non-human animal(s) produced and/or maintained by the methods as described herein to produce a plurality of offspring.
  • 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 embryos are identical to A and B of the method of the invention for generating non-human animals from mutagenized embryos.
  • Oocytes and sperm cells are subject to In Vitro Fertilization (FVF).
  • FVF In Vitro Fertilization
  • the resulting embryos comprising 1 cell-stage embryos (e.g., zygotes), 2 cell-stage embryos or
  • Blastomeres' nucleic acid or trophoectodermal cells' 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 or trophoectodermal cell samples (A-I) or directly transferred into foster mothers (Embryo transfer) after the mutation identification on nucleic acid isolated from the blastomer samples or trophoectodermal cell samples (A-2).
  • Gl offspring is delivered, hi A-I, 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.
  • the Gl embryo is selected based on the results of the mutation identification on nucleic acid isolated from the blastomeres or trophoectodermal cells (bypassing the "frozen archive” and the DNA "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.
  • Gl offspring Gl Offspring "living archive”
  • tissue sample dissection for DNA collection or "archive”
  • Germ cells are collected to establish a germ cell "frozen archive”.
  • the DNA 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-I). 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”, “DNA archive”).
  • embryo inter alia includes 1 cell-stage embryos, preferably fertilized oocytes (i.e., zygotes). It also includes subsequent cell-stage embryos, including 2 cell-stage embryos and 4 cell-stage embryos.
  • organs 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.
  • treating an embryo with a mutagen and “treated embryo” as used herein refers to the contact or exposure of an embryo in vitro with a mutagen of choice.
  • phenotype refers to one or more morphological, physiological, behavioral and/or biochemical traits possessed by a cell or organism that result from its genotype.
  • alteration of the phenotype refers to a non- human animal of the present invention displaying one or more readily observable abnormalities compared to the wild-type animal.
  • 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.
  • the animal shows a loss of function phenotype.
  • the animal shows a gain of function phenotype.
  • 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.
  • capable of sexually reproducing refers to a non- human animal or non-human animals capable of sexually reproducing via gametes fusion, that is fusion of sperm and egg cell.
  • mature sperm cells or "spermatozoa” mean sperm cells isolated from cauda epidydimis or vas deferens.
  • chimeric or “chimera” as used herein mean that the developing embryo mutagenized at the developmental stage of a 1 cell-stage embryo, e.g., a zygote, will be a 50% chimera with approximately 50% of its cells having the same mutations. In case of mutagenesis of a 2 cell— stage embryo or 4 cell-stage embryo, the embryo will be 25% or 12,5% chimeric, respectively, for a particular mutation.
  • embryo capable of producing a mutated non-human animal refers to an at least 1-cell stage embryo that is capable of developing into a sexually reproducing animal as described herein, e.g., upon (reimplantation into a foster mother for development.
  • an embryo may be an embryo resulting from fertilization, e.g., by AI 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.
  • germ cells capable of producing a mutated non-human animal inter alia refers to sperm cells, e.g., spermatogonia, spermatides, or mature sperm cells, which are capable of fertilizing, e.g., via IVF, 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 IVF.
  • sperm cells e.g., spermatogonia, spermatides, or mature sperm cells, which are capable of fertilizing, e.g., via AI or in the course of mating, an oocyte in vivo, or are capable of developing into such mature sperm cells.
  • oocytes capable of being fertilized in vivo, e.g., via AI or in the course of mating.
  • nucleic acid refers to DNA, such as genomic DNA, or cDNA, and also RNA.
  • RNA 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.
  • mutation 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.
  • mutation also includes chromosomal rearrangements.
  • Insertid mutation as used herein means a mutation introduced by chemical or physical mutagens.
  • 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.
  • archive 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 an embryonic developmental stage thereof, as well as embryos or germ cells or the non-human animal itself.
  • non-transgenic 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.
  • blastomer refers to any cell resulting from dissection of an embryo.
  • trophoectodermal cell refers to any extra embryonic 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.
  • This method may comprise contacting an oocyte with sperm cells for fertilization (either via In Vitro Fertilization, (IVF), via Artificial Insemination (AI), or via normal mating) and allowing it to develop into an embryo, e.g., a 1 cell-stage embryo (zygote), a 2 cell- stage embryo, or a 4 cell-stage embryo.
  • IVF In Vitro Fertilization
  • AI Artificial Insemination
  • normal mating e.g., a 1 cell-stage embryo (zygote), a 2 cell- stage embryo, or a 4 cell-stage embryo.
  • the method further comprises the subsequent treatment of the embryo thus obtained with a mutagen, e.g., a chemical or physical mutagen, preferably ENU (see Example 2).
  • a mutagen e.g., a chemical or physical mutagen, preferably ENU (see Example 2).
  • ENU a chemical or physical mutagen
  • the replication of the DNA treated e.g., with ENU, produces two different DNA molecules in the diploid zygote having multiple mutations in the genome.
  • the developing embryo is a chimeric embryo.
  • 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 the homologous DNA position of the sequence will be the wild type sequence.
  • a two cell-stage embryo is produced with one cell being heterozygous for a specific mutation and the other cell bearing the wild-type sequence at the same position.
  • the developing embryo will be a 50% chimera with respect to a particular mutation
  • hi case of the treatment of a 2 cell-stage embryo, e.g. with ENU the percentage of cells bearing one identical mutation is reduced to 25%. Therefore, the developing embryo will be a 25% chimera with respect to a particular mutation.
  • the method further comprises allowing the embryo (the 1 cell-stage embryo, e.g., the zygote, or the 2 cell-stage embryo) 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 one or more germ cells of the non-human animal (see Example 7).
  • the embryo the 1 cell-stage embryo, e.g., the zygote, or the 2 cell-stage embryo
  • the embryo the 1 cell-stage embryo, e.g., the zygote, or the 2 cell-stage embryo
  • the method further comprises allowing the 4 cell-stage embryo to develop into an at least 8-cell stage embryo or into a non-human animal capable of sexually reproducing; and storing the embryo or one or more germ cells of the non-human animal (see Example 7).
  • the embryos treated with the mutagen may be derived, e.g., from the fertilization of oocytes with sperm cells.
  • the sperm cells may furthermore be from the same or from a different non-human animal species compared to the non-human animal species from which the oocyte is derived.
  • the sperms and/or 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.
  • said fertilization comprises In Vitro Fertilization (IVF).
  • IVF In Vitro Fertilization
  • a resulting zygote, 2 cell-stage embryo, or 4 cell-stage embryo is subjected to in vitro treatment with a mutagen.
  • a treated zygote or 2 cell-stage embryo 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.
  • 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.
  • a 4 cell-stage embryo treated in vitro with a mutagen, is allowed to develop into an at least 8 cell-stage embryo.
  • This embryo is dissected and one ore more cells, preferably at least two cells, for example four cells, wherein these cells are, e.g., blastomers 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, e.g., Examples 3 and 6).
  • the remaining embryo e.g., a morula or a blastocyst, preferably an at least 4-cell embryo, is stored, preferably frozen or stored in an appropriate culture medium.
  • 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 4-cell embryo into a non-human animal foster mother.
  • 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.
  • the embryo treated with the mutagen is allowed to develop into a non-human animal capable of sexually reproducing, e.g., via embryo transfer into a foster mother (see, e.g., Example 8).
  • 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.
  • 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 (e.g., as in Example 6).
  • 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 IVF 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.
  • this step comprises the IVF 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).
  • 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, one or more of its germ cells.
  • the non- human animal carrying the mutation in the gene of interest thus identified may be further bread to produce a plurality of offspring generations carrying said mutation.
  • the resulting non-human animal as described herein may be non-transgenic.
  • said non-human animal is a vertebrate, e.g., a mammal, a fish, or a bird.
  • 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.
  • the method comprises contacting and fertilizing a plurality of oocytes and allowing to develop into embryos for subsequent treatment of said embryos with a mutagen, e.g., a chemical or physical mutagen as described herein, preferably ENU.
  • a mutagen e.g., a chemical or physical mutagen as described herein, preferably ENU.
  • Treated embryos are further allowed to develop into a plurality of subsequent cell-stage embryos, e.g., treated 1 cell-stage embryos, e.g., treated zygotes, or treated 2 cell-stage embryos are allowed to develop into at least 4 cell-stage embryos, or into non-human animals capable of sexually reproducing; and treated 4 cell- stage embryos are allowed to develop into at least 8 cell-stage embryos, or non-human animals capable of sexually reproducing.
  • a mutagen e.g., a chemical or physical mutagen as described herein, preferably ENU.
  • Treated embryos are further allowed to develop into a plurality
  • 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 a plurality of the non-human animals, e.g., for 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 embryos treated with a mutagen in vitro ("living archive").
  • 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.
  • the invention encompasses mutagenesis of embryos 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).
  • 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
  • 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).
  • mutations are 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.
  • 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), 3 H 2 O, Urethane (UR), Bleomycine, Nitrogen Mustard, Vincristine, Dimethylnitros
  • the chemical mutagens mainly cause single nucleotide alterations.
  • 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.
  • ENU offers the opportunity of obtaining a very large number of mutations in vivo, which gives tremendous power to ENU mutagenesis.
  • 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.
  • 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.
  • Physical mutagens e.g., radiation mutagenesis via gamma-radiation, X-ray radiation, or neutrons, may also be used in accordance with the invention.
  • 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.
  • 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., embryonic cells as in the present invention.
  • Embryo biopsy and subsequent single-cell genetic analysis of the embryonic cells allow screening of embryos at the preimplantation stage of development.
  • 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.
  • PGD 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 Nowshari 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).
  • a biopsy may be taken from the blastocyst stage, where the embryo contains up to 300 cells.
  • 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.
  • 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.
  • nucleic acid samples may be DNA or RNA, preferably genomic DNA.
  • 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).
  • tissue sample e.g., the one or more, preferably two, cells isolated from the embryo or the non- human animal
  • the tissue sample are cultured in vitro under appropriate conditions in order to allow proliferation of such cells, thereby increasing the amount of tissue and nucleic acid derivable therefrom (see, e.g., Example 5).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 of a non-human animal 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, hi a preferred embodiment, the individual is a human. Said embryo or non-human animal is preferably a mouse, rat, cattle, or pig. hi 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 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).
  • TGCE Temperature Gradient Capillary Electrophoresis
  • the amplified nucleic acid sample e.g., the genomic DNA
  • 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).
  • 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.
  • TGCE Temporal Gradient Capillary Electrophoresis
  • RevealSystem RevealSystem
  • heteroduplices 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).
  • SSCP single strand conformation polymorphism
  • fSSCP fluorescent SSCP
  • SSCP Denaturing Gradient Gel Electrophoresis
  • Cleavage of Mismatches Constant Denaturing Capillary Electrophoresis
  • 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.
  • 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 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.
  • embryos are stored, e.g., for reimplantation.
  • Long-term storage may comprise freezing of the embryos.
  • Protocols for freezing such as cryopreservation, may be applied to embryos at the pronucleate, cleavage, blastocyst, or morula stage of development and depend on the slow diffusion of the cryoprotectant through the zona pellucida (see Example 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 M16 medium at 37°C and 5% CO 2 , 5%O 2 , and 90% N 2 (cf. Example 3).
  • the embryos can be stored in KSOM- Medium (Lawitts and Biggers.; 1991) at 37°C and 5%C0 2 , 5% O 2 and 90% N 2 .
  • the cells which are stored in accordance with the invention may, e.g. comprise one or more cells of an embryo as described herein.
  • such cells may comprise, e.g., one or more cells of a non-human animal, e.g., cells from its tissues or organs.
  • cells are preferably kept in culture medium, e.g., for mouse blastomer cells in DMEM (Dulbecco's modified Eagle's medium; Invitrogen GmbH, Düsseldorf Germany), containing 15% FCS, 2 mM Glutamine, 0.1 mM ⁇ -mercaptoethanol, 50 mg/ml penicillin/streptomycin at 37°C, under 5% CO 2 and >90 % humidity in an incubator.
  • cells are preferably 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.
  • freezing e.g., mouse sperm cells
  • the males are preferably aged between 3-5 month, because the spermatozoa of younger males sometimes are less viable.
  • males are separated from each other two weeks before freezing, have proven fertility, and have not mated for more than one week.
  • 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, hi this time, the spermatozoa should disperse from the tissue (grey clouds).
  • the dish is shaken carefully.
  • the desired number of samples e.g., 10 aliquots per 15 ⁇ l
  • a 1 ml syringe is connected with the French straw and approx. 100 ⁇ l 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 0 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 suitable method of long-term storage of sperm cells according to the invention is air-drying 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).
  • ICSI intracytoplasmatic sperm injection
  • 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).
  • 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 (IVF), i.e., incubation of sperms with oocytes outside of a non-human organism (cf., e.g., Example 2).
  • 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
  • Reimplantation according to the present invention comprise the reimplantation or transfer of a mutagenized embryo at its various developmental stages, e.g. zygotes, morulae, blastocysts.
  • embryos from the one cell to the morula stage e.g., day 0.5 - 2.5 days post coitus (dpc)
  • dpc day 0.5 - 2.5 days post coitus
  • embryos from the one cell to the morula stage e.g., day 0.5 - 2.5 days post coitus (dpc)
  • dpc day 0.5 - 2.5 days post coitus
  • High quality mature sperms i.e., spermatozoa
  • 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).
  • testis structures were washed briefly in 0.9% NaCl at room temperature, transferred into 500 ⁇ l of HTF fertilization medium (Quinn et al., 1985), and cut into 5 pieces allowing the sperms to flush out. Testis structures were removed.Incubation in HTF medium was for 20 min. at 37°C in an incubator with an atmosphere of 5% CO 2 in air. Capacitated sperm cells were subject to in vitro fertilization, as described later in this Example.
  • the oocyte donor females were sacrificed 14 hours after Ovogest injection. After desinfection 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, Kunststoff, Germany). Oviducts from all the female mice were collected in one culture dish. Collected oviducts were transferred to a culture dish filled with 400 ⁇ l 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% CO 2 in air until in vitro fertilization. In Vitro Fertilization
  • the oocyte cumulus complexes were transferred into fertilization dishes containing capacitated spermatozoa.
  • the total volume of spermatozoa/oozyte cumulus complex in HTF was 200 ⁇ l, overlaid with mineral oil to prevent drying.
  • Oocytes and spermatozoa were incubated for 4 to 6 hours in an incubator (37°C, 5% CO 2 in air) for fertilization and ENU mutagenesis (see Example 2).
  • 1 cell stage embryos, 2 cell-stage embryos, and 4 cell-stage embryos derived from in vitro fertilization are subjected to in vitro ENU mutagenesis (see Example 2).
  • EXAMPLE 2 ENU Treatment of Non-Human Embryos
  • Zygotes, 2 cell-stage embryos, or 4 cell-stage embryos, respectively, all derived from in vitro fertilization, are subjected to in vitro ENU mutagenesis.
  • ENU is dissolved in Soerenson Buffer, pH 6.0 for preparation of an ENU stock solution.
  • Soerenson buffer 9.078 g of BCH 2 PO 4 (Merck KgaA, Darmstadt, Germany), (Stock A) , and 11.976 g Of Na 2 HPO 4 (Merck KgaA, Darmstadt, Germany), (Stock B) is dissolved in each 1000 ml of distilled H 2 O. 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, Kunststoff, 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.
  • IVF IVF is stopped after 4-6 hours 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 ⁇ l 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.
  • the prepared oocytes are transferred into a fresh culture dish filled with 200 ⁇ l of KSOM- medium and are covered with equilibrated lightweight paraffin oil (equilibrated over night with KSOM-medium). For overnight incubation the culture dish is placed into an incubator adjusted to 37°C and an atmosphere of 5% CO 2 in air. The following day, the number of 2- cell embryos is examined microscopically.
  • Blastomer dissection of a mouse 8-cell embryo, derived from an ENU treated zygote may be performed according to a method described in Liu et al., 1993.
  • zygotes are prepared as described in Example 1 and are treated with ENU as described in Example 2.
  • Treated zygotes are then cultured in 15 ⁇ l droplets of Ml 6 medium containing 5 mg/ml BSA and covered with lightweight paraffin oil (embryo tested; Sigma Aldrich Chemie GmbH; Kunststoff, Germany). After culturing of the treated zygotes for approximately 48 h in an incubator at 37°C in an atmosphere containing 5% CO 2 , 5% O 2 , and 90% N 2 , 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 ⁇ m and in inner diameter of 30-50 ⁇ m 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 ⁇ m.
  • a bevel angle of 45° is made with a Narishige 6-4 Microgrider.
  • the inner diameter of the aspiration pipette is about 13-15 ⁇ m.
  • the holding and aspiration pipettes are connected to 800 ⁇ l Narishige micrometer syringes by plastic tubing filled with lightweight paraffin oil.
  • An 8-cell embryo is transferred into 10 ⁇ l 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.
  • 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% CO 2 , 5% O 2 , and 90% N 2 . Survival of the biopsy is assessed 1 h later under the inverted microscope at magnification of x 100 or x200.
  • Example 7 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
  • PEP Primer Extension Preamplification
  • PCR buffer 25 mM MgCl 2 /gelatin (1 mg/ml)/100 mM Tris-HCl, pH 8.3
  • 3 ⁇ l dNTP mixture each 2 mM
  • 1 ⁇ l Taq polymerase each 2 mM
  • the volume is raised to 60 ⁇ l 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 ⁇ l 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.
  • both dissected blastomeres are directly transferred to one well of a gelatin-coated (0.2% gelatin (Invitrogen GmbH, Düsseldorf Germany) in PBS (Invitrogen GmbH, Düsseldorf Germany)) 96-well plate, each well filled with 100 ⁇ l of ES cell medium (DMEM (Dulbecco's modified Eagle's medium; Invitrogen GmbH, Düsseldorf Germany), containing 15% FCS, 2 mM Glutamine, 0.1 mM ⁇ -mercaptoethanol, and 50 mg/ml penicillin/streptomycin).
  • DMEM Disulbecco's modified Eagle's medium
  • FCS 2 mM Glutamine
  • 0.1 mM ⁇ -mercaptoethanol 50 mg/ml penicillin/streptomycin
  • the cells are trypsinized, resuspended in PBS buffer, collected by centrifugation (5 min., lOOOxg, 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, Kunststoff, Germany)) for nucleic acid preparation. Nucleic acid is subjected to mutation detection (see Example 6).
  • genomic DNA is used to PCR amplify DNA fragments of the myostatin gene with myostatin-specif ⁇ c 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 ⁇ l) 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 via previous in vitro culturing for sample material enrichment (see Example 5), or both.
  • each cycle 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.
  • a typical temperature profile for denaturation/renaturation is:
  • TGCE TGCE
  • 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 7O 0 C.
  • 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.
  • 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.
  • 6-cell dissected treated embryo Rapid-freezing of biopsied mouse embryos (e.g., 6-cell dissected treated embryo) is performed by a method described in Liu et al., 1993.
  • 6-cell dissected treated embryos grown from in vitro fertilized eggs (as described in Example 1) which were subjected to subsequent in vitro ENU mutagenesis in the zygotic developmental stage
  • Example 2 (as described in Example 2), and which were dissected as described in Example 3, are cryopreserved by a slow freezing procedure with 1.5 M 1,2- ⁇ ropanediol (PROH) adapted from the procedure described by Lasalle et al., 1985.
  • the cryoprotectant solution is prepared by adding 1.5 M PROH (Fluka AG, VeI, Leuven, Belgium) and 0.1 M sucrose
  • a single biopsied embryo is transferred into 30 ⁇ l of the cold cryoprotectant mixture and then transferred into 0.25 ml plastic straws (Type -2 A 175, Industrie de Ia Medecine Veterinaire, L'Aigle, France).
  • the straws are carefully labeled and put into the freezing chamber of a programmable biological freezer (Planer R294, HVL, 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 0 C at a cooling rate of-2°C/min.
  • the induction of the extracellular ice crystal formation is done at -6 0 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 ⁇ l 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 M 16 culture medium droplets under lightweight paraffin oil.
  • 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.
  • 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.
  • Embryos for embryo transfer are selected depending on the results of pre- implantation genetic diagnosis.
  • 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, Kunststoff, Germany) on a warming plate at 37°C.
  • 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 6 cell-stage embryos are collected with a transfer pipette in approximately 50 ⁇ l of M2 medium.
  • 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).
  • 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.
  • 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 5 superovulation (see in Example 1).
  • sperms of Example 1 are concentrated to 2x10 6 to 5x10 6 sperms in a 50 ⁇ l volume for artificial insemination.
  • the 50 ⁇ l 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 i o 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. 15 After insemination with 50 ⁇ l 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.
  • Cheung VG Nelson SF.: Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA.
  • GD 1992. Preclinical models for human pre-embryo biopsy and genetic diagnosis. I. Efficiency and normalcy of mouse pre-embryo development after different biopsy techniques. Fertil Steril.,57(2):425-430. Tominaga K, and Hamada Y, 2004. Efficient production of sex-identified and cryosurvived bovine in- vitro produced blastocysts. Theriogenology, 61 (6):1181-91.

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Abstract

L'invention concerne, entre autres, un procédé de génération d'animaux mutants non humains faisant appel à la mutagénèse d'embryons combinée au transfert d'embryons ultérieur. Le procédé consiste à traiter un embryon d'un animal non humain à l'aide d'un mutagène in vitro, puis à stocker l'embryon ou à permettre à l'embryon de se développer dans un animal non humain pouvant se reproduire sexuellement. Ledit animal non humain peut ensuite être conservé ou une ou plusieurs cellules germinales dudit animal peuvent être stockées. L'invention concerne également une archive comprenant des embryons ou des cellules germinales obtenus par le procédé de l'invention, ainsi que l'utilisation du/des embryon(s) ou de la/des cellule(s) germinale(s) obtenus ou pouvant être obtenus par le procédé de l'invention pour produire un animal non humain.
PCT/EP2005/007015 2004-06-30 2005-06-29 Procedes de production de betail et de modeles pathologiques ameliores pour la recherche therapeutique Ceased WO2006002902A2 (fr)

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CN109619031A (zh) * 2018-12-28 2019-04-16 安徽汇康畜牧科技有限公司 皖鹅一号新品种科学培育方法
US12507679B2 (en) 2021-04-16 2025-12-30 Auburn University Engineered catfish and uses thereof

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US6943025B2 (en) * 2001-01-10 2005-09-13 Children's Medical Center Corporation Method for identifying genes involved in cell proliferation

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109619031A (zh) * 2018-12-28 2019-04-16 安徽汇康畜牧科技有限公司 皖鹅一号新品种科学培育方法
US12507679B2 (en) 2021-04-16 2025-12-30 Auburn University Engineered catfish and uses thereof

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