WO2012158982A2 - Méthodes pour effectuer une transgenèse d'organismes et de cellules souches spermatogoniales (ssc) à l'aide de vecteurs de transposons - Google Patents

Méthodes pour effectuer une transgenèse d'organismes et de cellules souches spermatogoniales (ssc) à l'aide de vecteurs de transposons Download PDF

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WO2012158982A2
WO2012158982A2 PCT/US2012/038461 US2012038461W WO2012158982A2 WO 2012158982 A2 WO2012158982 A2 WO 2012158982A2 US 2012038461 W US2012038461 W US 2012038461W WO 2012158982 A2 WO2012158982 A2 WO 2012158982A2
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organism
transposon
sscs
genetically modified
spermatogonial stem
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PCT/US2012/038461
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WO2012158982A3 (fr
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Eric M. Ostertag
John S. CRAWFORD
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Transposagen Biopharmaceuticals, Inc.
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Publication of WO2012158982A2 publication Critical patent/WO2012158982A2/fr
Publication of WO2012158982A3 publication Critical patent/WO2012158982A3/fr

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    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/061—Sperm cells, spermatogonia
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1082—Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/03—Animals modified by random mutagenesis, e.g. using ENU, chemicals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells

Definitions

  • Gene modification is a process whereby a specific gene, or a fragment of that gene, is altered. This alteration of the transgenic gene may result in a change in the level of R A and/or protein that is encoded by that gene.
  • the modified gene may be studied in the context of a cell, or, more preferably, in the context of a genetically modified organism.
  • Genetically modified organisms are among the most useful research tools in the biological sciences, as well as agricultural, pharmaceutical and biotechnology applications.
  • An example of a genetically modified organism is a transgenic organism which harbors a transgene (e.g. heterologous or foreign gene) that results altered function to the gene and protein.
  • transgenesis has also been shown to be productive in identifying new drugs and drug targets.
  • Genetically modified organisms exhibiting clinically relevant phenotypes are valuable for drug discovery and development and for drug target identification.
  • transgenic somatic or germ cells facilitate the production of genetically modified offspring or cloned organisms having a phenotype of interest.
  • Such organisms have a number of uses, for example as models of physiological disorders (e.g., of human genetic diseases) that are useful for screening the efficacy of candidate therapeutic compounds or compositions for treating or preventing such physiological disorders.
  • identifying the gene(s) responsible for the phenotype provides potential drug targets for modulating the phenotype and, when the phenotype is clinically relevant, for therapeutic intervention.
  • Transposons unlike homologous recombination and site specific homing endonucleases do not require sequence specificity between the transposon donor and the site of integration. The ability of transposons to integrate and deliver DNA sequences in "cut and paste” or "copy and paste” mechanism provides a great system for transgenesis in mammals.
  • SSCs are sperm stem cells that maintain
  • SSCs are readily and efficiently genetically modified by a variety of transposon vectors. Genetically modified SSCs can be expanded and transplanted into recipient males. The recipient male is bred with wild type females to produce genetically modified offspring.
  • transposon vector technologies have previously not been used to produce genetic modifications in many cells which can then be used to generate genetically modified organisms. While transposon transgenesis have been described in other cells and cell lines, spermatogonial stem cells (SSCs) have not previously been genetically modified with a variety of transposon technologies.
  • SSCs spermatogonial stem cells
  • SSCs spermatogonial stem cells
  • this invention relates to methods for transposon based transgenesis in spermatogonial stem cells (SSCs) of many species of mammals.
  • SSCs spermatogonial stem cells
  • Transposon based transgenesis of SSCs allow for the efficient production of genetically modified organisms in many species of mammals.
  • Transposon based transgenesis of cells results in altered function of gene(s) or gene product(s) and genetically modified organisms, and cell or tissue culture models are produced from these engineered cells.
  • Modified cells and organisms include transgenic cells and organisms..
  • the invention relates to genetically modified organisms created by transposon based transgenesis including but not limited to mammals rats, mice, pigs, rabbits, guinea pigs, dogs, non-human primates as well as the descendants and ancestors of such organisms.
  • the invention describes methods for generating
  • SSCs spermatogonial stem cells
  • the invention provides kits that are used to produce
  • kits transposon based transgenic spermatogonial stem cells (SSCs) which can be used to generate genetically modified organisms.
  • the kits typically include one or more transposon technology such as piggyBac.
  • the kit may also contain one or more sets of spermatogonial stem cells (SSCs) for genetic modification as well as media and conditions necessary for growing stem cells.
  • the kits may include instructions for (i) introducing the transposons into the SSCs (ii) identifying SSCs which have been modified (iii) growing transposon modified cells in media or conditions necessary and to numbers required for cells to produce genetically modified organisms (iv) using the grown cells to produce a genetically modified organism (v) identifying which organisms or progeny harbor the transposon mutation of interest.
  • composition comprising of one or more spermatogonial stem cell(s) (SSCs), wherein the SSCs comprise of one of the following genetic modifications (i) one or more transgenic insertions (ii) an addition of a heterologous nucleic acid sequence (iii) wherein one or more of the genetic modifications are caused by transposon technology.
  • SSCs spermatogonial stem cell(s)
  • the heterologous nucleic acid sequence is chosen from a selectable marker or an orthologous gene.
  • the heterologous nucleic acid sequence is chosen from one or more tissue specific expression transgenic insertions.
  • the heterologous nucleic acid sequence is chosen from one or more cell line specific expression transgenic insertions. [0017] In some embodiments of the invention, the heterologous nucleic acid sequence is chosen from one or more germline specific expression transgenic insertions.
  • the heterologous nucleic acid sequence is chosen from one or more somatic cell specific expression transgenic insertions.
  • the heterologous nucleic acid sequence is chosen from one or more Cre recombinase expressing transgenic insertions.
  • the heterologous nucleic acid sequence is chosen from one or more transposase expressing transgenic insertions.
  • the one or more spermatogonial stem cells is derived from the germline lineage of an animal.
  • the one or more spermatogonial stem cells further comprise at least one inverted tandem repeat of a transposon or a variant thereof.
  • the transposon technology consists of the transposon families IS630-Tcl -mariner (ITm), hobo-Ac-Tam (hAT) and piggyBac (PB).
  • the transposon technology consists of the transposons Sleeping Beauty, T 2, piggyBac, Minos, Frog Prince, Mariner-Like Elements (MLE), Mimarl, Hsmarl, Mosl, ISYIOO, Tn7.ln some embodiments, one or more of the above-listed transposons is not used.
  • the transposon technology consists of the transposons Hermes, Pokey, Tn5, Tn916, Tel /mariner, S elements, Quetzal elements, Txr elements, Tcl-like transposon subfamilies, Tc-3 like transposons, ICEStl elements, maT, and P -elements.
  • an organism comprising one or more spermatogonial stem cells
  • the one or more spermatogonial stem cells comprise one or more of the following genetic modifications (i) one or more transgenic insertions (ii) an addition of a heterologous nucleic acid sequence (iii) one or more of the genetic modifications are caused by transposon technology.
  • the one or more stem cells further comprises
  • a composition comprising one or more spermatogonial stem and: (a) a transposon integrates into a nucleic acid sequence within the genome of the one or more spermatogonial stem cells; or (b) a nucleic acid sequence that encodes a transposon integrates into a nucleic acid sequence within the genome of the one or more spermatogonial stem cells; the one or more spermatogonial stem cells is derived from the germline lineage of an animal.
  • the stem cell is a spermatogonial stem cell derived from a rat.
  • the spermatogonial stem cell is genetically modified by transposons piggyBac or Sleeping Beauty, the spermatogonial stem cell is not derived from a rat.
  • the stem cell is a spermatogonial stem cell derived from a minipig.
  • the one or more spermatogonial stem cells further comprise at least one inverted tandem repeat of a transposon or a variant thereof.
  • the one or more spermatogonial stem cells further comprise: (a) one or more nucleic acid sequences at least 70% homologous to a nucleic acid sequence chosen from:
  • TCAACTG SEQ ID NO:2
  • the spermatogonial stem cell is genetically modified by a fragment of nucleic acid sequence 70%> homologous to SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID N0:4, the spermatogonial stem cell is not derived from a rat.
  • composition comprising one or more progeny of an organism, the one or more progeny comprise any one or more of the one or more mutations (i), (ii), and (iii).
  • the one or more progeny further comprise at least one inverted tandem repeat of a transposon or variant thereof.
  • the composition is a colony of mammals.
  • the organism is an animal.
  • the organism is a mini pig.
  • the organism is a rat.
  • the organism is genetically modified by transposons piggyBac or Sleeping Beauty, the organism is not a rat.
  • the organism is chosen from a mouse, pig, rabbit, dog, cat, goat, non-human primate, mini pig, ferret, farm animals, fish, chicken, and bird.
  • the organism is chosen from a salmonoid, carp, tilapia, or tuna.
  • the organism is an insect.
  • a mammal comprising one or more
  • the one or more spermatogonial stem cells comprise one or more of the following genetic modifications (i) one or more transgenic insertions (ii) an addition of a
  • heterologous nucleic acid sequence (iii) one or more of the genetic modifications are caused by transposon technology.
  • the one or more spermatogonial stem cells are transplanted from an in vitro culture.
  • the mammal further comprises a nucleic acid that comprises a transposon sequence that is at least 70% homologous to: SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and/or SEQ ID NO:4.
  • the mammal further comprises a nucleic acid that comprises a transposon sequence that is at least 70% homologous to: SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and/or SEQ ID NO:4, the mammal is not a rat.
  • the mammal is mini pig
  • the mammal is a rat.
  • the mammal is a sterile male mini pig.
  • the mammal is a sterile male rat.
  • the mini pig is DAZL deficient or DAZL-
  • the rat is DAZL deficient or DAZL-/-
  • organisms comprising:
  • At least one organism comprising one or more spermatogonial stem cells the one or more spermatogonial stem cells comprise one or more of the following genetic modifications (i) one or more transgenic insertions (ii) an addition of a heterologous nucleic acid sequence (iii) one or more of the genetic modifications are caused by transposon technology and (b) progeny of the organism of subpart (a).
  • the heterologous nucleic acid is a
  • the at least one organism and the progeny further comprise at least one inverted tandem repeat of a transposon or variant thereof.
  • the at least one organism and the progeny further comprise a nucleic acid that comprises a transposon sequence that is at least 70% homologous to: SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and/or SEQ ID NO:4.
  • the at least one organism and the progeny further comprise a nucleic acid that comprises a transposon sequence that is at least 70% homologous to: SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and/or SEQ ID NO:4, the organism and progeny are not rats.
  • genetically modified organisms comprising:
  • step (c) implanting one or more spermatogonial stem cells from the culture of step (b) into an organism.
  • the organism is capable of passing at least one integration of a gene of interest to progeny by germline transmission.
  • the genetically modified organism is a mammal.
  • the genetically modified organism is a mini pig.
  • the genetically modified organism is a rat.
  • the organism is genetically modified by transposons piggyBac or Sleeping Beauty, the organism is not a rat.
  • the genetically modified organism is a sterile male mini pig.
  • the genetically modified organism is a sterile male rat.
  • the method further comprises: breeding the organism implanted with the one or more spermatogonial stem cells with another animal to generate one or more progeny that comprise the mutated gene of interest.
  • the progeny are mammals.
  • genetically modified organisms comprising:
  • step (c) implanting the at least one spermatogonial stem cell comprising at least one integration of a gene of interest from the culture of step (b) into a first organism.
  • a method for breeding a colony of genetically modified organisms by transposons piggyBac or Sleeping Beauty, genetically modified organisms that make up the colony are not rats.
  • the first and second organisms are mammals.
  • the first organisms are mini pigs.
  • the second organisms are rats.
  • a method of manufacturing a first filial generation of genetically modified organisms comprising two or more distinct subsets of organisms comprising:
  • the first filial generation of genetically modified organisms comprises two or more sets of organisms, each set comprising a distinct mutation of interest derived from a haplotype of distinct spermatogonial stem cells transplanted into a parent of the organism.
  • the organism is a mammal.
  • a kit comprising: (a) a transposon or a nucleic acid sequence that encodes a transposon that integrates a nucleic acid sequence of a gene of interest; and (b) an instruction manual comprising directions
  • kits comprising of the culture media for the one or more spermatogonial stem cells.
  • kits comprising of spermatogonial stem cells genetically modified by transposons piggyBac and Sleeping Beauty, the spermatogonial stem cells are not derived from a rat.
  • Figure 1 Schematic of spermatogonial stem cells (SSCs) separated into multiple colonies are genetically modified with different transposons. The genetically modified SSCs are selected and pooled together to form a pool of SSCs containing different genetic modifications relating to the production of different genetically modified organisms using a single recipient male.
  • SSCs spermatogonial stem cells
  • FIG. 2 A schematic of a colony of wild type spermatogonial stem cells (SSCs) showed in cell culture.
  • Figure 3 Schematic for propagation of SSCs in cell culture
  • Figure 4 piggy Bac transposon based transgenesis vector which includes a multiple cloning site (MCS) for introduction of the gene of choice.
  • MCS multiple cloning site
  • Figure 5 Schematic for transplantation of genetically modified rat SSC
  • the genetically modified SSCs are used to produce genetically modified rats by mating recipient males with wild type (WT) females.
  • Figure 6 Schematic for transfection of transgenesis vector and fluorescent marker gene.
  • Figure 7 Vector map of the piggy Bac based rasH2 transgenic construct.
  • Table 1 Reagents for the production of SSC medium.
  • Table 2 Reagents for passaging and propagation of SSCs.
  • Transposon based genetic modification includes but is not limited to one or more transgene insertions.
  • the transposon mediated genetic modification of SSCs includes Sleeping Beauty, TU2, piggyBac, Minos, Frog Prince, Mariner-Like Elements (MLE), Mimarl, Hsmarl, Mosl, ISY100, Tn7.
  • the transposon mediated genetic modification of SSCs includes Hermes, Pokey, Tn5, Tn916, Tel /mariner, S elements, Quetzal elements, Txr elements, Tel -like transposon subfamilies, Tc-3 like transposons, ICEStl elements, maT, and - elements.
  • the invention includes methods for the use of transposon mediated genetically modified spermatogonia or spermatogonial stem cells (SSCs) are expanded and grown to adequate numbers and transplanted into azoospermic genetically deficient male organisms for germline transmission of the transposon mediated mutation which is used to produce a genetically modified organism.
  • SSCs transposon mediated genetically modified spermatogonia or spermatogonial stem cells
  • Ranges may be expressed herein as from “about” one particular value, and/or to
  • deoxyribonucleotide nucleotide or DNA sequence within a gene, chromosome or genome of an organism.
  • insertion or “integration” or “knock- in” which is meant an alteration in the nucleic acid sequence that may replace the endogenous, normal or wild-type allele with an exogenous allele.
  • the exogenous allele includes but is not limited to a full length gene of the same or a different species, a section of a gene of the same or different species, a replacement cassette and reporter or selection genes and markers.
  • Knock-in mutations can be produced by homologous recombination, site-specific deletion, repair mechanism provocation via targeting proteins, as well as site specific targeted DNA transposons.
  • a "coding sequence” or a sequence “encoding” an expression product such as a
  • RNA, polypeptide, protein, or enzyme is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme.
  • a coding sequence for a protein may include a start codon (usually ATG) and a stop codon.
  • "Complementary,” as used herein, refers to the subunit sequence complementarity between two nucleic acids, e.g., two DNA molecules. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position.
  • two nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs).
  • a “deletion mutation” means a type of mutation that involves the loss of genetic material, which may be from a single base to an entire piece of chromosome. Deletion of one or more nucleotides in the DNA could alter the reading frame of the gene; hence, it could result in a synthesis of a nonfunctional protein due to the incorrect sequence of amino acids during translation.
  • “Derived from the germline of a animal or plant” is meant genetic material or nucleotides or DNA or genes or chromosomes or genomes or transgenes or mutations that may be passed on to offspring or next generation of which an organism is derived from.
  • Embryo is a multicellular diploid eukaryote in early stage of development.
  • Embryonic stem cell is a stem cell derived from the inner mass of the blastocyst or early stage embryo.
  • exon is meant a region of a gene which includes sequences which are used to encode the amino acid sequence of the gene product.
  • express and expression mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence.
  • a DNA sequence is expressed in or by a cell to form an "expression product” such as a protein.
  • the expression product itself e.g. the resulting protein, may also be said to be “expressed”.
  • An expression product can be characterized as intracellular, extracellular or secreted.
  • intracellular means something that is inside a cell.
  • extracellular means something that is outside a cell.
  • a substance is "secreted” by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.
  • gene also called a "structural gene” means a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes, and may or may not include introns and regulatory DNA sequences, such as promoter sequences, 5'- untranslated region, or 3 '-untranslated region which affect for example the conditions under which the gene is expressed.
  • Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription.
  • gene trap is meant to mean a DNA sequence which can delivered using a
  • genetically modified is meant a gene or genetic sequence that is altered from its native state (e.g. by insertion mutation, deletion mutation, nucleic acid sequence mutation, or other mutation), or that a gene product is altered from its natural state (e.g. by delivery of a transgene that works in trans on a gene's encoded mRNA or protein, such as delivery of inhibitory RNA or delivery of a dominant negative transgene).
  • a "germ cell” is a cell that gives rise to the gametes of an organism.
  • the germ cell is often a stem cell which can differentiate into gametes as well as other biological cell types.
  • a germ cell includes but is not limited to pluripotent cells, totipotent cells, spermatogonial stem cells (SSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) embryos, germ cells, primordial germ cells (PGCs), plant tube cells, pollen cells, and spores.
  • heterologous refers to a combination of elements not naturally occurring.
  • heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.
  • the heterologous DNA includes a gene foreign to the cell.
  • a heterologous expression regulatory element is such an element operatively associated with a different gene than the one it is operatively associated with in nature.
  • the term "homology” refers to the subunit sequence identity or similarity between two polymeric molecules e.g., between two nucleic acid molecules, e.g., between two DNA molecules, or two polypeptide molecules.
  • a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two polypeptide molecules is occupied by phenylalanine, then they are identical at that position.
  • the homology between two sequences is a direct function of the number of identical positions, e.g., if half (e.g., 5 positions in a polymer 10 subunits in length) of the positions in two polypeptide sequences are identical then the two sequences are 50% identical; if 70% of the positions, e.g., 7 out of 10, are matched or homologous, the two sequences share 70%> identity.
  • the polypeptide sequences ACDEFG and ACDHIK share 50%> identity
  • the nucleotide sequences CAATCG and CAAGAC share 50%> identity are examples of the polypeptide sequences ACDEFG and ACDHIK share 50%> identity
  • nucleotide sequences CAATCG and CAAGAC 50%> identity.
  • Homologous recombination is the physical exchange of DNA expedited by the breakage and reunion of two non-sister chromatids. In order to undergo recombination the DNA duplexes must have complimentarity. The molecular mechanism is as follows: DNA duplexes pair, homologous strands are nicked, and broken strands exchange DNA between duplexes. The region at the site of recombination is called the hybrid DNA or heteroduplex DNA. Second nicks are made in the other strand, and the second strand crosses over between duplexes. After this second crossover event the reciprocal recombinant or splice recombinant is created.
  • a "induced pluripotent stem cell" or (iPSC) cell is an adult cell that has been reprogrammed back to an embryonic like state. iPSCs can differentiate into many different cell types as well as produce genetically modified organisms.
  • knock-out an alteration in the nucleic acid sequence that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80% compared to the unaltered gene.
  • the alteration may be an insertion, deletion, frameshift mutation, or missense mutation.
  • the alteration is an insertion or deletion, or is a frameshift mutation that creates a stop codon.
  • knock-in is meant an alteration in the nucleic acid sequence that replaces the endogenous, normal or wild-type allele with an exogenous allele.
  • the exogenous allele includes but is not limited to a full length gene of the same or a different species, a section of a gene of the same or different species, a replacement cassette and reporter or selection genes and markers.
  • Knock-in mutations can be produced by homologous recombination, site specific deletion, repair mechanism provocation via targeting proteins, as well as site specific targeted DNA transposons.
  • Standard Animal or “pig” are breeds of inbred or outbred swine which can be used for research.
  • a "modifying agent” or “mutagen” is meant to be a physical or biological or chemical agent that changes genetic material or nucleotides, DNA, genes, chromosomes, genomes or organisms. Modifying agents can include natural and engineered proteins such as transposons.
  • a “mutation” is a detectable change in the genetic material in the organism, which is transmitted to the organism's progeny.
  • a mutation is usually a change in one or more deoxyribonucleotides, the modification being obtained by, for example, adding, deleting, inverting, or substituting nucleotides.
  • Exemplary mutations include but are not limited to a deletion mutation, an insertion mutation, a nonsense mutation or a missense mutation.
  • the terms “mutation” or “mutated” as used herein are intended to denote an alteration in the "normal” or "wild-type" nucleotide sequence of any nucleotide sequence or region of the allele.
  • normal and wild-type are intended to be synonymous, and to denote any nucleotide sequence typically found in nature.
  • mutated and normal are thus defined relative to one another; where a cell has two chromosomal alleles of a gene that differ in nucleotide sequence, at least one of these alleles is a “mutant” allele as that term is used herein.
  • Nucleic Acid sequence mutation is a mutation to the DNA of a gene that
  • a point mutation which affects a single nucleotide can result in a transition (purine to purine or pyrimidine to pyrimidine) or a transversion (purine to pyrimidine or pyrimidine to purine).
  • a point mutation that changes a codon to represent a different amino acid is a missense mutation. Some point mutations can cause a change in amino acid so that there is a premature stop codon; these mutations are called nonsense mutations.
  • a mutation that inserts or deletes a single base will change the entire downstream sequence and are known as frameshift mutations. Some mutations change a base pair but have no effect on amino acid representation; these are called silent mutations. Mutations to the nucleic acid of a gene can have different consequences based on their location (intron, exon, regulatory sequence, and splice joint).
  • phenotype means any property of a cell or organism.
  • a phenotype can simply be a change in expression of an mRNA or protein.
  • Phenotypes also include, but are in no way limited to, cellular, biochemical, histological, behavioral, or whole organismal properties that can be detected by the artisan.
  • Phenotypes include, but are not limited to, cellular transformation, cell migration, cell morphology, cell activation, resistance or sensitivity to drugs or chemicals, resistance or sensitivity to pathogenic protein localization within the cell (e.g. translocation of a protein from the cytoplasm to the nucleus), profile of secreted or cell surface proteins, (e.g., bacterial or viral) infection, post-translational modifications, protein localization within the cell (e.g.
  • translocation of a protein from the cytoplasm to the nucleus profile of secreted or cell surface proteins, cell proliferation, signal transduction, metabolic defects or enhancements, transcriptional activity, cell or organ transcript profiles (e.g., as detected using gene chips), apoptosis resistance or sensitivity, animal behavior, organ histology, blood chemistry, biochemical activities, gross morphological properties, life span, tumor susceptibility, weight, height/length, immune function, organ function, any disease state, and other properties known in the art.
  • the effects of mutation of one or more genes in a cell or organism can be determined by observing a change in one or more given phenotypes (e.g., in one or more given structural or functional features such as one or more of the phenotypes indicated above) of the mutated cell or organism compared to the same structural or functional feature(s) in a corresponding wild-type or (non- mutated) cell or organism (e.g., a cell or organism that in which the gene(s) have not been mutated).
  • a change in one or more given phenotypes e.g., in one or more given structural or functional features such as one or more of the phenotypes indicated above
  • the mutated cell or organism compared to the same structural or functional feature(s) in a corresponding wild-type or (non- mutated) cell or organism (e.g., a cell or organism that in which the gene(s) have not been mutated).
  • plasmid is meant a circular strand of nucleic acid capable of autosomal replication in plasmid-carrying bacteria.
  • the term includes nucleic acid which may be either DNA or RNA and may be single- or double-stranded.
  • the plasmid of the definition may also include the sequences which correspond to a bacterial origin of replication.
  • Pluripotent cells are stem cells that are capable of differentiating into any of the germ layers and can produce any type of fetal and adult cell.
  • a "promoter sequence” is a DNA regulatory region capable of binding RNA
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site
  • the promoter may be operatively associated with other expression control sequences, including enhancer and repressor sequences.
  • regulatory sequence is defined herein as including promoters
  • enhancers and other expression control elements such as polyadenylation sequences, matrix attachment sites, insulator regions for expression of multiple genes on a single construct, ribosome entry/attachment sites, introns that are able to enhance expression, and silencers.
  • reporter gene any gene which encodes a product whose expression is detectable.
  • a reporter gene product may have one of the following attributes, without restriction: fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., lacZ or luciferase), or an ability to be specifically bound by a second molecule (e.g., biotin or an antibody-recognizable epitope).
  • selectable marker is meant a gene product which may be selected for or against using chemical compounds, especially drugs.
  • Selectable markers often are enzymes with an ability to metabolize the toxic drugs into non-lethal products.
  • the pac (puromycin acetyl transferase) gene product can metabolize puromycin
  • the dhfr gene product can metabolize trimethoprim (tmp)
  • the bla gene product can metabolize ampicillin (amp).
  • Selectable markers may convert a benign drug into a toxin.
  • the HSV tk gene product can change its substrate, FIAU, into a lethal substance.
  • Another selectable marker is one which may be utilized in both prokaryotic and eukaryotic cells.
  • the neo gene for example, metabolizes and neutralizes the toxic effects of the prokaryotic drug, kanamycin, as well as the eukaryotic drug, G418.
  • selectable marker gene as used herein is meant a gene or other expression cassette which encodes a protein which facilitates identification of cells into which the selectable marker gene is inserted
  • SSC serotonin-derived sperm stem cell
  • Totipotent cells are cells that have the ability to divide and differentiate into any cell type including extrembryonic cells.
  • transgenic any cell or organism which includes a nucleic acid
  • transgenic mice represent another embodiment of the invention, other transgenic mammals including, without limitation, transgenic rodents (for example, hamsters, guinea pigs, rabbits,) and transgenic pigs, cattle, sheep, and goats are included in the definition.
  • transfection means the introduction of a foreign nucleic acid into a cell.
  • transformation means the introduction of a "foreign” (i.e.
  • extrinsic or extracellular gene DNA or RNA sequence to an ES cell or pronucleus, so that the cell will express the introduced gene or sequence to produce a desired substance in a genetically modified organism.
  • transposition is meant the process of one DNA sequence insertion into another (location) without relying on sequence homology.
  • the DNA element can be transposed from one chromosomal location to another or from introduction of exogenous DNA and inserted into the genome.
  • a "transposition event” is used herein to refer to the translocation of a DNA transposon either from one location on the chromosomal DNA to another or from one location on introduced exogenous DNA to another on the chromosomal DNA.
  • transposon vector is meant a linear strand of DNA capable of integrating into a second strand of DNA which may be linear or may be a circularized plasmid.
  • Transposons often have insertion sequences, or remnants thereof, at their extremities, and are able to integrate into sites within the second strand of DNA selected at random, or nearly random.
  • Preferred transposons have a short (e.g., less than 200) base pair repeat at either end of the linear DNA.
  • transposable elements is meant any genetic construct including but not limited to any gene, gene fragment, or nucleic acid that can be integrated into a target DNA sequence under control of an integrating enzyme, often called a transposase.
  • Transposon families is meant to mean the families of transposons that make up all transposons based on their characteristics.
  • Transposon mediated genetic modification or “transposon mediated modification” or “transposon mediated mutation” or “transposon based genetic modification” or “transposon based modification” or “transposon based mutation” is meant to mean any genetic modification generated by a transposon.
  • vector is used interchangeably with the terms “construct”, “cloning vector” and “expression vector” and means the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, (e.g. ES cell or pronucleus) so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence including but not limited to plasmid, phage, transposons, retrotransposons, viral vector, and retroviral vector.
  • non- viral vector is meant any vector that does not comprise a virus or retrovirus.
  • a "variant” is a nucleotide, set of nucleotides, DNA, RNA, gene, chromosome, genome, cell or organism which differs.
  • the variant may differ in nucleotide sequence, gene expression, RNA expression, protein expression and function, genotype, phenotype and characteristics.
  • Nucleotide sequences may be variants of the sequences in transposon inverted tandem repeats (ITRs) (shown in table 3) that are at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% homologous to known ITRs shown in table 3.
  • ITRs transposon inverted tandem repeats
  • a "vector sequence” as used herein, refers to a sequence of DNA comprising at least one origin of DNA replication and at least one selectable marker gene.
  • eukaryotic or prokaryotic cell including, but not limited to, haploid, diploid, triploid, tetraploid, or aneuploid.
  • the cell is diploid.
  • Cells in which the methods of the present invention can be advantageously used include stem cells.
  • the cells are spermatogonial stem cells (SSCs).
  • the invention comprises of methods to produce a transgenic or otherwise genetically modified cell using a transposon vector.
  • the transposon vector integrates into spermatogonial stem cells (SSCs) which are used to generate at least hemizygous, heterozygous or homozygous genetically modified organisms.
  • SSCs spermatogonial stem cells
  • the transposon mediated mutation creates a transgenic mutation in SSCs.
  • SSCs are saturated with transposon introduced transgenes.
  • the transgene expresses Cre recombinase, or tissue specific Cre recombinase, or cell type specific Cre recombinase, or germline specific Cre recombinase or somatic specific Cre recombinase.
  • the transgene expression is a tissue specific transposase, or cell type specific transposase, or germline specific transposase or somatic specific transposase.
  • transgenic cells can be within the
  • tissue explants e.g., in vivo or in situ
  • transgenic tissues or cells isolated from the organism using art-known methods and transgenic genes according to the present methods.
  • the tissues or cells are either maintained in culture (e.g., in vitro), or re -implanted into a tissue or organism (e.g., e vivo).
  • compositions wherein the integrating enzyme is a transposase.
  • the transposase of the composition is not limited and to any one transposase and can be selected from at least the group consisting of Sleeping Beauty (SB), Tn7, Tn5, mosl, piggybac, Himarl, Hermes, Tol2 element, Pokey, Minos, S elements, P-element, ICEStl, Quetzal elements, Tn916, maT, Tcl/mariner and Tc3.
  • transposase of the composition is not limited and to any one transposase and can be selected from at least the group consisting of Sleeping Beauty (SB), Tn7, Tn5, Tn916, Tcl/mariner, Minos and S elements, Quetzal elements, Txr elements, maT, mosl, piggybac, Himarl, Hermes, Tol2 element, Pokey, P-element, and Tc3. Additional transposases may be found throughout the art, for example, U.S. Pat. No. 6,225,121, U.S. Pat. No. 6,218,185 U.S. Pat. No. 5,792,924 U.S. Pat. No. 5,719,055, U.S. Patent Application No. 20020028513, and U.S. Patent
  • compositions of the invention can include transposases not yet identified.
  • transposons include, but are not limited to the following.
  • Transposons unlike homologous recombination and site specific homing endonucleases do not require any sequence between the transposon donor and the site of integration.
  • the ability of transposons to integrate and deliver DNA sequences in "cut and paste” or "copy and paste” mechanism provides a great system for transgenesis in mammals and spermatogonial stem cells (SSCs).
  • SSCs spermatogonial stem cells
  • the major transposon families are known as IS630-Tcl -mariner (ITm), hobo-Ac-Tam (hAT) and piggyBac (PB).
  • ITRs inverted terminal repeats
  • a transposase enzyme which removes the transposon from its location (e.g. plasmid) and inserts the transposon into a new target site (e.g. the genome of a mammal).
  • the present invention utilizes the transposon piggyBac (PB), and sequence configurations outside of PB, for use as a mobile genetic element as described in U.S. Pat. No. 6,962,810.
  • PB transposon piggyBac
  • the Lepidopteran transposon piggyBac is capable of moving within the genomes of a wide variety of species, and is gaining prominence as a useful gene transduction vector.
  • the transposon structure includes a complex repeat configuration consisting of an internal repeat (IR), a spacer, and a terminal repeat (TR) at both ends, and a single open reading frame encoding a transposase.
  • the Lepidopteran transposable element piggyBac transposes via a unique cut- and-paste mechanism, inserting exclusively at 5' TTAA 3' target sites that are duplicated upon insertion, and excising precisely, leaving no footprint (Elick et al, 1996b; Fraser et al, 1996; Wang and Fraser 1993).
  • the present invention utilizes the Sleeping Beauty(S ) transposon system for genome manipulation as described, for example, in U.S. Pat. No. 7,148,203.
  • the system utilizes synthetic, salmonid- type Tel -like transposases (SB) with recognition sites that facilitate transposition.
  • SB synthetic, salmonid- type Tel -like transposases
  • the transposase binds to two binding-sites within the inverted repeats of salmonid elements, and appears to be substrate-specific, which could prevent cross- mobilization between closely related subfamilies of fish elements.
  • To 12 is a member of the hAT family, is 4.7-kbp in length and has ITRs of 17 and
  • the transposase consists of four exons and catalyzes the transposition reaction of Tol2 transposons.
  • the Tol2 transposon is capable of delivering large amounts of DNA during transposition.
  • Minos anew transposable element from Drosophila hydei, is a member of the Tcl-like family of transposons. Nucl. Acids Res. 19:6646; Merriman P J, Grimes C D, Ambroziak J, hackett D A, Skinner P, and Simmons M J. (1995) S elements: a family of Tcl-like transposons in the genome of Drosophila melanogaster. Genetics 141 : 1425-1438).
  • Frog Prince FP is in the same family as SB and was also reconstructed from TLE sequences isolated from the frog Rana pipiens.
  • Mariner-Like Elements are within the ITm family and consist of five
  • Natural MLE's have short ITRs flanked by an intronless ORF encoding its transposase.
  • the major MLEs are known as Himarl, Hsmarl and Mosl.
  • ISY100 is a member of the ITm superfamily and was characterized in the genome of Synechocystis sp. ISY100 transposase features are similar to TLE.
  • Tn7 is a bacterial transposon with site specificity in E.coli. Tn7 requires four protein for integration and specificity; TnsA and TnsB make up the transposase, TnD is the target selector and TnsC is a non-sequence specific DNA binding protein.
  • Tn5 pre-cleavage synaptic complex. J Mol Biol 302:49-63
  • Tn7 Kuduvalli P N, Rao J E, Craig N L. (2001) Target DNA structure plays a critical role in Tn7 transposition. EMBO J 20:924-932), Tn916 (Marra D, Scott J R. (1999)
  • Tc3 Tu Z. Shao H. (2002) Intra- and inter-specific diversity of Tc-3 like transposons in nematodes and insects and implications for their evolution and transposition. Gene 282: 133-142
  • ICEStl Bosset V et al. (2002) The ICEStl element of Streptococcus thermophilus belongs to alarge family of integrative and conjugative elements that exchange modules and change their specificity of integration. Plasmid. 48(2): 77-97), maT, and P-element (Rubin G M and Spradling A C. (1983) Vectors for P element mediated gene transfer in Drosophila. Nucleic Acids Res. 11 :6341-6351). These references are incorporated herein by reference in their entirety for their teaching of the sequences and uses of transposons and transposon ITRs.
  • Transposons relates to generating transgenic insertions at higher efficiency due to the unique nature of SSCs, including but not limited to chromatin structure and methylation patterns.
  • Transposons relates to generating multiple transgenic insertions in the same SSC or SSC line which relates to generating genetically modified organisms with multiple transgenic insertions in fewer experimental steps and in a shorter timeframe than is possible with other systems.
  • Transposons relates to generating multiple transgenic insertions in the same SSC or SSC line in multiple and consecutive experiments or transfections which relates to generating genetically modified organisms with multiple transgenic insertions in fewer experimental steps and in a shorter timeframe than is possible with other systems.
  • Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions is fewer experimental steps and in a shorter timeframe than is possible with other systems. (Figure 1).
  • the separate SSCs or SSC lines may be two or more.
  • Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions is fewer experimental steps and in a shorter timeframe than is possible with other systems. (Figure 1).
  • the separate SSCs or SSC lines may be three or more.
  • Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions is fewer experimental steps and in a shorter timeframe than is possible with other systems. ( Figure 1).
  • the separate SSCs or SSC lines may be four or more.
  • Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions is fewer experimental steps and in a shorter timeframe than is possible with other systems. (Figure 1).
  • the separate SSCs or SSC lines may be five or more.
  • Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions is fewer experimental steps and in a shorter timeframe than is possible with other systems. (Figure 1).
  • the separate SSCs or SSC lines may be six or more.
  • Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions is fewer experimental steps and in a shorter timeframe than is possible with other systems. (Figure 1).
  • the separate SSCs or SSC lines may be seven or more.
  • Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions is fewer experimental steps and in a shorter timeframe than is possible with other systems. (Figure 1).
  • the separate SSCs or SSC lines may be eight or more.
  • Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions is fewer experimental steps and in a shorter timeframe than is possible with other systems. (Figure 1).
  • the separate SSCs or SSC lines may be nine or more.
  • Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions is fewer experimental steps and in a shorter timeframe than is possible with other systems. (Figure 1).
  • the separate SSCs or SSC lines may be ten or more.
  • Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions is fewer experimental steps and in a shorter timeframe than is possible with other systems. (Figure 1).
  • the separate SSCs or SSC lines may be eleven or more.
  • Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions is fewer experimental steps and in a shorter timeframe than is possible with other systems. (Figure 1).
  • the separate SSCs or SSC lines may be twelve or more.
  • Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions is fewer experimental steps and in a shorter timeframe than is possible with other systems. (Figure 1).
  • the separate SSCs or SSC lines may be thirteen or more.
  • Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions is fewer experimental steps and in a shorter timeframe than is possible with other systems. (Figure 1).
  • the separate SSCs or SSC lines may be fourteen or more.
  • Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions is fewer experimental steps and in a shorter timeframe than is possible with other systems. (Figure 1).
  • the separate SSCs or SSC lines may be fifteen or more.
  • separate pools or lines of genetically modified SSCs which may be used to generate a genetically modified organism, does not increase the amount of effort, time, and resources used, as well as does not decrease the efficiency of genetically modified organism production.
  • Multiple separate and distinct genetically modified SSCs may be transplanted into a single sterile recipient.
  • the mixed population of distinct genetically modified SSCs which are derived from separate SSC pools from two or more pools to fifteen or more pools mature within the sterile recipient.
  • the sterile recipient is then bred with multiple wild type females which may be two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more.
  • These multiple females produce offspring which have incorporated the desired mutation into their germline.
  • increasing the number distinct or separate pools or lines of genetically modified SSCs, which may be used to generate a genetically modified organism does not increase the amount of effort, time, and resources used, as well as does not decrease the efficiency of genetically modified organism production.
  • the sterile recipient rat may be a recipient for multiple rounds of separate or distinct genetically modified SSCs.
  • the sterile rat may be a recipient of fifteen or more different genetically modified SSCs and breed with twenty or more wild type females to produce fifteen or more separately genetically modified organisms.
  • the sterile male may be treated to eliminate the first round of genetically modified SSCs and become a recipient of another round of fifteen or more separately or distinct genetically modified SSCs, breed with twenty or more wild type females to produce fifteen or more separate genetically modified organisms.
  • the sterile male may be a recipient of mixed populations of fifteen or more genetically modified SSCs and breed twenty or more wild type females two times or more, three times or more, four times or more, or five times or more.
  • increasing the number distinct or separate pools or lines of genetically modified SSCs, which may be used to generate a genetically modified organism does not increase the amount of effort, time, and resources used, as well as does not decrease the efficiency of genetically modified organism production.
  • Increasing the number of genetically modified SSCs does not require the effort and resources of other stem cell systems such as embryonic stem (ES) cells or embryos.
  • ES embryonic stem
  • Increasing the amount of genetically modified ES cells for genetically modified organism production requires an increase in the number of technical steps such as blastocyst injections, as well as the number of oviduct transfer surgeries.
  • the SSC system may produce fifteen or more separate genetically modified stem cell populations for genetically modified organism production in a single step, while in order to produce fifteen or more separately genetically modified ES cells, fifteen or more separate steps must be performed on all levels of the procedure, which include but are not limited to blastocyst injection, oviduct transfer, zygote production, preparation of DNA, DNA microinjection, reimplantation of injected zygotes or breeding chimeric progeny.
  • genetic modification of SSCs using Transposons relates to generating genetically modified organisms without requiring the steps required in producing genetically modified organisms from alternative stem cells, including but not limited to embryonic stem cells, embryo's, induced pluripotent stem (iPS) cells, somatic stem cells. Genetic modification in alternative stem cells includes but is not limited to zygote production, preparation of DNA, DNA microinjection, reimplantation of injected zygotes or breeding chimeric progeny.
  • the stem cells of the present invention comprise one or more transposons, one or more inverted tandem repeats (ITRs) (shown in table 3) of a transposon of variants thereof.
  • ITRs inverted tandem repeats
  • the present invention comprise one or more transposons, one or more inverted tandem repeats (ITRs) of a transposon wherein the variant sequence inverted tandem repeats are at least 70 % homologous to known ITRs and known transposon elements (shown in table 3).
  • ITRs inverted tandem repeats
  • the present invention comprise one or more transposons, one or more inverted tandem repeats (ITRs) of a transposon wherein the variant sequence inverted tandem repeats are at least 75 % homologous to known ITRs and known transposon elements (shown in table 3).
  • ITRs inverted tandem repeats
  • the present invention comprise one or more transposons, one or more inverted tandem repeats (ITRs) of a transposon wherein the variant sequence inverted tandem repeats are at least 80 % homologous to known ITRs and known transposon elements (shown in table 3).
  • ITRs inverted tandem repeats
  • the present invention comprise one or more transposons, one or more inverted tandem repeats (ITRs) of a transposon wherein the variant sequence inverted tandem repeats are at least 85 % homologous to known ITRs and known transposon elements (shown in table 3).
  • ITRs inverted tandem repeats
  • the present invention comprise one or more transposons, one or more inverted tandem repeats (ITRs) of a transposon wherein the variant sequence inverted tandem repeats are at least 90 % homologous to known ITRs and known transposon elements (shown in table 3).
  • ITRs inverted tandem repeats
  • the present invention comprise one or more transposons, one or more inverted tandem repeats (ITRs) of a transposon wherein the variant sequence inverted tandem repeats are at least 95 % homologous to known ITRs and known transposon elements (shown in table 3).
  • ITRs inverted tandem repeats
  • the present invention comprise one or more transposons, one or more inverted tandem repeats (ITRs) of a transposon wherein the variant sequence inverted tandem repeats are at least 96 % homologous to known ITRs and known transposon elements (shown in table 3).
  • ITRs inverted tandem repeats
  • the present invention comprise one or more transposons, one or more inverted tandem repeats (ITRs) of a transposon wherein the variant sequence inverted tandem repeats are at least 97 % homologous to known ITRs and known transposon elements (shown in table 3).
  • ITRs inverted tandem repeats
  • the present invention comprise one or more transposons, one or more inverted tandem repeats (ITRs) of a transposon wherein the variant sequence inverted tandem repeats are at least 98 % homologous to known ITRs and known transposon elements (shown in table 3).
  • ITRs inverted tandem repeats
  • the present invention comprise one or more transposons, one or more inverted tandem repeats (ITRs) of a transposon wherein the variant sequence inverted tandem repeats are at least 99 % homologous to known ITRs and known transposon elements (shown in table 3).
  • ITRs inverted tandem repeats
  • DMEM-high glucose + Sodium Bicarbonate Medium contains Dulbecco's Modified Eagle's Medium-high glucose (Sigma, D5648); 1.5g Sodium
  • Bicarbonate (Sigma, S5761), 1L sterile water which are filtered using a 0.2um filter unit and stored at 4C;
  • SSC Feeder Medium contains 225mL DMEM-high glucose + sodium bicarbonate; 25mL Heat Inactivated Fetal Bovine Serum: FBS (Tissue Culture Biologicals, 104), which are filtered using a 0.2um filter unit and stored at 4C; 0.1% Gelatin is generated by dissolving 1 g gelatin from Porcine Skin- Type A (Sigma, G1890) in 1L ultrapure water.
  • GDNF Recombinant GDNF
  • R&D Systems 512-GF-010
  • lOOug/mL lOOng/uL
  • BSA O.OOlg BSA-Calbiochem 126609 in lOmL Sigma D8537 lx PBS-sterile filtered
  • rR-GDNF is pipetted up and down to mix, but not vortexed.
  • rbH-FGF Fibroblast Growth Factor- Basic Human
  • rbH-FGF Recombinant Fibroblast Growth Factor- Basic Human
  • rbH-FGF Recombinant Fibroblast Growth Factor- Basic Human
  • rbH-FGF is supplied at 25ug, then reconstituted to 25ug/mL (25ng/uL) by adding lmL- lx PBS/ 0.1% BSA (O.OOlg BSA-Calbiochem 126609 in lOmL Sigma D8537 lx PBS-sterile filtered).
  • rbH-FGF is pipetted up and down to mix but not vortexed.
  • Dilute 2- Mercaptoethanol (Sigma M3148) is prepared by adding 4.7uL stock to 6mL DHF12 (Sigma D8437).
  • SG Medium Spermatogonial Culture Medium
  • rR-GDNF final concentration is 20 ng/ml
  • rbH-FGF 20 ng/ml 2-mercaptoethanol 100 ⁇
  • L-glutamine 4mM final concentration media's overall final concentration glutamine in 6mM
  • B27 Supplement minus vitamin A lx.
  • feeder cell lines coat dish with 0.1% gelatin and incubate at 37C -1 hour and wash lx with lx PBS.
  • Thaw IRR mouse embryonic fibroblasts (Global Stem) by placing frozen vial at 37C immediately after removing from liquid nitrogen until ice crystals disappear. Transfer contents into 9mL of 37C DR4 Feeder medium. Spin at 1000 rpm for 5 minutes, discard supernatant, and resuspend in SSC Feeder medium. Plate on gelatin coated surface in SSC Feeder medium for 16-48 hr.
  • Spermatogonia are easily distinguished during counting as the predominant population of smaller, round cells with smooth surfaces, as compared to occasionally observed, larger and often irregular shaped irradiated MEFs. Typically, 2-4 x 106spermatogonia can be harvested from a single, 10 cm dish ( Figure 3).
  • Freezing Medium (SG Freezing Medium) by adding DMSO (Sigma, D2650) at a concentration of 10% (v/v) in SG Medium. Filter-sterilize and cool the prepared freezing medium on ice prior to use. Prepare a 5100 Cryo 1°C Freezing
  • plasmid DNA such as selection fluorescent markers and homologous recombination vectors using Lipofectamine 2000 requires a number of reagents and methods.
  • Transposon constructs and plasmids used for transfection into SSCs may contain a promoter, multiple cloning site (MCS), drug resistance gene, or fluorescent marker gene (Figure 4).
  • Reagents include undifferentiated spermatogonia, SG Medium (pre-warmed),
  • Opti-MEM (cat. no. 31985-062; Invitrogen, Inc.), Lipofectamine 2000 (cat. no. 11668-019; Invitrogen), highly purified transposon construct and plasmid DNA containing selection markers or homologous recombination vectors in TE buffer at 1-2 ⁇ g/ ⁇ l, Gelatin-coated plates, and plates with fresh MEF feeder layers.
  • transposon construct and plasmid DNA in Opti-MEM as follows: In a 1.5 ml microfuge tube, dilute 1 ⁇ g DNA/100 ⁇ - ⁇ . In a separate 1.5 ml micro fuge tube, dilute 2 2000/100 ⁇ - ⁇ . Incubate tubes separately for 5-10 min. Combine contents of each tube together and incubate at room temperature for at least 20 minutes (but no longer than 6 hr) to obtain the Transfection Mixture. During this incubation step, proceed to harvesting cells for transfection. Harvest cultures of proliferating spermatogonia grown on MEFs.
  • spermatogonia maintained on MEF feeder layers
  • 40 ⁇ volume of the Transfection Mixture is used to transfect -2x105 spermatogonia in a total transfection volume of 200 ⁇ .
  • mix the transfection by gently pipetting cells up and down two times midway through the incubation period.
  • wash spermatogonia by first suspending the transfection suspension to 20 times its volume using fresh culture medium (i.e. 4 ml medium/200 ⁇ transfection reaction), and then pellet the cells for 5 min at 400 x g. Discard the supernatant fluid, and wash the pellet(s) two additional times using fresh culture medium at an equivalent of the 20x volume/wash. After the third wash, suspend the cell pellet in fresh medium and then plate transfected cells onto fresh MEF feeder layers for selection of genetically modified spermatogonial lines.
  • Clonal selection for genetically modified SSCs is done by using the following reagents: established, proliferating line of spermatogonial stem cells, geneticin selective antibiotic: G418 (cat no 11811-031, Invitrogen Inc.), DNA Constructs expressing a resistance gene that selects for survival in G418 containing medium (i.e. neomycin phosphotransferase gene), fibroblast feeder cell line expressing a resistance gene that selects for survival in G418 containing medium.
  • G418 proliferating line of spermatogonial stem cells
  • G418 cat no 11811-031, Invitrogen Inc.
  • DNA Constructs expressing a resistance gene that selects for survival in G418 containing medium i.e. neomycin phosphotransferase gene
  • fibroblast feeder cell line expressing a resistance gene that selects for survival in G418 containing medium.
  • transposon construct plasmid DNA or a selectable marker
  • the treated spermatogonia are plated directly into SG Medium at an equivalent of -3x105 spermatogonia/well (9.5 cm2) in a 6-well plate containing freshly prepared MEFs.
  • the transfected (or virally transduced) spermatogonia are then allowed to proliferate in cell number for -18 days after transfection with plasmid DNA.
  • the culture medium is replenished every two days; and, fresh MEFs are spiked onto cultures of the transfected spermatogonia after -10 days.
  • cultures are harvested and then passaged onto freshly prepared MEFs in SG medium and maintained for an additional 2-3 days before initiating clonal selection in SG medium containing -75 ⁇ g/ml G418 (Invitrogen, Inc.). After initiating selection, cultures are fed fresh SG medium containing G418 every two days during an 8-10 day selection period. Thereafter, cells are fed every two days using SG medium alone to expand clonally enriched lines of spermatogonia that can be used to produce transgenic organisms, as described in the following sections.
  • genetic modification of SSCs produced using transposon relates to generating multiple transgenic insertions in the same SSC or SSC line, which relates to generating genetically modified organisms with multiple transgenes.
  • the embodiment relates to transfection with multiple transposon constructs integrating multiple DNA sequences and locations within the same SSC or SSC line in a single transfection.
  • the embodiment of the invention relates to clonal selection and screening for multiple transgene insertions in single SSCs or SSC lines.
  • genetic modification of SSCs produced using transposons relates to generating multiple transgenic insertions in the same SSC or SSC line in multiple and consecutive experiments or trans fections.
  • the embodiment of the invention relates to clonal selection and screening for multiple transgenic insertions in single SSCs or SSC lines.
  • the embodiment of the invention relates to generating genetically modified organisms or a colony of genetically modified organisms with multiple transgenic insertions in single SSCs or SSC lines.
  • genetic modification of SSCs produced using transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions.
  • SSCs or SSC lines are separated by including, but not limited to, different media, colonies or transfection dishes. The separated SSCs or SSC lines undergo one or more experiments or transfections. The separate SSCs or SSC lines are then brought together for production of multiple genetically modified organisms in a single injection into a recipient male followed by a single breeding step.
  • genetic modification of SSCs produced using Transposons relates to generating multiple transgenic insertions in the same SSC or SSC line, which relates to generating genetically modified organisms with multiple transgenic insertions.
  • the embodiment relates to transfection with multiple transposon constructs targeting multiple DNA sequences and locations within the same SSC or SSC line in a single transfection.
  • the embodiment of the invention relates to clonal selection and screening for multiple transgenic insertions in single SSCs or SSC lines.
  • genetic modification of SSCs produced using Transposons relates to generating multiple transgenic insertions in the same SSC or SSC line in multiple and consecutive experiments or transfections.
  • the embodiment of the invention relates to clonal selection and screening for multiple transgenic insertions in single SSCs or SSC lines.
  • the embodiment of the invention relates to generating genetically modified organisms or a colony of genetically modified organisms with multiple transgenic insertions in single SSCs or SSC lines.
  • genetic modification of SSCs produced using Transposons relates to generating multiple transgenic insertions in separate SSCs or SSC lines followed by pooling or combining separate SSCs or SSC lines and injecting into a single recipient male, which relates to generating multiple genetically modified organisms containing one or more transgenic insertions.
  • SSCs or SSC lines are separated by including, but not limited to, different media, colonies or transfection dishes. The separated SSCs or SSC lines undergo one or more experiments or transfections. The separate SSCs or SSC lines are then brought together for production of multiple genetically modified organisms in a single injection into a recipient male followed by a single breeding step.
  • the SSCs are derived from an organism.
  • the SSCs may be collected by spermatocyte harvest, the SSCs may be selected and purified using laminin selection, and propagated, cryopreserved and validated by cell surface marker identification.
  • the SSCs are derived from a tissue sample.
  • the SSCs may be collected by spermatocyte harvest, the SSCs may be selected and purified using laminin selection, and propagated, cryopreserved and validated by cell surface marker identification.
  • the SSCs are derived from cells.
  • the SSCs may be collected by spermatocyte harvest, the SSCs may be selected and purified using laminin selection, and propagated, cryopreserved and validated by cell surface marker identification.
  • the SSCs used for production of organisms are derived from an organism or tissue with a well-characterized disease state.
  • the SSCs are used for the production of organisms, which may be further genetically modified.
  • the SSCs used for production of organisms are derived from a well-characterized disease state wherein the disease state is a metabolic disorder, which is not limited to diabetes.
  • the SSCs are used for the production of organisms which may be further genetically modified.
  • the SSCs used for production of organisms are derived from a well-characterized disease state wherein the disease state is an oncology disorder, which is not limited to prostate cancer.
  • the SSCs are used for the production of organisms which may be further genetically modified.
  • the SSCs used for production of organisms are derived from a well-characterized disease state wherein the disease state is an autoimmune disorder, which is not limited to arthritis.
  • the SSCs are used for the production of organisms which may be further genetically modified.
  • the SSCs used for production of organisms are derived from a well-characterized disease state wherein the disease state is a cardiovascular disorder, which is not limited to atherosclerosis.
  • the SSCs are used for the production of organisms which may be further genetically modified.
  • the SSCs used for production of organisms are derived from a well-characterized disease state wherein the disease state is a neurodegenerative disorder, which is not limited to Alzheimer's disease.
  • the SSCs are used for the production of organisms which may be further genetically modified.
  • the SSCs used for production of organisms are derived from a well-characterized disease state wherein the disease state is a behavioral disorder, which is not limited to Schizophrenia.
  • the SSCs are used for the production of organisms which may be further genetically modified.
  • the SSCs used for production of organisms are derived from induced pluripotent stem (iPS) cells from a well, characterized disease state wherein the disease state is a metabolic disorder, which is not limited to diabetes.
  • the SSCs are used for the production of organisms which may be further genetically modified.
  • the SSCs used for production of organisms are derived from induced pluripotent stem (iPS) cells from a well- characterized disease state wherein the disease state is an oncology disorder, which is not limited to prostate cancer.
  • iPS induced pluripotent stem
  • the SSCs are used for the production of organisms which may be further genetically modified.
  • the SSCs used for production of organisms are derived from induced pluripotent stem (iPS) cells from a well- characterized disease state wherein the disease state is an autoimmune disorder, which is not limited to arthritis.
  • the SSCs are used for the production of organisms which may be further genetically modified.
  • the SSCs used for production of organisms are derived from induced pluripotent stem (iPS) cells from a well- characterized disease state wherein the disease state is a cardiovascular disorder, which is not limited to atherosclerosis.
  • the SSCs are used for the production of organisms which may be further genetically modified.
  • the SSCs used for production of organisms are derived from induced pluripotent stem (iPS) cells from a well- characterized disease state wherein the disease state is a neurodegenerative disorder, which is not limited to Alzheimer's disease.
  • the SSCs are used for the production of organisms which may be further genetically modified.
  • the SSCs used for production of organisms are derived from induced pluripotent stem (iPS) cells from a well- characterized disease state wherein the disease state is a behavioral disorder, which is not limited to
  • the SSCs are used for the production of organisms which may be further genetically modified.
  • the SSCs used for production of organisms are derived from induced pluripotent stem (iPS) cells from a well- characterized genetic background.
  • iPS induced pluripotent stem
  • the SSCs used for production of organisms are derived from induced pluripotent stem (iPS) cells from a well- characterized genetic background wherein the genetic background is associated with different established strains of organism.
  • iPS induced pluripotent stem
  • the SSCs used for production of organisms are derived from induced pluripotent stem (iPS) cells from a well- characterized genetic background wherein the genetic background is associated with known ethnic or regional genetic make-ups.
  • iPS induced pluripotent stem
  • SSCs containing transgenic insertions are generated to
  • SSCs containing transgenic insertions mutations are generated to produce genetically modified mammals.
  • SSCs containing transgenic insertions are generated to produce genetically modified rodents.
  • SSCs containing transgenic insertions are generated to produce genetically modified rats.
  • SSCs containing transgenic insertions comprised of transposons piggyBac and Sleeping Beauty do not produce genetically modified rats.
  • SSCs containing transgenic insertions are generated to produce genetically modified mice.
  • SSCs containing transgenic insertions are generated to produce genetically modified pigs
  • SSCs containing transgenic insertions are generated to produce genetically modified rabbits
  • SSCs containing transgenic insertions are generated to produce genetically modified guinea pigs.
  • SSCs containing transgenic insertions are generated to produce genetically modified dogs.
  • SSCs containing transgenic insertions are generated to produce genetically modified cats.
  • SSCs containing transgenic insertions are generated to produce genetically modified goats.
  • SSCs containing transgenic insertions are generated to produce genetically modified chickens.
  • SSCs containing transgenic insertions are generated to produce genetically modified non-human primates.
  • SSCs containing transgenic insertions are generated to produce genetically modified ferrets.
  • SSCs containing transgenic insertions are generated to produce genetically modified birds.
  • SSCs containing transgenic insertions are generated to produce genetically modified farm animals.
  • SSCs containing transgenic insertions are generated to produce genetically modified fish.
  • SSCs containing transgenic insertions are generated to produce genetically modified slamonoids.
  • SSCs containing transgenic insertions are generated to produce genetically modified carp.
  • SSCs containing transgenic insertions are generated to produce genetically modified tilapia.
  • SSCs containing transgenic insertions are generated to produce genetically modified tuna.
  • the invention provides kits that are used to produce
  • kits typically include one or more genetic engineering technology, such as Transposons.
  • the kit may also contain one or more sets of stem cells for genetic modification.
  • the stem cells may include, but is not limited to spermatogonial stem cells (SSCs), as well as media and conditions necessary for growing SSCs.
  • the kits may include exogenous sequences for genomic introduction, such as but not limited to reporter genes or selectable markers.
  • kits may include instructions for (i) introducing the Transposons into the stem cells (ii) identifying stem cells which have been genetically modified (iii) growing genetically modified stem cells in media or conditions necessary and to numbers required for stem cells to produce genetically modified organisms (iv) using the grown stem cells to produce a genetically modified organism (v) identifying which organisms or progeny harbor the genetic modification of interest.
  • the invention provides a kit which includes a mixed
  • the mixed population of genetically modified SSCs may be provided in suitable quantities for direct injection into a sterile male recipient for the production of multiple genetically modified organisms in a single step.
  • the mixed population of separate or distinct genetically modified SSCs may consist of at least two genetically modified SSCs, at least two genetically modified SSCs, at least three genetically modified SSCs, at least four genetically modified SSCs, at least five genetically modified SSCs, at least six genetically modified SSCs, at least seven genetically modified SSCs, at least eight genetically modified SSCs, at least nine genetically modified SSCs, at least ten genetically modified SSCs, at least twenty genetically modified SSCs, at least thirty genetically modified SSCs, at least forty genetically modified SSCs, at least fifty genetically modified SSCs, at least one hundred genetically modified SSCs, at least one thousand genetically modified SSCs, at least ten thousand genetically modified SSCs, at least thirty thousand
  • the invention provides a kit which includes one or more sets of SSCs for genetic modification.
  • the sets of SSCs may be derived from well-characterized organisms with different disease states.
  • the SSCs may contain multiple transgene insertions, which may be derived from genetic modification or naturally or by any method.
  • the kit may include the media and conditions to grow disease state SSCs, as well as the sterile recipient male for the production of genetically modified organisms.
  • the invention provides a kit which includes the necessary tools for the derivation of SSC lines from an organism or tissue sample, as well as the necessary tools to genetically modify the derived SSC and produce a genetically modified organism from the derived SSCs.
  • the kit may include cell collection tools such as spermatocytes for harvest, and SSC selection tools such as laminin selection, and SSC propagation and cryopreservation tools as well as SSC validation tools which may include cell surface marker staining.
  • the kit may also include media and conditions for growing the SSCs, tools for genetic modification of the SSCs as well as sterile recipient males for production of genetically modified organisms from the SSCs.
  • the invention provides a kit which includes SSCs which have been generated from induced pluripotent stem (iPS) cells.
  • the iPS cells may be derived from well characterized different genetic backgrounds including disease states as well as regional, strain, ethnic genetic backgrounds.
  • the kit may also include media and conditions for growing the SSCs, tools for genetic modification of the SSCs as well as sterile recipient males for production of genetically modified organisms from the SSCs.
  • Germline transmission from genetically modified SSCs can be carried out by using the following reagents: Disposable Pasteur Pipettes (cat. no. 13-678-20C, Thermo Fisher Scientific Inc.), 30G Precision Glide Needles (cat. no. 305106, BD, Inc.), 1 ml Syringes (cat. no. 309602, BD Inc.), Busulfan (cat. no. 154906, MP Biomedicals), Dimethyl Sulfoxide (DMSO) (cat. no. 317275, Calbiochem), Trypan Blue (cat. no. T6146-25G, Sigma Inc.), Triadine Prep Solution, (10% povidone iodine solution, cat. no.
  • PBS Dulbecco's phosphate-buffered saline (PBS; cat. no.D8537, Sigma Inc.) 200 mg/L KC1 (w/v), 200 mg/L KH2P04 (w/v), 8 g/L NaCl (w/v), 1.15 g/L Na2HP04 (w/v)., Kimwipes (cat. no.
  • Reusable Warming Pad (cat. no. TPZ- 1215EA, Kent Scientific Corporation), 10 ml Syringes (cat. no. 309604, BD, Inc.), Acepromazine (cat. no. 038ZJ03, Vedco), Rompun (cat. no. LA33806A, Lloyd Laboratories) Ketaset (cat. no. 440761, Fort Dodge Animal Health), Buprenex Injectable (cat. no. 12496-0757-1, Reckitt Benckiser), Shaving Razors - Stainless Steel Surgical Prep Blades (cat. no. 74-0001, Personna), Suture Thread; Spool Suture (cat. no.
  • busulfan-treated wildtype Sprague Dawley organisms, or male-sterile DAZL- deficient, organisms at 24 days of age can be used as recipients for spermatogonial lines.
  • 14 - 16 days prior to the transplantation procedure which is performed at 24 days of age.
  • each organism is administered a single dose of busulfan (12.5 mg/kg, i.p. for wildtype organisms; 12.0 mg/kg for D ⁇ ZZ-deficient organisms), and then housed in a quiet, clean and well ventilated location within an approved animal facility.
  • a 4 mg/ml working stock of busulfan in 50% DMSO is prepared by first dissolving busulfan in 100% DMSO at 8 mg/ml, and then adding and equal volume of filter-sterilized, deionized water.
  • spermatogonia On the day of transplantation, genetically modified spermatogonia are harvested from culture and suspended in ice cold, culture medium (i.e. either SG medium or DHF12-FBS-2ME) at concentrations ranging from 4-6x105 spermatogonia/ 100 ⁇ .
  • culture medium i.e. either SG medium or DHF12-FBS-2ME
  • concentrations ranging from 4-6x105 spermatogonia/ 100 ⁇ .
  • the cellular suspension is transferred to a sterile microfuge tube and maintained on ice until the time of transplantation.
  • the cell suspension is supplemented with a 20%> volume of a filter-sterilized, 0.04%> trypan blue solution made fresh in PBS the same day.
  • the first busulfan-treated recipient male is anesthetized by intraperitoneal (i.p.) injection of a cocktail containing 100 mg/ml ketaset, 20 mg/ml rompun, and 10 mg/ml acepromazine at 0.1 ml/lOOg body weight to achieve a surgical plane of anesthesia (as demonstrated by the lack of a pedal reflex in the toe pinch test).
  • the recipient is layed on its back.
  • the abdominal skin is then opened just rostral to the pelvis, and the testis is exposed.
  • the efferent ductules leading into the rete testis are then accessed by blunt dissection using micro-dissection forceps.
  • the ductules are further dissected up to the base of their respective testis to yield visible access to the rete, which will be the site of injection.
  • the harvested spermatogonial suspension is mixed gently by pipetting up and down -5 times with a p200 tip and then -70-80 ⁇ of the suspension is loaded into a 100 ⁇ glass capillary injection needle (-50 ⁇ opening) using a flame pulled, transfer pipette (i.e. made from Pasteur pipettes) and rubber squeeze bulb.
  • the injection needle containing spermatogonia is manually inserted into the rete of the testis, and the cells are transferred into the testis by injection using a stationary 10 ml syringe (i.e.
  • each organism is administered a single dose of buprenorphine hydrochloride (25 ⁇ g/kg) (Buprenex Injectable, Reckitt Benckiser) as it starts to regain consciousness. An additional dose is given every 6-12 hr for the next 48 hr upon signs of discomfort or pain. Wound clips are removed at 12-14 days post- surgery. The recipients are then housed together for -60 days prior to initiating breeding studies.
  • buprenorphine hydrochloride 25 ⁇ g/kg
  • Reckitt Benckiser Borenex Injectable, Reckitt Benckiser
  • Recipient males transplanted with spermatogonial lines are paired with wild-type females of similar age at 60-70 days post-transplantation.
  • the first Fl progeny are born between 100 and 150 days post-transplantation and recipients can continue to sire litters for greater than 300 days post-transplantation due to the long-term spermatogenesis colony forming potential of laminin-binding spermatogonia.
  • Transgenic progeny from recipient-founders and wild-type females are identified by genomic PCR and/or Southern Blot analysis using probes specific to the mutation of interest.
  • SSCs Spermatogonial stem cells
  • SSCs spermatogonial stem cells
  • spermatogenesis spermatogonial stem cells
  • DAZL germ-line specific gene product
  • the genetically modified germ line recipient males are then bred with wild type females to produce offspring that harbor the genetic mutation (Production and Use of Rat Spermatogonial Stem Cell Lines (PCT/US2009/066275, WO/2010/065550).
  • Embryonic stem cells are a pluripotent cell derived from the inner mass of the blastocyst or early stage embryo. Genetically modified ESCs from a donor are microinjected into a recipient blastocyst. Recipient blastocysts containing genetically modified ES cells are implanted into pseudopregnant surrogate females. The progeny, some of which have a genetic modification to the germline can then be established, and lines homozygous for the genetic modification can be produced by interbreeding.
  • Induced pluripotent stem cells are artificially derived pluripotent cells from a less or non pluripotent cell, typically a somatic cell. There are multiple methods for which iPS cells can be "reprogrammed" to a pluripotent state from non
  • pluripotent cells including the expression of reprogramming factors.
  • Genetically modified iPS cells from a donor are microinjected into a recipient blastocyst.
  • Recipient blastocysts containing genetically modified ES cells are implanted into pseudopregnant surrogate females.
  • the progeny some ofwhich have a genetic modification to the germline can then be established, and lines homozygous for the genetic modification can be produced by interbreeding.
  • Somatic stem cells or adult stem cells are potent cells found in organs after
  • Somatic stem cells can be isolated from organs and tissues and have the potential to differentiate into many cell types of that organ and organism.
  • cord blood stem cells can be isolated from umbilical cord blood (CBEs) (McGuckin et al. (2008) Nature Protocols. 3, 6, 1046-1055. These cells are then expanded and used in the production of genetically modified organisms.
  • CBEs are known as "embryonic-like" due to the expression of similar markers as embryonic stem cells.
  • CBEs are a very small fraction of the cells present in umbilical cord blood. The CBE fraction is depleted of hematopoietic stem cells which stimulate hematopoietic commitment.
  • CBEs are plated at high concentrations (10 million cells per 1 ml) in TPOFLK medium which is supplemented with extracellular matrix (ECM) proteins.
  • ECM extracellular matrix
  • the ECM proteins are essential for cell survival and aggregate formation similar to embryoid bodies which promotes cell-cell interactions and secretion of growth factors.
  • Dynamic cell culture conditions are maintained based on cell phenotype: formation of floating aggregates, size and number of cell aggregates, cell adhesion and differentiation.
  • Genetically modified somatic stem cells from a donor are microinjected into a recipient blastocyst.
  • fresh or frozen cleavage stage embryos, produced from in vitro fertilization (IVF) can be cultured to blastocyst stage.
  • the inner cell masses are isolated to produce ES cell lines that are capable of undifferentiated proliferation in vitro.
  • Recipient blastocysts containing genetically modified somatic stem cells are implanted into pseudopregnant surrogate females.
  • the progeny some of which have a genetic modification to the germline can then be established, and lines homozygous for the genetic modification can be produced by interbreeding.
  • genetically modified somatic stem cells can be reprogrammed into iPS cells in order to produce genetically modified organisms.
  • An embryo is a multicellular diploid eukaryote in early stage of development.
  • Embryos can be genetically modified in vitro or in vivo. Embryos containing genetic mutations may be implanted into pseudopregnant surrogate females. The progeny which have a genetic modification to the germline can then be established, and lines homozygous for the genetic modification can be produced by interbreeding.
  • compositions comprising rat spermatogonial stem cell line of a predetermined genetic background.
  • the description provides stem cell lines wherein the predetermined genetic background is Sprague-Dawley or Fisher 344.
  • the description provides stem cell lines wherein the transplanted haplotype comprises an internal tandem repeat (ITR) from a transposon.
  • ITR internal tandem repeat
  • the description provides stem cell lines wherein the transplanted haplotype comprises at least one ITR from a transposon selected from the piggyBac ITR, Sleeping Beauty ITR, or a combination thereof.
  • the description provides stem cell lines wherein the stem cell line is in culture between about 158 and about 205 days.
  • the description provides stem cell lines wherein the stem cell line has a doubling time of between about 8 and about 9 days. [00301] In some embodiments, the description provides stem cell lines wherein the stem cell line expands no less than about 20,000 times as compared to the number of cells seeded in culture.
  • the description provides stem cell lines wherein the stem cell line is frozen at -196 degrees Celsius.
  • compositions comprising a stem cell line of claims 1 to 8 and a culture medium.
  • compositions further comprises
  • the culture medium comprises Dulbecco's Modified Eagle Medium (DMEM), Ham's F12 nutrient mixture, glial cell-derived neurotrophic factor (GDNF), Fibroblast Growth Factor-2 (FGF2), 2-mercaptoethanol, L-glutamine, and B27-minus vitamin A supplement solution, wherein said composition supports in vitro culturing of spermatogonial stem cells.
  • DMEM Dulbecco's Modified Eagle Medium
  • GDNF glial cell-derived neurotrophic factor
  • FGF2 Fibroblast Growth Factor-2
  • 2-mercaptoethanol 2-mercaptoethanol
  • L-glutamine and B27-minus vitamin A supplement solution
  • the description provides male rats of a
  • predetermined genetic background comprising a transplanted haplotype derived from a rat spermatogonial stem cell line, wherein said rat is sterile absent the presence of the transplanted haplotype.
  • the description provides rats wherein the male rat is
  • the description provides librarys of cells of a
  • spermatogonial stem cell line that is derived from rat testes, wherein said library contains a plurality of transposon-mediated gene knockout or knockin mutants.
  • compositions comprising
  • the description provides methods for culturing rat spermatogonial stem cells isolated from rat somatic testis cells and culturing the rat spermatogonial stem cells in the culture medium described above.
  • the description provides isolated rat spermatogonial stem cell lines of a predetermined genetic background for use in introducing a transgene or mutation into the genome of a male rat of a predetermined genetic background, wherein said rat is sterile absent the presence of the transplanted haplotype and wherein the haplotype comprises a transposon sequence or a fragment thereof.
  • the description provides isolated rat spermatogonial stem cell lines for the use described above wherein the rat stem cell line is genetically modified with a transposon prior to transplanting the rat spermatogonial stem cell line into the testes of a male rat.
  • the methods used in the present invention are comprised of a combination of spermatogonial stem cell (SSC) introduction methods, transposon based genetic modification methods, and generation of transposons mediated genetically modified organisms from SSCs.
  • SSC spermatogonial stem cell
  • transposon based genetic modification methods spermatogonial stem cell
  • transposons mediated genetically modified organisms from SSCs.
  • the invention may include but is not limited to the methods described below.
  • the transposon mediated mutation is produced in one introduction method.
  • SSCs SSCs. These SSCs can proliferate in cell culture and be genetically modified without affecting their ability to differentiate into other cell types including germ line cells. SSCs can be injected into the rete testis of a recipient animal. The progeny which have a genetic modification to the germ line can then be established, and lines homozygous for the genetic modification can be produced by interbreeding (figure 5).
  • genetic modification of rat spermatogonial stem cells is not carried out by transposons piggyBac of Sleeping Beauty.
  • transgenic spermatogonial stem cells are produced transposons to generate genetically modified organisms.
  • Preparing SSCs for transposon mediated modification involves preparing feeder cell lines, and sub- culturing SSC lines.
  • Preparing feeder cells may be carried out by thawing embryonic fibroblasts (EF), and placing on gelatin coated surface in SSC feeder medium.
  • Sub-culturing SSCs may be carried out by seeding SSCs on EF medium. A 1 : 1 to 1 :2 split passage is required before expanding into larger SSC numbers.
  • spermatogonia Once established after the first several passages on EFs cultures of spermatogonia are passaged at ⁇ 1 :3 dilutions onto a fresh monolayer of EFs. For passaging, cultures are first harvested by gently pipetting them free from the EFs. After harvesting, the "clusters" of spermatogonia are dissociated by gentle trituration. Spermatogonia are easily distinguished during counting as the predominant population of smaller, round cells with smooth surfaces, as compared to occasionally observed, larger and often irregular shaped irradiated EFs.
  • the invention pertains to transposon mediated transgenesis generated in
  • SSCs spermatogonial stem cells
  • Transposon mediated transgenic SSCs are used to produce a genetically modified organism.
  • Transposon vectors may include transgenic vectors which deliver transgenes (figure 4)
  • transposon mutagenesis technology is expressed in SSCs generating transgenesis.
  • the transposon mutagenesis technology may be introduced into SSCs via transfection using lipofetamine.
  • a transfection mixture may be prepared by mixing transfectamine with the transposon transgenesis technology. After harvesting undifferentiated SSCs, add transfection mixture to the cell suspension, incubate, wash and plate the SSCs onto fresh EF feeder layers.
  • the stem cells of the present invention comprise one or more transposons, one or more inverted tandem repeats (ITRs) (shown in table 3) of a transposon of variants thereof.
  • ITRs inverted tandem repeats
  • the present invention comprise one or more transposons, one or more inverted tandem repeats (ITRs) of a transposon wherein the variant sequence inverted tandem repeats are at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% homologous to known ITRs and and known transposon elements (shown in table 3).
  • ITRs inverted tandem repeats
  • clonal selection of transposon modified SSCs may be carried out by first plating treated spermatogonia.
  • the genetically modified SSCs are allowed to proliferate in cell number replenishing the medium with fresh EFs.
  • Selection for genetically modified SSCs may be carried out in several methods. Selection using a reporter gene or selectable or cell sorting and mutation screening.
  • the invention pertains to transposon mediated mutations generated in
  • SSCs spermatogonial stem cells
  • transposon mediated genetically modified SSCs are provided.
  • the method for producing such organisms involves germline transmission of the genetically modified SSCs. Wild type and genetically sterile or DAZL deficient organisms are prepared for transplantation of transposon mediated genetically modified SSCs into seminiferous tubules. A cellular suspension of genetically modified SSCs is transferred to a sterile microfuge. Genetically sterile recipients are layed on back. The abdominal skin is then opened just rostral to the pelvis, and the testis exposed. The efferent ductules leading into the rete testis are then accessed by blunt dissection using micro-dissection forceps.
  • the ductules are further dissected up to the base of their respective testis to yield visible access to the rete, which will be the site of injection.
  • the injection needle containing transposon mediated genetically modified SSCs are manually inserted into the rete of the testis, and the cells are transferred into the testis by injection.
  • the injected testis is then carefully placed back into the abdominal cavity and the same procedure can be performed on the contra-lateral testis to achieve more optimal breeding.
  • the abdominal wall (sutured) and skin (wound clips) are surgically closed. Recipient males transplanted with transposon mediated genetically modified SSCs are paired with wild-type females to produce transposon mediated genetically modified organisms.
  • Transposons can be used to genetically modify minipig spermatogonial stem cells
  • SSCs SSCs
  • Figure 2 A schematic of wild type SSCs in colony are shown in Figure 2.
  • a piggybac (PB) vector was constructed which contained the human rasH2 gene under a promoter for the generation of transgenic minipigs ( Figure 7).
  • the PB vector will be co-transfected with a plasmid expressing PB transposase into minipig spermatogonial stem cells (SSCs) (schematic figure 6). Based on the selectable marker SSC colonies were screened for PB transgenesis.
  • SSCs minipig spermatogonial stem cells
  • SSCs minipig spermatogonial stem cells
  • SSCs minipig spermatogonial stem cells
  • PB transposon mediated rasH2 transgenic SSCs will be split and propagated to adequate amounts for implantation into sterile or DAZL deficient recipient males (schematic figure 3).
  • the proper surgery and injection of genetically modified SSCs will be carried out into the minipig seminiferous tubules.
  • the genetically modified SSCs will be allowed to mature within the recipient male.
  • PB vector used to create knockout mutations For review, we re-introduce the PB "mutagen," which we call GTv3, that will be used to create gene trap mutations (Figure 8). GTv3 contains a neo expression module to enable selection of piggyBac-transduced cells for G418 resistance. In addition to the tdTomato gene trap, two independent recombinase sites (FRT and FLEx) are now included to mediate sequential irreversible inversions to enable the production of conditional knockout mutations.
  • FRT and FLEx two independent recombinase sites
  • the probe binds to newly synthesized PCR products during the annealing step, and during the extension step the elongating DNA strand releases the probe from the neo hybridization primer which now can detected by the RealPlex machine.
  • the level of fluorescence is directly related to the level of neo specific product amplified at each cycle. 20ng of each DNA sample (standards and unknowns) were used in the qPCR reactions (all samples were tested in triplicate). The fluorescence levels from the released Taqman probes are measured for Ct (cycle threshold) values.

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Abstract

L'invention concerne des méthodes de transgenèse reposant sur des transposons, dans des cellules souches spermatogoniales (SSC) de plusieurs espèces de mammifères.
PCT/US2012/038461 2011-05-17 2012-05-17 Méthodes pour effectuer une transgenèse d'organismes et de cellules souches spermatogoniales (ssc) à l'aide de vecteurs de transposons WO2012158982A2 (fr)

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Publication number Priority date Publication date Assignee Title
CN105238818A (zh) * 2015-09-29 2016-01-13 潘雨堃 一种在体内精原干细胞基因组中引入随机插入突变的方法
CN108378019A (zh) * 2018-03-13 2018-08-10 洛阳轩智生物科技有限公司 一种人精原干细胞的冻存液
CN111850037A (zh) * 2019-04-30 2020-10-30 潘雨堃 一种piggyBac转座子高通量遗传筛选方法

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US20060288438A1 (en) * 1999-07-21 2006-12-21 Yoshihiro Ozeki Novel miniature inverted-repeat transposable elements (MITEs)-like element and transcriptional activation element
US20080279838A1 (en) * 2005-04-08 2008-11-13 Csaba Miskey Reconstructed Human Mariner Transposon Capable of Stable Gene Transfer Into Chromosomes in Vertebrates
WO2010065550A2 (fr) * 2008-12-01 2010-06-10 Board Of Regents University Of Texas System Production et utilisation de lignées de cellules souches spermatogoniales de rat
US20100287628A1 (en) * 2009-04-23 2010-11-11 Ostertag Eric M Genetically Modified Rat Models for Cancer

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US20060288438A1 (en) * 1999-07-21 2006-12-21 Yoshihiro Ozeki Novel miniature inverted-repeat transposable elements (MITEs)-like element and transcriptional activation element
US20080279838A1 (en) * 2005-04-08 2008-11-13 Csaba Miskey Reconstructed Human Mariner Transposon Capable of Stable Gene Transfer Into Chromosomes in Vertebrates
WO2010065550A2 (fr) * 2008-12-01 2010-06-10 Board Of Regents University Of Texas System Production et utilisation de lignées de cellules souches spermatogoniales de rat
US20100287628A1 (en) * 2009-04-23 2010-11-11 Ostertag Eric M Genetically Modified Rat Models for Cancer

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105238818A (zh) * 2015-09-29 2016-01-13 潘雨堃 一种在体内精原干细胞基因组中引入随机插入突变的方法
CN108378019A (zh) * 2018-03-13 2018-08-10 洛阳轩智生物科技有限公司 一种人精原干细胞的冻存液
CN111850037A (zh) * 2019-04-30 2020-10-30 潘雨堃 一种piggyBac转座子高通量遗传筛选方法

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