WO2012158564A1 - Compositions et procédés permettant d'augmenter la fréquence de la recombinaison homologue - Google Patents

Compositions et procédés permettant d'augmenter la fréquence de la recombinaison homologue Download PDF

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WO2012158564A1
WO2012158564A1 PCT/US2012/037650 US2012037650W WO2012158564A1 WO 2012158564 A1 WO2012158564 A1 WO 2012158564A1 US 2012037650 W US2012037650 W US 2012037650W WO 2012158564 A1 WO2012158564 A1 WO 2012158564A1
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zscan4
cells
cell
nucleic acid
seq
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Minoru S.H. Ko
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The United States Of America As Represented By The Secretary, Department Of Health & Human Services
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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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors

Definitions

  • compositions and methods for enhancing or prolonging the pluripotency of a stem cell and the use of such pluripotent stem cells.
  • This disclosure further concerns compositions and methods for increasing the frequency of homologous recombination in mammalian cells.
  • ES cells are prototypical pluripotent cells, which are derived from the inner cell mass of blastocysts (Martin, Proc Natl Acad Sci USA 78:7634-7638, 1981; Evans and Kaufman, Nature 292: 154-156, 1981). ES cells have an unusual capacity of
  • ZFN zinc finger nuclease
  • Zscan4 Zinc finger and scan domain-containing protein 4
  • 2-cell stage embryos and ES cells Falco et al, Dev
  • Disclosed herein is the finding that increasing the frequency of Zscan4 activation in mouse ES cells not only enhances, but maintains their developmental potency in long-term cell culture.
  • expression of Zscan4 enhances homologous recombination in mammalian cells.
  • nucleic acid molecules including vectors, encoding a Zscan4- ERT2 fusion protein.
  • Recombinant Zscan4-ERT2 fusion proteins are also provided.
  • Compositions and cells (such as ES cell or iPS cells) comprising the Zscan4-ERT2 nucleic acid molecules and fusion proteins are also provided herein.
  • nucleic acid molecules including vectors, encoding a Zscan4 protein with a C-terminal truncation of at least one zinc finger domain, referred herein to as Zscan4-AC.
  • Recombinant Zscan4-AC proteins are also provided.
  • Compositions and cells (such as ES cell or iPS cells) comprising the Zscan4-AC nucleic acid molecules and proteins are also provided herein.
  • the methods include contacting the stem cell or stem cell population with a Zscan4-ERT2 nucleic acid molecule, fusion protein or composition as disclosed herein. In other embodiments, the methods include contacting the stem cell or stem cell population with a Zscan4-AC nucleic acid molecule, protein or composition as disclosed herein.
  • the method includes contacting the cell with a Zscan4-ERT2 nucleic acid molecule, vector or fusion protein, or composition thereof, as disclosed herein.
  • the disclosed method can be carried out in vitro or in vivo.
  • FIG. 1 depicts that the constitutive expression of a Zscan4c-ERT2 fusion protein increases the number of Zscan4 + ES cells.
  • FIG. 1A is a schematic of the structure of a Zscan4c- ERT2 fusion protein.
  • Zscan4c contains one SCAN domain and four C2H2 zinc finger domains.
  • FIG. IB are fluorescence microscopy images of MC1-ZE3 cells, in which a Zscan4 promoter drives the expression of Emerald marker (left), MC1-ZE3-ZERT2 clone #15 cells, in which the Zscan4c-ERT2 fusion protein is constitutively expressed, cultured in the absence of Tmx
  • FIG. 1C is a graph showing flow-cytometry analysis of MC1-ZE3 ES cells (left, control) and MC1-ZE3-ZERT2 #15 ES cells (right) in the absence or presence of 1 ⁇ Tmx.
  • FIG. 1C is a graph showing flow-cytometry analysis of MC1-ZE3 ES cells (left, control) and MC1-ZE3-ZERT2 #15 ES cells (right) in the absence or presence of 1 ⁇ Tmx.
  • ID is a graph showing the results of quantitative RT-PCR analysis of endogenous Zscan4 expression measured by using PCR primer pairs specific for 3' -UTR of Zscan4 in MC1-ZE3 ES cells (left, control) and MC1-ZE3-ZERT2 #15 ES cells (right) in the absence or presence of 1 ⁇ Tmx.
  • IE is a series of images of V6.5 parental ES cells (passage number 14), V6.5 ZERT2 #2 (p.20), V6.5 ZERT2 #7 (p.21), V6.5 ZERT2 #10 (p.20), V6.5 ZERT2 #18 (p.22) ES cell colonies after whole- mount RNA in situ hybridization of a Zscan4 full-length probe, which detects both endogenous and exogenous Zscan4 RNAs (upper panel) or a Zscan4 3' -UTR probe, which detects only endogenous Zscan4 RNAs (lower panel).
  • IF is a schematic showing comparisons of global expression profiles between V6.5 ZERT2 #18 ES cells and Em + ES cells (upper panel), and between Tmx " and Tmx + conditions of V6.5 ZERT2 #18 ES cells (lower panel).
  • Zscan4-related genes Zscan4c, BC061212, Tmeme92, and Tcstvl/3) are already upregulated in the V6.5 ZERT2 #18 ES cells even without Tmx.
  • FIG. 2 depicts Zscan4 lacking the C-terminus increases the number of Zscan4 + cells.
  • FIG. 2A is a schematic showing the structure of Zscan4c, Zscan4c-ERT2, Zscan4c-AC and Zscan4c-AN proteins.
  • Zscan4c-AC was made by deleting four Zinc finger domains at the C- terminus of Zscan4c protein.
  • Zscan4c-AN was made by deleting the SCAN domain at the
  • FIGS. 2B-2G are fluorescence microscopic images of ZDC- MC1-ZE16 #3, #4, #20 for Zscan4c-AC and ZDN-MC1-ZE16 #5, #8, #15 for Zscan4c-AN.
  • the results demonstrate that the expression of Zscan4c-AC increases the number of Zscan4 + cells, whereas the expression of Zscan4c-AN does not change the number of Zscan4 + cells.
  • FIG. 3 depicts that the constitutive expression of a Zscan4c-ERT2 fusion protein increases and prolongs developmental potency of ES cells.
  • FIG. 3A shows representative coat colors of chimeric mice generated by injecting various ES cells into blastocysts. The higher chimerism represents the higher contribution of injected ES cells to mice, indicating the higher developmental potency of ES cells.
  • FIG. 3B is a graph showing the percent distribution of chimerism levels among "n" number of mice born from various ES cell lines.
  • FIG. 4 depicts that tetraploid (4N) complementation assays confirm the higher potency of ES cells expressing a Zscan4c-ERT2 fusion protein.
  • FIG. 4A is a table showing development of 4N blastocysts injected with multiple ES cells (10-15 ES cells): V6.5 parental ES cells (passage 18), V6.5 ZERT2 #7 (passage 22), V6.5 ZERT2 #10 (passage 22), V6.5 ZERT2 #18 (passage 19), and freshly isolated TA1 ES cells (passage 3).
  • FIG. 18 is a table showing development of 4N blastocysts injected with multiple ES cells (10-15 ES cells): V6.5 parental ES cells (passage 18), V6.5 ZERT2 #7 (passage 22), V6.5 ZERT2 #10 (passage 22), V6.5 ZERT2 #18 (passage 19), and freshly isolated TA1 ES cells (passage 3).
  • FIG. 4B is a table showing development of 4N blastocysts injected with single ES cells: V6.5 parental ES cells (passage 18), V6.5 ZERT2 #2 (passage 21), V6.5 ZERT2 #18, and freshly isolated TA1 ES cells (passage 4).
  • FIG. 4C is an image of the embryos examined: only properly developed embryos were counted (the group on the right).
  • FIG. 4D is a pair of images of two live embryos derived from single V6.5 ZERT2 #18 ES cells shown in FIG. 4A.
  • FIG. 4E shows a proposed model of ES cell potency.
  • FIGS. 5A-5C depict tables providing lists of genes upregulated in MC1-ZE7 Em + cells compared to Em " cells.
  • FIG. 6 depicts the generation and characterization of V6.5 ZERT2 ES cell clones.
  • FIG. 6A is a graph showing results of qRT-PCR analysis of Zscan4 expression levels by a primer pair detecting RNA from both endogenous Zscan4 and exogenous Zscan4 (transcripts from a pCGA-Zscan4-ERT2).
  • the primer sequences are 5'-AGTCTGACTGATGAGTGCTTGAAGCC- 3' (SEQ ID NO: 15) and 5 ' -GGCCTTGTTGCAGATTGCTGTTG-3 ' (SEQ ID NO: 16). Data were normalized by the expression of H2A, using primers 5'- TTGCAGCTTGCTATACGTGGAGATG-3 ' (SEQ ID NO: 17) and 5'-
  • FIG. 6B is a graph of growth curves of V6.5 ZERT2 #18 ES cells in the presence (Tmx+) or absence of Tamoxifen (Tmx-).
  • Tmx+ the presence of Tamoxifen
  • Tmx- Tamoxifen
  • FIG. 6C is a series of images showing morphologies of cells in each culture condition.
  • FIG. 7A depicts a scatter plot showing genes expressed differentially between V6.5 ZERT2 #18 ES cells and control V6.5 #2 ES cells.
  • FIG. 7B depicts a scatter plot showing genes expressed differentially between V6.5 ZERT2 #18 ES cells cultured for 2 days in the presence of Tmx and control V6.5 #2 ES cells.
  • FIG. 8 is a table listing the top 50 genes upregulated in V6.5 ZERT2 #18 ES cells compared to V6.5 #2 ES cells.
  • FIG. 9 is a table listing the top 50 genes upregulated in V6.5 ZERT2 #18 ES cells cultured in the presence of Tmx for 2 days compared to V6.5 #2 ES cells.
  • FIG. 10 depicts the derivation of new Fl (C57BL/6J X 129S6/SvEvTac) hybrid ES cell lines.
  • FIG. 10A is a table showing blastocysts obtained by mating C57BL/6J females with 129S6/SvEvTac males. Blastocysts were cultured in vitro on the mouse embryo fibroblasts (MEFs) feeders in 15% KSR medium (Invitrogen) supplemented with 50 nM PD98059 (MEK1 inhibitor). *Outgrowths showed undifferentiated (U), differentiated (D), and mixed (U/D) cellular phenotypes.
  • FIG. 10B is table providing a summary of ES derivation results.
  • FIG. 11 depicts the results of testing developmental potency of newly derived Fl hybrid ES cell lines by tetraploid complementation assays.
  • FIG. 11A is a table of six ES cell lines that showed undifferentiated cellular phenotypes when injected into tetraploid (4N) mouse blastocysts. Success rates of obtaining live embryos at E13.5 varied among ES cell lines, ranging from 15% to 60%. Clone #10 was selected for its highest success rate (named TA1 ES cell line) and was used for subsequent studies.
  • FIG. 11B is a series of representative images of 4N placentas and El 3.5 embryos derived from the TA1 ES cell line. Normal appearance of female and male gonads dissected from these embryos indicates their germline competence.
  • FIG. 12A is a schematic presentation of random integration events. Because a drug selectable marker is under the control of a strong and constitutive promoter (CAG), after the drug selection, cells with a vector integrated into any random genome locations can proliferate and form colonies.
  • FIG. 12B is a schematic presentation of homologous recombination events.
  • FIG. 12C is a schematic representation of the detailed structure of a vector for homologous recombination.
  • FIG. 12D is a graph showing the rate of random integration events with a pCAG-EGFP-IRES-Zeo-pA vector and the rate of homologous recombination events with a pOct4-IRES-HygtkpA vector in V6.5 wild type ES cells. Rates were calculated by the colony number after the drug selection by the number of survived cell colonies after electroporation without the drug selection. Bars indicate the standard errors among the replicate.
  • FIG. 13A is a schematic showing CGZ3 ES cells were established by transfecting
  • V6.5 ES cells sequentially with a pCAG-CreGR-IRES-His-pA plasmid and a pfloxedCAG-
  • Zscan4ERT2-IRES-puro-pA plasmid In GCZ3 cells without dexamethasone (Dex-), a Zscan4-
  • ERT2 fusion protein is constitutively expressed.
  • FIG. 13B is a schematic of experimental procedures: CGZ3 ES cells (4 x 10 cells) were split into four 10cm dishes with or without Dex, cultured for 2 days, passaged into six 10cm dishes, and cultured for 3 days. Cells were harvested from each culture condition and transfected with 100 ⁇ g of a pBRCAG-EGFP-IRES-hyg-pA vector for the random integration or a pOct4-IRES-HygtkpA vector for the homologous recombination. After electroporation, cells were cultured in Dex- conditions for 7 days with the drug selection.
  • FIG. 13B is a schematic of experimental procedures: CGZ3 ES cells (4 x 10 cells) were split into four 10cm dishes with or without Dex, cultured for 2 days, passaged into six 10cm dishes, and cultured for 3 days. Cells were harvested from each culture condition and transfected with 100 ⁇ g of a pBRCAG-EGFP-IRES-
  • FIG. 13C is a set of images showing cell colonies visualized by Leishman's staining.
  • FIG. 13D is a graph showing the number of colonies by random integration and homologous recombination. Bars indicate the standard error among replications.
  • FIG. 13E is a pair of images showing surviving colonies after electroporation. To normalize the plasmid integration efficiency by the electroporation conditions, cells were plated after electroporation in a series of 1 ⁇ 2 dilutions and cultured for 7 days without drug selection. Colonies were visualized by Leishman's staining and scored.
  • FIG. 13F is a graph showing the number of colonies that survived after electroporation. Bars indicate the standard error among replications.
  • FIG. 13C is a set of images showing cell colonies visualized by Leishman's staining.
  • FIG. 13D is a graph showing the number of colonies by random integration and homologous recombination. Bars indicate the standard error among replications.
  • FIG. 13G is a graph showing the frequency of gene integration per survived cell. Rates of gene integration were calculated by dividing the colony numbers after the drug selection (FIG. 13D) by the number of survived colonies without the drug selection (FIG. 13F). Bars indicate the standard errors among replications.
  • FIG. 13H is a summary table showing the comparison of the gene integration efficiency.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID Nos: 1 and 2 are nucleotide and amino acid sequences of human ZSCAN4.
  • SEQ ID Nos: 3 and 4 are nucleotide and amino acid sequences of mouse Zscan4a.
  • SEQ ID Nos: 5 and 6 are nucleotide and amino acid sequences of mouse Zscan4b.
  • SEQ ID Nos: 7 and 8 are nucleotide and amino acid sequences of mouse Zscan4c.
  • SEQ ID Nos: 9 and 10 are nucleotide and amino acid sequences of mouse Zscan4d.
  • SEQ ID Nos: 11 and 12 are nucleotide and amino acid sequences of mouse Zscan4e.
  • SEQ ID Nos: 13 and 14 are nucleotide and amino acid sequences of mouse Zscan4f.
  • SEQ ID NOs: 15-18 are nucleotide sequences of primers used for qRT-PCR analysis of Zscan4 and H2A.
  • SEQ ID NO: 19 is the nucleotide acid sequence of plasmid
  • SEQ ID NO: 20 is the nucleotide sequence of plasmid pPyCAG-hZscan4-ERT2.
  • SEQ ID NO: 21 is the amino acid sequence of human ERT2.
  • SEQ ID NO: 22 is the amino acid sequence of a mouse Zscan4c-ERT2 fusion protein.
  • SEQ ID NO: 23 is the amino acid sequence of a human ZSCAN4-ERT2 fusion protein.
  • SEQ ID NO: 24 is the nucleotide sequence of plasmid pCAG-Zscan4-AC.
  • SEQ ID NO: 25 is the amino acid sequence of mouse Zscan4c-AC (lacking all four zinc finger domains).
  • Administration To provide or give a subject an agent, such as an ES cell or population of ES cells, by any effective route.
  • An exemplary route of administration includes, but is not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous or intraarterial).
  • Agent Any protein, nucleic acid molecule, compound, cell, small molecule, organic compound, inorganic compound, or other molecule of interest.
  • Contacting Placement in direct physical association; includes both in solid and liquid form. As used herein, “contacting” is used interchangeably with “exposed.” In some cases, “contacting” includes transfecting, such as transfecting a nucleic acid molecule into a cell.
  • Degenerate variant A polynucleotide encoding a polypeptide, such as a Zscan4 polypeptide, that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the
  • Differentiation refers to the process by which a cell develops into a specific type of cell (for example, muscle cell, skin cell etc.). Differentiation of embryonic stem cells refers to the development of the cells toward a specific cell lineage. As a cell becomes more differentiated, the cell loses potency, or the ability to become multiple different cell types.
  • Embryonic stem (ES) cells Pluripotent cells isolated from the inner cell mass of a developing blastocyst.
  • ES cells can be derived from any organism, such as a mammal.
  • ES cells are produced from mice, rats, rabbits, guinea pigs, goats, pigs, cows, non- human primates or humans.
  • Human and murine derived ES cells are exemplary.
  • ES cells are pluripotent cells, meaning that they can generate all of the cells present in the body (bone, muscle, brain cells, etc.). Methods for producing murine ES cells can be found, for example, in U.S.
  • Methods for producing human ES cells can be found, for example, in U.S. Patent No. 6,090,622, PCT Publication No. WO 00/70021 and PCT Publication No. WO
  • ES cell lines are known in the art and are publically available.
  • NIH National Institutes of Health
  • Human Embryonic Stem Cell Registry provides a list of a number of human ES cell lines that have been developed (a list can be found online at the NIH Office of Extramural Research website at
  • a molecule "encapsulated" in a nanoparticle refers to a molecule (such as Zscan4-ERT2 fusion protein) that is either contained within the nanoparticle or attached to the surface of the nanoparticle, or a combination thereof.
  • ERT2 A protein comprising a mutated ligand binding domain of the human estrogen receptor that does not bind its natural ligand (17P-estradiol) at physiological concentrations, but is highly sensitive to nanomolar concentrations of tamoxifen or its metabolite 4-hydroxy-tamoxifen (40HT) (Feil et ah, Biochem Biophys Res Commun 237(3):752-757, 1997).
  • An exemplary amino acid sequence for ERT2 is set forth herein as SEQ ID NO: 21, and the corresponding coding sequence is set forth herein as nucleotides 3989-4936 of SEQ ID NO: 19.
  • ES cell therapy A treatment that includes administration of ES cells to a subject.
  • the ES cells are Zscan4 + ES cells.
  • Functional fragment or variant (of Zscan4) The disclosed Zscan4 polynucleotides and polypeptides (such as those set forth as SEQ ID NOs: 1-14) include functional fragments and variants of Zscan4 that retain Zscan4 biological activity. Functional fragments and/or variants of Zscan4 generally comprise at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence identity with one of the Zscan4 sequences set forth as SEQ ID NOs 1-14. When less than the entire sequence is being compared for sequence identity, fragments will typically possess at least 80% sequence identity over the length of the fragment, and can possess, for example, sequence identities of at least 85%, 90%, 95% or 99%.
  • Fusion protein A protein generated by expression of a nucleic acid sequence engineered from nucleic acid sequences encoding at least a portion of two different (heterologous) proteins. To create a fusion protein, the nucleic acid sequences must be in the same reading frame and contain no internal stop codons.
  • the fusion protein is a Zscan4- ERT2 fusion protein. In some examples, the fusion protein comprises a linker between the two different proteins.
  • Genome stability The ability of a cell to faithfully replicate DNA and maintain integrity of the DNA replication machinery.
  • An ES cell with a stable genome generally defies cellular senescence, can proliferate more than 250 doublings without undergoing crisis or transformation, has a low mutation frequency and a low frequency of chromosomal abnormalities (e.g., relative to embryonal carcinoma cells), and maintains genomic integrity.
  • Long telomeres are thought to provide a buffer against cellular senescence and be generally indicative of genome stability and overall cell health.
  • Chromosome stability e.g., few mutations, no chromosomal rearrangements or change in number
  • a loss of genome stability is associated with cancer, neurological disorders and premature aging. Signs of genome instability include elevated mutation rates, gross chromosomal rearrangements, alterations in chromosome number, and shortening of telomeres.
  • heterologous polypeptide or polynucleotide refers to a polypeptide or polynucleotide derived from a different source or species.
  • a mouse Zscan4 peptide expressed in a human ES cell is heterologous to that ES cell.
  • Homologous recombination A type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. For example, homologous recombination occurs during meiosis when paired chromosomes from two parents align so that similar DNA sequences from the paired chromosomes cross over each other.
  • Host cells Cells in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication.
  • Induced pluripotent stem (iPS) cells A type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inducing a "forced" expression of certain genes.
  • iPS cells can be derived from any organism, such as a mammal. In one embodiment, iPS cells are produced from mice, rats, rabbits, guinea pigs, goats, pigs, cows, non-human primates or humans. Human and murine derived iPS cells are exemplary.
  • iPS cells are similar to ES cells in many respects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability.
  • iPS cells are typically derived by transfection of certain stem cell-associated genes (such as Oct-3/4 (Pouf51) and Sox2) into non-pluripotent cells, such as adult fibroblasts. Transfection can be achieved through viral vectors, such as retroviruses, lentiviruses, or adenoviruses.
  • stem cell-associated genes such as Oct-3/4 (Pouf51) and Sox2
  • transfection can be achieved through viral vectors, such as retroviruses, lentiviruses, or adenoviruses.
  • cells can be transfected with Oct3/4, Sox2, Klf4, and c-Myc using a retroviral system or with OCT4, SOX2, NANOG, and LIN28 using a lentiviral system.
  • iPS from adult human cells are generated by the method of Yu et al. (Science 318(5854): 1224, 2007) or Takahashi et al. (Cell 131(5):861-72, 2007).
  • Isolated An isolated nucleic acid has been substantially separated or purified away from other nucleic acid sequences and from the cell of the organism in which the nucleic acid naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA.
  • isolated thus encompasses nucleic acids purified by standard nucleic acid purification methods.
  • nucleic acids prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • isolated proteins have been substantially separated or purified from other proteins of the cells of an organism in which the protein naturally occurs, and encompasses proteins prepared by recombination expression in a host cell as well as chemically synthesized proteins.
  • isolated cells have been substantially separated away from other cell types.
  • Linker One or more nucleotides or amino acids that serve as a spacer between two molecules, such as between two nucleic acid molecules or two peptides (such as in a fusion protein). In some examples a linker is 1 to 100 amino acids, such as 1 to 50 or 5 to 10 amino acids.
  • Nanoparticle A particle less than about 1000 nanometers (nm) in diameter.
  • Exemplary nanoparticles for use with the methods provided herein are made of biocompatible and biodegradable polymeric materials.
  • the nanoparticles are PLGA nanoparticles.
  • a "polymeric nanoparticle” is a nanoparticle made up of repeating subunits of a particular substance or substances.
  • Poly(lactic acid) nanoparticles are examples of poly(lactic acid) nanoparticles.
  • nanoparticles having repeated lactic acid subunits having repeated lactic acid subunits.
  • poly(glycolic acid) nanoparticles are nanoparticles having repeated glycolic acid subunits.
  • Non-human animal Includes all animals other than humans.
  • a non-human animal includes, but is not limited to, a non-human primate, a farm animal such as swine, cattle, and poultry, a sport animal or pet such as dogs, cats, horses, hamsters, rodents, such as mice, or a zoo animal such as lions, tigers or bears.
  • the non-human animal is a transgenic animal, such as a transgenic mouse, cow, sheep, or goat.
  • the transgenic non-human animal is a mouse.
  • a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked nucleic acid sequences are contiguous and where necessary to join two protein coding regions, in the same reading frame.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.
  • Pluripotent/pluripotency A "pluripotent" cell is a cell that can form all of an organism's cell lineages (endoderm, mesoderm and ectoderm), including germ cells, but cannot form an entire organisms autonomously.
  • enhancing or prolonging pluripotency refers to increasing the pluripotent capacity or duration of pluripotency of a stem cell.
  • Polypeptide A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L- optical isomer or the D-optical isomer can be used, the L-isomers being preferred.
  • the terms "polypeptide” or "protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins.
  • the term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced.
  • polypeptide fragment refers to a portion of a polypeptide which exhibits at least one useful epitope.
  • functional fragments of a polypeptide refers to all fragments of a polypeptide that retain an activity of the polypeptide, such as a Zscan4.
  • Biologically functional fragments can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell, including affecting cell proliferation or differentiation.
  • An “epitope” is a region of a polypeptide capable of binding an immunoglobulin generated in response to contact with an antigen.
  • substantially purified polypeptide refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • the polypeptide is at least 50%, for example at least 80% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • the polypeptide is at least 90% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • the polypeptide is at least 95% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • a Zscan4 polypeptide (or Zscan4 fusion protein, such as Zscan4-ERT2), or other polypeptides disclosed herein, includes at most two, at most five, at most ten, at most twenty, or at most fifty conservative substitutions.
  • the immunologic identity of the protein may be assessed by determining whether it is recognized by an antibody; a variant that is recognized by such an antibody is immunologically conserved. Any cDNA sequence variant will preferably introduce no more than twenty, and preferably fewer than ten amino acid substitutions into the encoded polypeptide.
  • Variant amino acid sequences may be, for example, at least 80%, 90% or even 95% or 98% identical to the native amino acid sequence (such as a native Zscan4 sequence or a Zscan4-ERT2 sequence, such as SEQ ID NO: 22 or 23).
  • Promoter Nucleic acid control sequences which direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription.
  • a promoter also optionally includes distal enhancer or repressor elements.
  • a "constitutive promoter” is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In contrast, the activity of an "inducible promoter” is regulated by an external signal or molecule (for example, a transcription factor).
  • Recombinant nucleic acid or polypeptide is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.
  • Sequence identity/similarity The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. This homology is more significant when the orthologous proteins or cDNAs are derived from species which are more closely related (such as human and mouse sequences), compared to species more distantly related (such as human and C. elegans sequences).
  • NCBI Basic Local Alignment Search Tool (Altschul et al, J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.
  • NCBI National Center for Biological Information
  • Stem cell A cell having the unique capacity to produce unaltered daughter cells (self- renewal; cell division produces at least one daughter cell that is identical to the parent cell) and to give rise to specialized cell types (potency).
  • Stem cells include, but are not limited to, ES cells, EG cells, GS cells, MAPCs, maGSCs, USSCs, adult stem cells and induced pluripotent stem cells.
  • stem cells can generate a fully differentiated functional cell of more than one given cell type. The role of stem cells in vivo is to replace cells that are destroyed during the normal life of an animal.
  • stem cells can divide without limit. After division, the stem cell may remain as a stem cell, become a precursor cell, or proceed to terminal differentiation.
  • a precursor cell is a cell that can generate a fully differentiated functional cell of at least one given cell type. Generally, precursor cells can divide. After division, a precursor cell can remain a precursor cell, or may proceed to terminal differentiation.
  • Subject Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals.
  • Subpopulation An identifiable portion of a population. As used herein, a
  • “subpopulation" of ES cells expressing Zscan4 is the portion of ES cells in a given population that has been identified as expressing Zscan4.
  • Telomere Refers to the end of a eukaryotic chromosome, a specialized structure involved in the replication and stability of the chromosome. Telomeres consist of many repeats of a short DNA sequence in a specific orientation. Telomere functions include protecting the ends of the chromosome so that chromosomes do not end up joined together, and allowing replication of the extreme ends of the chromosomes (by telomerase). The number of repeats of telomeric DNA at the end of a chromosome decreases with age.
  • Transfecting or transfection refers to the process of introducing nucleic acid into a cell or tissue. Transfection can be achieved by any one of a number of methods, such as, but not limited to, liposomal-mediated transfection, electroporation and injection.
  • Vector A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication (DNA sequences that participate in initiating DNA synthesis).
  • an expression vector contains the necessary regulatory sequences to allow transcription and translation of inserted gene or genes.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art. Vectors include, for example, virus vectors and plasmid vectors.
  • Zscan4 A group of genes that have previously identified as exhibiting 2-cell specific expression and ES cell-specific expression (PCT Publication No. WO 2008/118957) and have been shown to promote telomere elongation and genome stability (Zalzman et ah, Nature
  • Zscan4 includes both human ZSCAN4 and mouse Zscan4.
  • Zscan4 refers to a collection of genes including three pseudogenes (Zscan4-psl, Zscan4-ps2 and Zscan4-ps3) and six expressed genes (Zscan4a, Zscan4b, Zscan4c, Zscan4d, Zscan4e and Zscan4f).
  • Zscan4 refers to Zscan4 polypeptides and Zscan4 polynucleotides encoding the Zscan4 polypeptides. Exemplary sequences are provided herein (see SEQ ID NOs: 1-14).
  • Zscan4-AC includes any mouse or human Zscan4 protein having a C-terminal deletion of at least one zinc finger domain.
  • the Zscan4-AC protein includes a deletion of at least two, such as three or all four zinc finger domains.
  • SEQ ID NO: 2 and SEQ ID NO: 8 provide the amino acid sequences of human ZSCAN4 and mouse Zscan4c, respectively, and delineate the N-terminal SCAN domain and C-terminal zinc finger (C2H2-type) domains.
  • the nucleotide and amino acid regions of each domain of human ZSCAN4 is listed in Table 1, and the nucleotide and amino acid regions of each domain of mouse Zscan4c is listed in Table 2.
  • Zscan4-ERT2 A fusion protein made up of a Zscan4 amino acid sequence and an ERT2 amino acid sequence.
  • Zscan4-ERT2 can also refer to a nucleic acid sequence encoding a Zscan4-ERT2 fusion protein.
  • Exemplary amino acid sequences for Zscan4 including SEQ ID NO: 2, 8, 10 and 14
  • ERT2 SEQ ID NO: 21
  • the Zscan4 sequence is a functional fragment or variant of a known Zscan4 sequence (such as SEQ ID NO: 2, 8, 10 or 14)
  • the ERT2 sequence is a functional fragment or variant of a known ERT2 sequence (such as SEQ ID NO: 21).
  • the Zscan4- ERT2 fusion protein comprises a linker (or spacer) between Zscan4 and ERT2.
  • Linkers are well known in the art and an appropriate linker can be selected by one of ordinary skill in the art.
  • the linker is encoded by the nucleotide sequence GCTAGC.
  • the gold standard for examining the pluripotency of stem cells is to see whether cells can contribute to the entire body of an animal. It is disclosed herein that increasing the frequency of Zscan4 activation in mouse ES cells not only enhances, but also maintains their developmental potency in long-term cell culture. As the potency increases, even a whole animal can be produced from a single ES cell injected into a 4N blastocyst at an unexpectedly high success rate. Although Zscan4-activated cells express genes that are also expressed in 2-cell stage mouse embryos, the transiently Zscan4- activated state of ES cells is not associated with the high potency of ES cells.
  • nucleic acid molecules encoding a Zscan4- ERT2 fusion protein are isolated nucleic acid molecules encoding a Zscan4- ERT2 fusion protein.
  • the Zscan4 is mouse Zscan4c or human ZSCAN4.
  • vectors comprising a Zscan4-ERT2 coding sequence, cells comprising such vectors (such as ES cells, iPS cells or other stem cells), and compositions that include the Zscan4- ERT2 encoding nucleic acid molecules or vectors.
  • nucleic acid molecules encoding a Zscan4AC protein a Zscan4 protein having a deletion of at least one zinc finger domain.
  • the Zscan4 is mouse Zscan4c or human ZSCAN4.
  • vectors comprising a Zscan4-AC coding sequence cells comprising such vectors (such as ES cells, iPS cells or other stem cells), and compositions that include the Zscan4-AC encoding nucleic acid molecules or vectors.
  • recombinant Zscan4-AC proteins cells comprising Zscan4-AC proteins and compositions that include the Zscan4-AC proteins.
  • compositions, Vectors and Cells Comprising Zscan4-ERT2
  • ERT2 is a mutated version of the ligand binding domain of human estrogen receptor. ERT2 does not bind its natural ligand (17P-estradiol) at physiological concentrations, but is highly sensitive to nanomolar
  • the nucleic acid molecule encoding the Zscan4-ERT2 fusion protein includes human ZSCAN4, mouse Zscan4c, mouse Zscan4d or mouse Zscan4f, or a functional fragment or variant thereof.
  • Functional fragments and variants of Zscan4 include, for example, any Zscan4 fragment or variant that retains one or more biological activities of Zscan4, such as the capacity to increase pluripotency of a stem cell, promote genomic stability or increase telomere length.
  • nucleic acid sequences for a variety of Zscan4 polynucleotides are known in the art (see, for example, PCT Publication No. WO 2008/118957) and are set forth herein, such as SEQ ID NO: 1 (human ZSCAN4), SEQ ID NO: 7 (mouse Zscan4c), SEQ ID NO: 9 (mouse Zscan4d) and SEQ ID NO: 13 (mouse Zscan4f).
  • Zscan4 nucleic acid sequences from other species are also publically available, including dog ZSCAN4 (GenBank Accession Nos. XM_541370 and XM_848557); cow ZSCAN4 (GenBank Accession No. XM_001789250); and horse ZSCAN4 (GenBank Accession No.
  • GenBank Accession numbers are herein incorporated by references as it appears in the GenBank database on February 22, 2011.
  • a fragment of a Zscan4 nucleic acid sequences includes at least 250, at least 500, at least 750, at least 1000, at least 1500, or at least 2000 consecutive nucleic acids of the Zscan4 polynucleotide.
  • a fragment of Zscan4 is a fragment that confers a function of Zscan4 when expressed in a cell of interest, such as, but not limited to, promoting pluripotency, enhancing genome stability and/or increasing telomere length.
  • the Zscan4 nucleic acid sequences contemplated herein include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the Zscan4 polypeptide encoded by the nucleotide sequence is functionally unchanged.
  • the Zscan4 nucleic acid sequence portion of the nucleic acid molecule encoding the Zscan4-ERT2 fusion protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1, 7, 9 or 13.
  • the Zscan4 nucleic acid sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 1, 7, 9 or 13.
  • the Zscan4 nucleic acid sequence consists of the nucleic acid sequence set forth in SEQ ID NO: 1, 7, 9 or 13.
  • the Zscan4 portion of the Zscan4-ERT2 fusion protein comprises mouse Zscan4c.
  • the Zscan4 nucleic acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 7.
  • the Zscan4 comprises human ZSCAN4.
  • the Zscan4 nucleic acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1.
  • the nucleic acid sequence encoding the ERT2 portion of the Zscan4-ERT2 fusion protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to nucleotides 3989-4936 of SEQ ID NO: 19.
  • the nucleic acid sequence encoding ERT2 comprises or consists of nucleotides 3989-4936 of SEQ ID NO: 19.
  • the nucleic acid molecule encoding the Zscan4-ERT2 fusion protein includes a linker sequence between the Zscan4 and ERT2 coding sequences.
  • Linkers are well known in the art and selection of an appropriate linker is well within the capabilities of one of ordinary skill in the art.
  • the linker is at least 2 amino acids (aa), at least 3, at least 5, at least 10, at least 20, at least 50 or at least 100 aa, such as 2 to 50 or 2 to 10 aa.
  • the linker includes the nucleic acid sequence GCTAGC (nucleotides 3983-3988 of SEQ ID NO: 19).
  • the nucleic acid molecule comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to nucleotides 2465-4936 of SEQ ID NO: 19.
  • the nucleic acid molecule comprises, or consists of, the sequence of nucleotides 2465-4936 of SEQ ID NO: 19.
  • the nucleic acid molecule comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to nucleotides 2479-4731 of SEQ ID NO: 20.
  • the nucleic acid molecule comprises, or consists of, the sequence of nucleotides 2479-4731 of SEQ ID NO: 20.
  • vectors that include a Zscan4-ERT2 encoding nucleic acid molecule disclosed herein.
  • Any suitable expression vector such as an expression (plasmid) vector (e.g., pPyCAG-BstXI-IP), or viral vector (e.g., an adenovirus, adenoassociated virus, lentivirus or retrovirus vector), is contemplated.
  • an expression vector e.g., pPyCAG-BstXI-IP
  • viral vector e.g., an adenovirus, adenoassociated virus, lentivirus or retrovirus vector
  • Numerous expression vectors and viral vectors are known in the art and the selection of an appropriate vector is well within the capabilities of one of ordinary skill in the art.
  • the vector comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 19 or SEQ ID NO: 20.
  • the vector comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 19 or SEQ ID NO: 20.
  • the nucleic acid sequence of the vector comprises, or consists of, SEQ ID NO: 19 or SEQ ID NO: 20.
  • the cell is a stem cell.
  • the stem cell is an ES cell or an iPS cell.
  • the origin of the stem cell can be from any suitable species.
  • the stem cell is a mouse, rat, human or non-human primate stem cell.
  • compositions comprising a nucleic acid molecule or vector encoding a Zscan4-ERT2 fusion protein are also provided herein.
  • the compositions may further include a carrier or diluent, such as a pharmaceutically acceptable carrier or diluent.
  • Zscan4-ERT2 fusion proteins encoded by the nucleic acid molecules and vectors described herein are further provided.
  • the recombinant Zscan4-ERT2 fusion protein includes human ZSCAN4, mouse Zscan4c, mouse Zscan4d or mouse Zscan4f, or a functional fragment or variant thereof.
  • Functional fragments and variants of Zscan4 include, for example, any Zscan4 fragment or variant that retains one or more biological activities of Zscan4, such as the capacity to increase pluripotency of a stem cell, promote genomic stability or increase telomere length.
  • Exemplary amino acid sequences for a variety of Zscan4 proteins are known in the art (see, for example, PCT Publication No. WO 2008/118957) and are set forth herein, such as SEQ ID NO: 2 (human ZSCAN4), SEQ ID NO: 8 (mouse Zscan4c), SEQ ID NO: 10 (mouse Zscan4d) and SEQ ID NO: 14 (mouse Zscan4f).
  • SEQ ID NO: 2 human ZSCAN4
  • SEQ ID NO: 8 mouse Zscan4c
  • SEQ ID NO: 10 mimouse Zscan4d
  • SEQ ID NO: 14 mouse Zscan4f
  • sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to these sequences and retain Zscan4 activity are contemplated and can be used in the methods provided herein.
  • Zscan4 amino acid sequences from other species are publically available, including dog ZSCAN4 (GenBank Accession Nos. XP_541370 and XP_853650); cow ZSCAN4 (GenBank Accession No. XP_001789302); and horse ZSCAN4 (GenBank Accession No. XP_001493994).
  • dog ZSCAN4 GenBank Accession Nos. XP_541370 and XP_853650
  • cow ZSCAN4 GenBank Accession No. XP_001789302
  • horse ZSCAN4 GenBank Accession No. XP_001493994.
  • Each of the above-listed GenBank Accession numbers is herein incorporated by references as it appears in the GenBank database on February 22, 2011.
  • a fragment of a Zscan4 protein includes at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450 or at least 500 consecutive amino acids of the Zscan4 polypeptide.
  • a fragment of Zscan4 is a fragment that confers a function of Zscan4, such as, but not limited to, increasing pluripotency, enhancing genome stability or increasing telomere length.
  • the Zscan4 protein portion of the Zscan4-ERT2 fusion protein includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 2, 8, 10 or 14.
  • the Zscan4 protein is a conservative variant of SEQ ID NO: 2, 8, 10 or 14, such that it includes no more than fifty conservative amino acid substitutions, such as no more than two, no more than five, no more than ten, no more than twenty, or no more than fifty conservative amino acid substitutions in SEQ ID NO: 2, 8, 10 or 14.
  • the Zscan4 peptide portion of the Zscan4-ERT2 fusion protein has an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2, 8, 10 or 14.
  • the Zscan4 comprises mouse Zscan4c.
  • the Zscan4c amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 8.
  • the Zscan4 portion comprises human ZSCAN4.
  • the ZSCAN4 amino acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 2.
  • the amino acid sequence of the ERT2 portion of the Zscan4- ERT2 fusion protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 21.
  • the amino acid sequence of ERT2 comprises or consists of SEQ ID NO: 21.
  • the Zscan4-ERT2 fusion protein includes a linker between the Zscan4 and ERT2 sequences.
  • Linkers are well known in the art and selection of an appropriate linker is well within the capabilities of one of ordinary skill in the art.
  • the linker includes the amino acid sequence Ala-Ser.
  • the amino acid sequence of the fusion protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 22.
  • the amino acid sequence of the Zscan4-ERT2 fusion protein comprises, or consists of, the amino acid sequence of SEQ ID NO: 22
  • the amino acid sequence of the fusion protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 23.
  • the amino acid sequence of the Zscan4-25 ERT2 fusion protein comprises, or consists of, the amino acid sequence of SEQ ID NO: 23.
  • isolated cells comprising a Zscan4-ERT2 fusion protein disclosed herein.
  • the cells are stem cells.
  • the stem cells are ES cells or iPS cells.
  • the origin of the stem cell can be from any suitable species.
  • the stem cell is a mouse, rat, human or non-human primate stem cell.
  • compositions comprising a Zscan4-ERT2 fusion protein are also provided herein.
  • the compositions may further include a carrier or diluent, such as a pharmaceutically acceptable carrier or diluent, for example saline.
  • Zscan4-AC isolated nucleic acid molecules encoding a Zscan4 protein with a C-terminal truncation
  • the C-terminally truncated Zscan4 comprises a deletion of at least one zinc finger domain.
  • the Zscan4-AC protein has a deletion of one, two, three or four zinc finger domains.
  • the nucleic acid molecule encoding the Zscan4-AC protein includes C-terminally truncated human ZSCAN4, mouse Zscan4c, mouse Zscan4d or mouse Zscan4f.
  • the Zscan4-AC protein is either human ZSCAN4 or mouse Zscan4c with a deletion of all four zinc finger domains.
  • the Zscan4- AC protein comprises the amino acid sequence of SEQ ID NO: 25 and/or is encoded by nucleotides 2465-3649 of SEQ ID NO: 24.
  • the Zscan4-AC nucleic acid sequences contemplated herein include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the Zscan4-AC polypeptide encoded by the nucleotide sequence is functionally unchanged.
  • the Zscan4-AC nucleic acid sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to nucleotides 2465-3649 of SEQ ID NO: 24.
  • the Zscan4-AC nucleic acid sequence comprises the nucleic acid sequence set forth as nucleotides 2465-3649 of SEQ ID NO: 24.
  • the Zscan4-AC nucleic acid sequence consists of the nucleic acid sequence set forth as nucleotides 2465-3649 of SEQ ID NO: 24.
  • the Zscan4-AC nucleic acid molecule is a human Zscan4AC nucleic acid molecule.
  • the human Zscan4-AC nucleic acid molecule comprises a deletion of at least nucleotides 1630-1950, nucleotides 1714-1950, nucleotides 1798- 1950 or nucleotides 1882-1950 of SEQ ID NO: 1.
  • the human Zscan4-AC nucleic acid molecule is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to nucleotides 1-1629, nucleotides 1-1713, nucleotides 1-1797 or nucleotides 1-1881 of SEQ ID NO: 1.
  • the human Zscan4-AC nucleic acid molecule comprises or consists of nucleotides 1-1629, nucleotides 1- 1713, nucleotides 1-1797 or nucleotides 1-1881 of SEQ ID NO: 1.
  • the Zscan4-AC nucleic acid molecule is a mouse Zscan4AC nucleic acid molecule.
  • the mouse Zscan4-AC nucleic acid molecule comprises a deletion of at least nucleotides 1383-1709, nucleotides 1470-1709, nucleotides 1554- 1709 or nucleotides 1638-1709 of SEQ ID NO: 7.
  • the mouse Zscan4-AC nucleic acid molecule is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to nucleotides 1-1382, nucleotides 1-1469, nucleotides 1-1553 or nucleotides 1-1637 of SEQ ID NO: 7.
  • the mouse Zscan4-AC protein comprises or consists of nucleotides 1-1382, nucleotides 1-1469, nucleotides 1-1553 or nucleotides 1-1637 of SEQ ID NO: 7.
  • vectors that include a Zscan4-AC encoding nucleic acid molecule disclosed herein.
  • Any suitable expression vector such as an expression (plasmid) vector (e.g., pPyCAG-BstXI-IP), or viral vector (e.g., an adenovirus, adenoassociated virus, lentivirus or retrovirus vector), is contemplated.
  • an expression (plasmid) vector e.g., pPyCAG-BstXI-IP
  • viral vector e.g., an adenovirus, adenoassociated virus, lentivirus or retrovirus vector
  • Numerous expression vectors and viral vectors are known in the art and the selection of an appropriate vector is well within the capabilities of one of ordinary skill in the art.
  • the vector comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 24.
  • the vector comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 24.
  • the nucleic acid sequence of the vector comprises, or consists of, SEQ ID NO: 24.
  • the cell is a stem cell.
  • the stem cell is an ES cell or an iPS cell.
  • the origin of the stem cell can be from any suitable species.
  • the stem cell is a mouse, rat, human or non-human primate stem cell.
  • compositions comprising a nucleic acid molecule or vector encoding a Zscan4AC protein are also provided herein.
  • the compositions may further include a carrier or diluent, such as a pharmaceutically acceptable carrier or diluent.
  • Zscan4-AC proteins encoded by the nucleic acid molecules and vectors described herein are further provided.
  • the recombinant Zscan4-AC protein includes C-terminally truncated human ZSCAN4, mouse
  • the Zscan4-AC protein includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 25.
  • the Zscan4-AC protein is a conservative variant of SEQ ID NO: 25, such that it includes no more than fifty conservative amino acid substitutions, such as no more than two, no more than five, no more than ten, no more than twenty, or no more than fifty conservative amino acid substitutions in SEQ ID NO: 25.
  • the Zscan4-AC protein has an amino acid sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 25.
  • the Zscan4-AC protein is a human Zscan4-AC protein.
  • the human Zscan4-AC protein comprises a deletion of at least amino acids 312-418, amino acids 340-418, amino acids 368-390 or amino acids 396-418 of SEQ ID NO: 2.
  • the human Zscan4-AC protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to amino acids 1-311, amino acids 1-339, amino acids 1-367 or amino acids 1-395 of SEQ ID NO: 2.
  • the human Zscan4-AC protein comprises or consists of amino acids 1-311, amino acids 1-339, amino acids 1-367 or amino acids 1-395 of SEQ ID NO: 2.
  • the Zscan4-AC protein is a mouse Zscan4-AC protein.
  • the mouse Zscan4-AC protein comprises a deletion of at least amino acids 395-503, amino acids 424-503, amino acids 452-503 or amino acids 480-503 of SEQ ID NO: 8.
  • the mouse Zscan4-AC protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to amino acids 1-394, amino acids 1-423, amino acids 1-451 or amino acids 1-479 of SEQ ID NO: 8.
  • the mouse Zscan4-AC protein comprises or consists of amino acids 1-394, amino acids 1-423, amino acids 1-451 or amino acids 1-479 of SEQ ID NO: 8.
  • the cells are stem cells.
  • the stem cells are ES cells or iPS cells.
  • the origin of the stem cell can be from any suitable species.
  • the stem cell is a mouse, rat, human or non-human primate stem cell.
  • compositions comprising a Zscan4-AC protein are also provided herein.
  • the compositions may further include a carrier or diluent, such as a pharmaceutically acceptable carrier or diluent, for example saline.
  • telomere length a stem cell or a stem cell population by promoting recurrent activation of Zscan4 in the stem cell or stem cell population.
  • the methods include contacting the stem cell or stem cell population with (i) a nucleic acid molecule encoding a Zscan4-ERT2 fusion protein or a composition thereof, (ii) a vector encoding a Zscan4-ERT2 fusion protein or a composition thereof, or (iii) a Zscan4-ERT2 fusion protein or a composition thereof.
  • the methods include contacting the stem cell or stem cell population with (i) a nucleic acid molecule encoding a Zscan4-AC protein or a composition thereof, (ii) a vector encoding a Zscan4-AC protein or a composition thereof, or (iii) a Zscan4-AC protein or a composition thereof.
  • a stem cell or stem cell population is contacted with an agent that promotes frequent activation of Zscan4.
  • the agent can be, for example, any nucleic acid molecule, polypeptide, small molecule or other compound that results in recurrent activation of Zscan4 in a cell.
  • the stem cell is an ES cell or an iPS.
  • the methods can be applied to stem cells of any species, for example, mouse, rat, human or non-human primate stem cells.
  • the method includes contacting the stem cell or stem cell population with a nucleic acid molecule or vector encoding a Zscan4-ERT2 fusion protein as disclosed herein. In other embodiments, the method includes contacting the stem cell or stem cell population with a Zscan4-ERT fusion protein disclosed herein.
  • the method includes contacting the stem cell or stem cell population with a nucleic acid molecule or vector encoding a Zscan4-AC protein as disclosed herein. In other embodiments, the method includes contacting the stem cell or stem cell population with a Zscan4-AC protein disclosed herein.
  • Methods of delivering a nucleic acid molecule into a cell are well known in the art.
  • "contacting" the stem cell with a nucleic acid molecule or vector includes transfection (such as liposomal-mediated transfection), electroporation, injection or any other suitable technique for introducing a nucleic acid molecule into a cell.
  • the Zscan4-ERT2 fusion protein or Zscan4-AC protein is encapsulated by a
  • nanoparticle to aid in delivery to the cells.
  • Suitable nanoparticles for use with the disclosed methods are known in the art and are described briefly below.
  • the nanoparticles for use with the methods described herein can be any type of biocompatible nanoparticle, such as biodegradable nanoparticles, such as polymeric nanoparticles, including, but not limited to polyamide, polycarbonate, polyalkene, polyvinyl ethers, and cellulose ether nanoparticles.
  • the nanoparticles are made of biocompatible and biodegradable materials.
  • the nanoparticles include, but are not limited to nanoparticles comprising poly (lactic acid) or poly(glycolic acid), or both poly (lactic acid) and poly(glycolic acid).
  • the nanoparticles are poly(D,L-lactic-coglycolic acid) (PLGA) nanoparticles.
  • biodegradable polymeric materials are contemplated for use with the methods described herein, such as poly(lactic acid) (PLA) and polyglycolide (PGA).
  • PVA poly(lactic acid)
  • PGA polyglycolide
  • Additional useful nanoparticles include biodegradable poly(alkylcyanoacrylate) nanoparticles (Vauthier et ah, Adv. Drug Del. Rev. 55: 519-48, 2003).
  • ES cells are injected into mouse blastocysts, transferred to uteri and the extent of ES cell potency is determined by the percent chimerism of the pups based on coat color.
  • a 4N complementation assay is performed. In this assay, ES cells are injected into a tetraploid (4N) blastocyst. Potency of the ES cells is determined by the ability of the ES cells to produce live embryos.
  • the pluripotency of a stem cell or a stem cell population is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%, as compared to the pluripotency of a stem cell or a stem cell population in the absence of increased Zscan4 activation frequency (such as in the absence of expression of an Zscan4-ERT2 fusion protein).
  • the method includes contacting the stem cell population with a nucleic acid molecule or vector encoding a Zscan4-ERT2 fusion protein disclosed herein. In other embodiments, the method includes contacting the stem cell population with a Zscan4-ERT fusion protein disclosed herein.
  • the method includes contacting the stem cell population with a nucleic acid molecule or vector encoding a Zscan4-AC protein disclosed herein. In other embodiments, the method includes contacting the stem cell population with a Zscan4-AC protein disclosed herein.
  • Zscan4 antibodies specific for Zscan4 (which are commercially available or can be produced according to standard procedures) can be used in immunological based assays to detect Zscan4 + cells.
  • fluorescence-activated cell sorting can be used to detect and quantify Zscan4+ cells in a population.
  • a Zscan4 reporter construct can be used to detect expression of Zscan4 (such as the pZscan4-Emerald vector as described in PCT Publication No. WO 2008/118957).
  • the increase in frequency of Zscan4 + cells in the population is an increase of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 50%, at least 75%, at least 90% or at least 100%.
  • the increase is relative to, for example, a population of cells that has not been contacted with a Zscan4-ERT2 nucleic acid or fusion protein, or a Zscan4-AC nucleic acid or protein (and thus has not undergone frequent activation of Zscan4).
  • Methods of promoting genome stability or increasing telomere length, or both, in a stem cell or a stem cell population are further provided.
  • the method includes contacting the stem cell or stem cell population with a nucleic acid molecule or vector encoding a Zscan4-ERT2 fusion protein disclosed herein.
  • the method includes contacting the stem cell or stem cell population with a Zscan4-ERT fusion protein disclosed herein.
  • the method includes contacting the stem cell or stem cell population with a nucleic acid molecule or vector encoding a Zscan4-AC protein disclosed herein. In other embodiments, the method includes contacting the stem cell or stem cell population with a Zscan4-AC protein disclosed herein.
  • genome stability is increased in a stem cell by at least 20%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 98%, for example relative to stem cell that has not been contacted with a Zscan4-ERT2 or Zscan4-AC protein or a nucleic acid encoding a Zscan4-ERT2 or Zscan4-AC protein (or compared to a value or range of values expected in a stem cell that has not undergone frequent activation of Zscan4).
  • Methods of measuring genome stability and telomere length are routine in the art, and the disclosure is not limited to particular methods. The particular examples provided herein are exemplary.
  • genome stability in a stem cell is measured by detecting cell proliferation.
  • Genome stability is increased if cell proliferation is increased, for example relative to a control cell (for example, a stem cell that has not been contacted with a Zscan4-ERT or Zscan4-AC protein or nucleic acid).
  • ES cell proliferation can be detected by growing ES cells in culture and measuring the doubling time of the cells after each passage.
  • genome stability is increased if crisis (e.g., cell death) does not occur at passage 8 or earlier.
  • genome stability in a stem cell is measured by performing karyotype analysis.
  • Genome stability is increased if the presence of karyotype abnormalities (such as chromosome fusions and fragmentations) is decreased or even absent, for example relative to a cell that has not undergone frequent activation of Zscan4.
  • karyotype analysis can be performed in stem cells by inducing metaphase arrests, then preparing metaphase chromosome spreads.
  • genome stability in stem cell is measured by measuring telomere sister chromatid exchange (T-SCE). Genome stability is increased if the presence of T-SCE is increased relative to a control (such as a stem cell that has not undergone frequent activation of Zscan4).
  • T-SCE can be measured in an stem cell by using telomere chromosome- orientation FISH (CO-FISH).
  • genome stability in stem cell is measured by measuring sister chromatid exchange (SCE). Genome stability is increased if the presence of SCE is decreased relative to a control, such as a stem cell that has not undergone frequent activation of Zscan4. For example, SCE can be measured in a stem cell by detecting SCE in a metaphase spread.
  • SCE sister chromatid exchange
  • telomere length is measured in stem cell. Telomere length is increased in a stem cell if the length of the telomeres is greater, for example relative to telomere length in a control cell that has not undergone frequent activation of Zscan4 (such as a cell that has not been contacted with a Zscan4-ERT2 or Zscan4-AC protein or nucleic acid). For example, telomere length can be detected in a stem cell by fluorescence in situ hybridization (FISH), quantitative FISH (Q-FISH), or telomere qPCR.
  • FISH fluorescence in situ hybridization
  • Q-FISH quantitative FISH
  • telomere qPCR quantitative FISH
  • HR homologous recombination
  • CGZ3 ES cells described above or any other mouse ES cells carrying the similar plasmid construct can be immediately used to increase the efficiency of making gene-manipulated ("knockout") mice.
  • CGZ3 ES cells can be transfected with any kind of a gene- targeting vector, which will be integrated into the desired region of the mouse genome with high efficiency.
  • Previously described methods require isolating and testing hundreds of ES cell colonies due to the extremely low efficiency of the homologous recombination-targeting event. This is a labor intensive step, which typically takes at least several months to complete.
  • the method disclosed herein will reduce the cost and time for this effort.
  • the same strategy can be applied to any type of mouse cell, which are difficult to use for gene manipulation by homologous recombination.
  • the strategy described above can also be applied to human ES, iPS, and other cell types.
  • the current paradigm for the therapeutic application of human ES and iPS cells requires the generation of patient- specific iPS cells from patient fibroblast cells, correcting the genetic defects such as disease-causing DNA mutations in iPS cells, differentiating the corrected iPS cells into desired cell types (such as neurons or cardiomyocytes) and transplanting these cells/tissues back into the patient.
  • desired cell types such as neurons or cardiomyocytes
  • correcting the mutations in iPS cells is currently considered a great technical challenge.
  • the method disclosed herein will solve this problem by dramatically increasing the efficiency of homologous recombination in human iPS cells.
  • Zscan4- mediated enhancement of homologous recombination can be applied to the efficient DNA correction for gene therapy.
  • a Zscan4-ERT2 -expressing vector or Zscan4-ERT2 protein e.g., in the nanoparticle-encapsulated form
  • a gene-targeting vector can be transiently transferred to the patients' tissue/organs to correct DNA mutations.
  • HR is increased by at least 25-fold, at least 50-fold, at least 75-fold, at least 90-fold, or even at least 100-fold, relative to the absence of Zscan4 (such as a Zscan-4 nucleic acid molecule, protein, or activator), such as Zscan4-ERT2.
  • Zscan4 such as a Zscan-4 nucleic acid molecule, protein, or activator
  • the method includes contacting the cell with a Zscan4-ERT2 nucleic acid molecule or fusion protein disclosed herein. In some embodiments, the method includes delivery of a vector encoding the Zscan4-ERT2 fusion protein.
  • the cell is a mouse cell or a human cell. In some examples, the cell is an ES cell or an iPS cell.
  • the nucleic acid molecule or vector encoding the Zscan4-ERT2 fusion protein, or the Zscan4- ERT2 fusion protein is encapsulated in a nanoparticle. The disclosed methods can be carried out in vitro or in vivo.
  • the method includes contacting the cell with a recombinant Zscan4 protein or a recombinant nucleic acid molecule encoding a Zscan4 protein, thereby enhancing efficiency of homologous recombination in the cell.
  • the Zscan4 protein comprises a Zscan4-ERT2 fusion protein.
  • a method of correcting a genetic mutation in a cell, tissue or organ of a subject by contacting the cell, tissue or organ with (1) a Zscan4-ERT2 fusion protein or a nucleic acid molecule encoding a Zscan4-ERT2 fusion protein; and (2) a gene-targeting vector, thereby correcting the genetic mutation in the cell, tissue or organ.
  • the Zscan4-ERT2 fusion protein is encapsulated in a nanoparticle.
  • the cell is isolated from the subject and the method is performed in vitro.
  • the method is an in vivo method.
  • the gene targeting vector can include any gene of interest containing a mutation in a cell, tissue or organ of a subject.
  • C57BL/6J (Olson et al., Cancer Res 63:6602-6606, 2003) were purchased from the Transgenic Core Laboratory of the Johns Hopkins University School of Medicine (Baltimore, MD).
  • V6.5 ES cells (Eggan et al, Proc Natl Acad Sci USA 98:6209-6214, 2001) derived from an Fl hybrid strain (C57BL/6 x 129/Sv) were purchased from Thermo Scientific Open Biosystem.
  • ES cell lines except for TA1 ES cell line (see below), were cultured at 37°C in 5% CO in the complete ES medium as previously described (Zalzman et al., Nature 464:858-863, 2010): DMEM (Gibco), 15% FBS (Atlanta Biologicals), 1000 U/ml leukemia inhibitory factor (LIF) (ESGRO,
  • TA1 ES cell lines were cultured as described above. For all cell lines, media was changed daily and cells were passaged every 2 to 3 days routinely.
  • KSOM medium for 3 days at 37°C in 5% CO .
  • Resulting blastocysts were transferred onto mouse embryo fibroblast (MEF) feeder cells treated with mitomycin C (Sigma) and cultured for 7 days in the complete ES medium (described above) after replacing 15% FBS with 15% KSR (Invitrogen) and adding 50 nM PD98059 (MEK1 inhibitor).
  • ICM inner cell mass
  • ACCUTASETM Inner cell mass
  • Newly derived ES cell lines were directly tested for their developmental potency by 4N-complementation (see below).
  • Zscan4 consist of 6 paralogous genes and 3 pseudogenes clustered on a -850 kb region of chromosome 7 (Falco et al, Dev Biol 307:539-550, 2007).
  • Zscan4a to Zscan4f the open reading frames (ORFs) of Zscan4c, Zscan4d, and Zscan4f are very similar to each other and encode a SCAN domain and four zinc finger domains (Falco et al, Dev Biol 307:539550, 2007).
  • ORFs open reading frames
  • mouse Zscan4c gene (Falco et al, Dev Biol 307:539-550, 2007) was fused with ERT2 (Feil et al, Proc Natl Acad Sci USA 93: 10887-10890, 1996) (314 a.a.) and cloned into Xhol/Notl sites of pPyCAG-BstXI-IP (Niwa et al, Gene 108: 193-199, 1991).
  • the resultant plasmid vector expresses Zscan4c-ERT2 fusion protein-IRES- puromycin-resistant protein under a strong CAG promoter.
  • ES cells were grown in 6- well plates.
  • 5 x 10 5 ES cells in suspension were transfected with 1 ⁇ g of a linearized pZscan4-Emerald vector (Zalzman et al., Nature 464:858-863, 2010) using EFFECTENETM (QIAGEN) according to manufacturer's protocol, and plated in 100 mm dishes. After selecting with 5 ⁇ g/ml blasticidin for 8 days, resulting ES cell colonies were picked, expanded, and frozen for further analysis.
  • a linearized pZscan4-Emerald vector Zalzman et al., Nature 464:858-863, 2010
  • EFFECTENETM QIAGEN
  • ES cell clones 5 x 10 5 ES cells in suspension were cotransfected with 0.5 ⁇ g of a linearized pCAG- Zscan4-ERT2 vector and 0.5 ⁇ g of PL452 (PGK promoter-Neo) (Liu et al., Genome Res 13:476- 484, 2003) using EFFECTENETM (QIAGEN) according to manufacturer's protocol, and plated in 100 mm dishes. After selecting with G418 for 8 days, resulting ES cell colonies were picked, expanded, and frozen for further analysis.
  • PGK promoter-Neo PL452
  • EFFECTENETM QIAGEN
  • Oligo-dT primers and SuperscriptTM III reverse transcriptase were used according to the manufacturer's instruction. Analysis was performed on the ABI 7300 Fast Real Time PCR system (Applied Biosystems). Data was normalized by Chuk (Falco et al., Reprod Biomed Online 13:394-403, 2006) with the AACt method (Livak et al, Methods 25:402-408, 2001).
  • Digoxigenin (DIG)- and biotin (BlO)-labeled RNA probes were transcribed from the PCR product templates using RNA Labeling Mix (Roche). Ethanol-precipitated probes were resuspended in water and quantified by RNA 6000 Nano Assay on a 2100 Bioanalyzer (Agilent Technologies). 10 5 cells/well were seeded in glass chamber slides, cultured for three days, fixed with PFA, and permeabilized with 0.5% TritonX-100. Cells were washed and incubated with 1 ⁇ g/ml DIG and BIO probes for 12 hours at 60°C in hybridization solution. Probes were detected by mouse anti-DIG antibody and by sheep anti-BIO, and visualized by fluorophore-conjugated secondary antibodies. Nuclei were stained with DAPI (blue).
  • DNA microarray analyses were carried out as described (Aiba et al., DNA Res 16:73-
  • Microarray data analyses were carried out by using an application developed in-house to perform ANOVA and other analyses (NIA Array Analysis software; online at lgsun.grc.nia.nih.gov/ANOVA/) (Sharov et ah, Bioinformatics 21:2548-2549, 2005).
  • CD1 females (Charles River, 8-12 week old) were used for superovulation by PMSG (Sigma) followed 48 hours later by hCG (Sigma) administration. After hCG administration, females were mated with males of the same strain and 2-cell embryos were collected by flushing oviducts. Recovered embryos were cultured in KSOM (Millipore) medium for 3 days at 37°C in 5% CO". Collected 2-cell embryos were directly transferred into 0.3 M mannitol solution and aligned automatically by alternate current (AC) pulse in an electrofusion chamber. Then two direct current (DC) pulses with 140V/mm were applied for 40 ⁇ 8 using LFlOl Electro Cell Fusion Generator.
  • AC alternate current
  • DC direct current
  • Fused embryos (4N) that had one blastomere were collected at 60 minutes cultivation and then culture continued in KSOM medium until they reached the blastocyst stage.
  • a single ES cell or 10-15 ES cells were injected into 2N or 4N blastocysts to assess their developmental potency and then transferred to E2.5 recipient females.
  • ES cells were cultured in the presence of 200 nM Tmx for 2-3 days before injection.
  • Em + cells showed a very similar gene expression profile to the Em " cells with only 161 differentially expressed genes (FIG. 5; see also PCT Publication No. WO 2008/118957 and Falco et al, Dev Biol 307:539-550, 2007).
  • Tcstvl and Tcstv3 two cell-specific transcript variant 1 and 3 genes (Struwe and Solter, GenBank accession AF067057.1 ; Zhang et al., Nucleic Acids Res 34:4780-4790, 2006) were among the most highly upregulated genes (FIG. 5).
  • RNA whole-mount in situ hybridization revealed
  • Zscan4 is a 2-cell embryo marker (Falco et al., Dev Biol 307:539-550, 2007)
  • 6 genes were selected from the list based on additional gene expression information in preimplantation embryos (Ko et al., Development 127: 1737-1749, 2000; Sharov et al., PLoS Biol 1:E74, 2003) and were examined for their expression profiles by qRT-PCR.
  • Transient Zscan4 + state is not associated with higher developmental potential
  • ES cells are thought to be equivalent to cells in the inner cell mass (ICM) of blastocysts (Nichols and Smith, Development 138:3-8, 2011; Yoshikawa et al., Gene Expr Patterns 6:213-224, 2006).
  • ICM inner cell mass
  • ES cells are a mixed population of -5% of 2-cell like cells and -95% of ICM-like cells.
  • the Zscan4 + state may represent high-potential true stem cells among the regular ES cell population.
  • V6.5 ZE cells (clone #17) were generated and their developmental potency was assessed by transfecting a pZscan4-Emerald vector into V6.5 ES cells derived from an Fl hybrid strain (C57BL/6 x 129/Sv), which has been extensively used for testing
  • Em + or Em cells were separated manually by pipetting, single ES cells were injected into 2N blastocysts, and the subsequent embryo development was observed. Based on the coat colors, it was found that Em " ES cells were able to contribute to the tissues of chimeric mice at a relatively high rate (31%), whereas Em + ES cells were not (0%). The results indicate that, contrary to expectations, Zscan4 + cells are not associated with high developmental potency compared to Zscan4 cells.
  • Zscan4-ERT2 increases the frequency of endogenous Zscan4 + cells in the absence of Tmx
  • ERT2 the tamoxifen (Tmx)inducible system was selected (Feil et al., Proc Natl Acad Sci USA 93: 10887-10890, 1996).
  • V6.5 ZERT2 V6.5 ZERT2
  • clone #18 was selected for the highest Zscan4 expression levels
  • clones #7 and #10 were selected for the second and third highest Zscan4 levels
  • clone #2 was selected with the background Zscan4 level (FIG. 6A).
  • clone #2 did not have any copies of the pCAG-Zscan4-ERT2 plasmid, and was thus used as a control (V6.5 #2).
  • Tmx + conditions slowed down the proliferation of ES cells (FIG. 6B) and made ES cells flatter (FIG. 6C).
  • the Tmx was removed from the medium after 10 passages in the Tmx+ conditions, the cell proliferation and morphology returned to normal (FIGS. 6B-6C), suggesting that effects of Tmx on the V6.5 ZERT2 cells were reversible.
  • Zscan4 protein lacking the C-terminus increases the number of Zscan4 + cells
  • FIG. 2A provides a schematic of the structure of Zscan4c, Zscan4c-ERT2, Zscan4c- AC and Zscan4c-AN proteins.
  • the amino acid sequence of Zscan4c-AC is set forth herein as SEQ ID NO: 25.
  • the mutated Zscan4c genes were placed under the strong and constitutive CAG promoter.
  • the sequence of the pCAG-Zscan4-AC vector is set forth herein as SEQ ID NO: 24.
  • Each vector was transfected into MC1-ZE16 ES cells (sister clones of MC1ZE3). Multiple independent clones were isolated: ZDC-MC1-ZE16 #3, #4, #20 for Zscan4c-AC; ZDN-MC1- ZE16 #5, #8, #15 for Zscan4c-AN. Fluorescence microscopy was performed on each cell clone.
  • FIGS. 2B-2G The images of ZDC-MC 1 -ZE 16 #3, #4, #20 and ZDN-MC1-ZE16 #5, #8, #15 are shown in FIGS. 2B-2G.
  • Zscan4-ERT2 enhanced and prolonged developmental potency of ES cells in the absence of Tmx
  • V6.5 parental ES cell line at its early passage showed 18% high, 29% moderate, and 41% low chimerism, which are within the standard range for Fl hybrid ES cell lines. It is known that the developmental potency of ES cells generally becomes lower after multiple passages and/or plasmid transfection/drug selection. As expected, compared to a V6.5 parental ES cell line, a control V6.5 #2 ES cell line, which did not carry Zscan4-ERT2 but was generated after transfection and drug selection, showed a slightly lower overall potency, which was further reduced over multiple passages (p21, p23, and p30) (FIG. 3B).
  • V6.5 ZERT2 #18 ES cells showed much higher developmental potency than parental V6.5 and control V6.5 #2 ES cells: 73% high and 27% moderate chimerism at passage 19 (FIG. 3B). Even more surprising was that such a high level of potency was maintained for an extended period of time and passages: for example, even at passage 30, more than 40% of pups derived from V6.5 ZERT2 #18 ES cells showed "high” chimerism, whereas none of the pups derived from control V6.5 #2 ES cells showed "high” chimerism (FIG. 3B). Five other ES cell lines of different genetic backgrounds and transgenes were tested, including a very early passage line from freshly isolated ES cells (TA1). Potency-wise none of these ES cell lines could even come close to V6.5 ZERT2 #18 cell lines (FIG. 3B).
  • MC2 ZERT2 #6 ES cells were generated by transfecting a Zscan4-ERT2 plasmid to an MC2 ES cell line (C57BL/6J) (Olson et al., Cancer Res 63:6602-6606, 2003).
  • V6.5 #2 ES cells did not produce any live embryos after injecting them into 77 tetraploid blastocysts (FIG. 4B).
  • V6.5 ZERT2 #18 cell 3 (7%) became complete embryos, 2 (5%) of which were alive at the time of dissection (FIGS. 4B and 4D).
  • This unusually high level of potency for V6.5 ZERT2 #18 ES cells was indeed comparable to that of early passage TA1 ES cells with 4% live embryos (FIG. 4B). Discussion
  • Zscan4 + cells Em + cells in the experiments described herein
  • ES cells steadily lose their average potency, irrespective of the occasional activation of Zscan4 (FIG. 4E, upper panel). More frequent activation of Zscan4 by the presence of Zscan4-ERT2 may maintain or even increase ES cell potency (FIG. 4E, lower panel).
  • ERT2-fusion proteins usually require Tmx for their activation. It is speculated that this may be related to a partial blocking of Zscan4 function, because the ERT2 domain is fused to the C-terminus of Zscan4, near four zinc-finger (C2H2) domains, whereas a SCAN domain is located at the N-terminus (Falco et al., Dev Biol 307:539-550, 2007).
  • FIG. 12 shows experimental procedures for random integration of a plasmid vector into the mouse genome (FIG. 12A) and homologous recombination/integration of a plasmid vector into the mouse genome (FIG. 12B).
  • the random integration vector (pBRCAG-EGFP- IRES-Hyg-pA) carries a CAG promoter, and thus can produce hygromycin-resistant cell colonies when integrated anywhere in the genome.
  • the homologous integration vector (pOct4- IRES-HygtkpA) does not carry a promoter sequence, and thus can produce hygromycin-resistant cell colonies only when integrated into a unique site in the mouse genome, which shares homology with the vector sequence (Oct4/Pou5f 1 locus; see FIG. 12C for the details of the vector design).
  • the efficiency of homologous integration/recombination is much lower than that of random integration. For example, the efficiency for the random integration was
  • the expression unit (a) constitutively produces Zscan4-ERT2 fusion protein.
  • Dex dexamethasone
  • the Cre-GR fusion protein becomes active and excises the expression unit (a), which is flanked by LoxP sites.
  • the Zscan4-ERT2 expression unit (a) can be completely removed from the ES cells, and thus, it will not interfere with the production of gene-targeted mice. 100% removal of transgene was confirmed in puromycin containing medium with 4-day Dex treatment.
  • CGZ3 ES cells were split into four 10 cm dishes with or without Dex, cultured for 2 days, passaged into six 10 cm dishes, and cultured for 3 days. As described above, culturing CGZ3 ES cells with Dex removed the Zscan4-ERT2 expression unit entirely. Therefore, Dex- conditions represent the presence of Zscan4-ERT2 expression, whereas Dex+ conditions represent the absence of Zscan4-ERT2 expression. Cells were harvested from each culture condition and transfected by electroporation with 100 ⁇ g of pBRCAG-EGFP-IRES-hyg-pA for random integration or pOct4-IRES-HygtkpA.

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Abstract

L'invention concerne une découverte selon laquelle l'augmentation de la fréquence de l'activation de Zscan4 dans des cellules ES murines non seulement améliore mais également conserve leur potentiel de développement dans une culture de cellules à long terme. Le potentiel augmentant, il est même possible de produire un animal à partir d'une seule cellule ES injectée dans un blastocyste avec un taux de réussite étonnamment élevé. Les études décrites dans la présente invention indiquent que les cellules ES acquièrent un potentiel plus élevé en passant par l'état d'activation de Zscan4 transitoire plus fréquemment que par l'état normal. Plus particulièrement, l'invention concerne des découvertes selon lesquelles la présence constitutive de Zscan4-ERT2, y compris en l'absence de son activateur habituel tamoxifène, peut augmenter la fréquence de l'activation de Zscan4 endogène dans des cellules ES, ce qui a pour effet d'augmenter le potentiel de développement des cellules ES. Ainsi, l'invention concerne des protéines hybrides Zscan4-ERT2 et des molécules d'acide nucléique et des vecteurs codant pour les protéines hybrides Zscan4-ERT2.
PCT/US2012/037650 2011-05-13 2012-05-11 Compositions et procédés permettant d'augmenter la fréquence de la recombinaison homologue WO2012158564A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014144932A2 (fr) 2013-03-15 2014-09-18 Elixirgen, Llc Procédés d'utilisation de zscan4 afin de rajeunir des cellules humaines

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060228798A1 (en) * 2002-11-27 2006-10-12 Catherine Verfaillie Homologous recombination in multipotent adult progenitor cells
US20100105043A1 (en) * 2007-03-26 2010-04-29 Ko Minoru S H Methods for modulating embryonic stem cell differentiation
WO2011028880A2 (fr) * 2009-09-04 2011-03-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédé d'amélioration de la stabilité du génome et de l'élongation du télomère dans des cellules souches embryonnaires

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060228798A1 (en) * 2002-11-27 2006-10-12 Catherine Verfaillie Homologous recombination in multipotent adult progenitor cells
US20100105043A1 (en) * 2007-03-26 2010-04-29 Ko Minoru S H Methods for modulating embryonic stem cell differentiation
WO2011028880A2 (fr) * 2009-09-04 2011-03-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédé d'amélioration de la stabilité du génome et de l'élongation du télomère dans des cellules souches embryonnaires

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZALZMAN ET AL.: "Zscan4 regulates telomere elongation and genomic stability in ES cells.", NATURE, vol. 464, no. 7290, 8 April 2010 (2010-04-08), pages 858 - 863 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014144932A2 (fr) 2013-03-15 2014-09-18 Elixirgen, Llc Procédés d'utilisation de zscan4 afin de rajeunir des cellules humaines
US10335456B2 (en) 2013-03-15 2019-07-02 Elixirgen Therapeutics, Inc. Methods of using ZSCAN4 for rejuvenating human cells
US10744183B2 (en) 2013-03-15 2020-08-18 Elixirgen Therapeutics, Inc. Methods of using ZSCAN4 for rejuvenating human cells
EP3782628A1 (fr) 2013-03-15 2021-02-24 Elixirgen Therapeutics, Inc. Procédés d'utilisation de zscan4 afin de rajeunir des cellules humaines
US11389504B2 (en) 2013-03-15 2022-07-19 Elixirgen Therapeutics, Inc. Methods of using ZSCAN4 for rejuvenating human cells
EP4410976A2 (fr) 2013-03-15 2024-08-07 Elixirgen Therapeutics, Inc. Procédés d'utilisation de zscan4 pour rajeunir des cellules humaines

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