Enhanced production of cloned mammals by zona pellucida-free homologous mammalian embryo aggregation
AssigneeThe Trustees of the University of Pennsylvania
InventorsMichele Boiani, Kenneth John McLaughlin, Hans R. Scholer
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Abstract
Compositions and methods are provided for the efficient and reproducible generation of clone animals of all developmental stages. Also provided are methods of use of the same in reproductive and therapeutic cloning protocols.
Claims28
A method for preparing a non-primate mammalian blastocyst capable of developing into a live-born animal, comprising:
- a) providing genetically identical reconstructed non-primate mammalian embryos;
- b) removing the zona pellucida of said reconstructed embryos;
- c) forming a homologous aggregate of at least two of the zona-free embryos of step b);
- and
- d) culturing the homologous embryo aggregate of c) under conditions which result in the formation of a non-primate mammalian blastocyst;
- wherein said reconstructed embryos are prepared from reconstructed non-primate mammalian oocytes comprising donor cell nuclei or donor nuclear DNA;
- wherein said donor cell is a non-primate mammalian embryonic stem cell or a non-primate mammalian embryonic germ cell;
- and wherein said at least two zona-free embryos in step c) are at the four-cell stage.
A method for preparing a non-primate blastocyst capable of developing into a live-born animal, comprising:
- a) providing genetically identical reconstructed embryos;
- b) removing the zona pellucida of said reconstructed embryos;
- c) forming a homologous aggregate of at least two of the zona-free embryos of step b);
- and
- d) culturing the homologous embryo aggregate of c) under conditions which result in the formation of a blastocyst;
- wherein said reconstructed embryos are prepared from reconstructed oocytes comprising donor cell nuclei or donor nuclear DNA;
- wherein said reconstructed embryo is from an animal selected from the group consisting of ovine, bovine, equine, goat, feline, canine, rabbit, murine, and porcine;
- and
- wherein said at least two zona-free embryos in step c) are at the four-cell stage.
A homologous, isolated non-primate mammalian embryo aggregate having increased biomass and excess extranuclear cytoplasmic factors which facilitate gene expression and blastocyst formation and enhance viability of said embryo, wherein said embryo aggregate lacks a zona pellucida, wherein said embryo aggregate comprises at least two genetically identical reconstructed non-primate mammalian embryos, and wherein said embryos are at the four-cell stage.
Dependencies are inferred from the claim text.