Somatic cell nuclear transfer methods
AssigneeNew York Stem Cell Foundation, Inc.
InventorDietrich M. Egli
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Abstract
The present invention provides methods for making reconstructed diploid human oocytes comprising the diploid genome of a human somatic cell, and also methods for making human nuclear transfer embryos, human embryonic stem cells, and human differentiated cells therefrom. The present invention also provides reconstructed human oocytes, human nuclear transfer embryos, human embryonic stem cells, and differentiated cells made using such methods, as well as compositions and kits useful in performing such methods.
Claims15
A method for producing a diploid human nuclear transfer embryo capable of developing into a blastocyst containing an inner cell mass, the method comprising:
- a. obtaining a diploid nuclear genome from a postnatal human somatic cell,
- b. obtaining an enucleated mature human oocyte,
- c. transferring the diploid nuclear genome into the enucleated mature human oocyte to form a reconstructed oocyte, wherein the transferring is performed in a medium that is calcium-free, and/or contains a calcium chelator, and/or contains a phosphatase inhibitor,
- d. subsequently contacting the reconstructed oocyte with a calcium ionophore, an inhibitor of translation, and an inhibitor meiotic kinases, to activate the reconstructed oocyte and promote entry into interphase, and
- e. contacting the reconstructed oocyte with a histone deacetylase inhibitor and/or a histone methylation inhibitor,
- thereby producing a diploid human nuclear transfer embryo capable of developing into a blastocyst containing an inner cell mass.
A kit for use in a nuclear transfer method, the kit comprising two or more of the following components:
- a. a nuclear transfer/cell fusion component, wherein the nuclear transfer/cell fusion component comprises a calcium free medium, a calcium chelator, a protein phosphatase inhibitor, and/or a fusogenic agent,
- b. an activation component, wherein the activation component comprises one or more of a calcium ionophore, a protein translation inhibitor, and a meiotic kinase inhibitor,
- c. an embryo culture component, wherein the embryo culture component comprises one or more of a histone deacetylase inhibitor, a histone methylation inhibitor, a protein translation inhibitor, and a meiotic kinase inhibitor, and
- d. an ES cell derivation component, wherein the ES cell derivation component comprises fetal bovine serum (FBS).
Dependencies are inferred from the claim text.