The present invention provides methods and compostions to improve the efficiency of somatic cell nuclear transfer (SCNT). There is increasing evidence that the epigenetic state of donor nuclei has a significant impact on potential of nuclear transfer embryos to develop into blastocysts, from which pluripotent stem cells are derived. Strategic application of histone agents, capable of altering epigenetic state such as methylation, allows zygotic activation and robust blastocyst generation.

The present invention provides novel methods for improving the efficiency of artificial activation of unfertilized mammalian oocytes by reducing the intracellular concentration of Zn.sup.2+ in the oocyte. The methods of the invention may additionally comprise a preceding step of increasing the intracellular concentration of Ca.sup.2+ in the oocyte prior to reduction of the intracellular Zn.sup.2+ concentration. The invention further provides unfertilized oocytes activated by the disclosed methods and viable mammalian animals produced from unfertilized oocytes activated by the disclosed methods.

The present invention provides methods and compostions to improve the efficiency of somatic cell nuclear transfer (SCNT) and the consequent production of nuclear transfer ESC (ntESC) and transgenic cells and/or non-human animals. More specifically, the present invention relates to the discovery that trimethylation of Histone H3-Lysine 9 (H3K9me3) in reprogramming resistant regions (RRRs) in the nuclear genetic material of donor somatic cells prevents efficient somatic cell nuclear reprogramming or SCNT. The present invention provide methods and compositions to decrease H3K9me3 in methods to improve efficacy of SCNT by exogenous or overexpression of the demethylase Kdm4 family and/or inhibiting methylation of H3K9me3 by inhibiting the histone methyltransferases Suv39h1 and/or Suv39h2.

Provided is a double-gene knockout vector system, a method for preparing porcine fibroblasts with both CD163 gene and CD13 gene being knocked-out, prepared porcine fibroblasts, and a method for preparing a gene-edited pig with both CD163 gene and CD13 gene being knocked-out. The vector system of the present disclosure comprises a CD163 gene knockout vector and a CD13 gene knockout vector. The CD163 gene knockout vector comprises a gene editing vector backbone and a DNA fragment ligated to the gene editing vector backbone, with a nucleotide sequence of the DNA fragment being shown in any one of SEQ ID NOs: 1-3. The CD13 gene knockout vector comprises a gene editing vector backbone and a DNA fragment ligated to the gene editing vector backbone, a nucleotide sequence of the DNA fragment being shown in any one of SEQ ID NOs: 4-6.

The present invention provides a novel oocyte maturation medium or/and embryo culture medium with a chemically defined supplement to produce matured oocytes at high efficiency. The inventive medium or supplement comprises three growth factors, namely, fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF-1) in a synergistic combination. Methods for oocyte and embryo culture are also provided.

Immunocompatible pluripotent stem cells (pSCs), which include cells compatible with different patient populations or patient-specific cells, find wide application in regenerative medicine therapies. Described herein are immunocompatible pSCs generated using techniques such as parthenogenesis resulting in cells possessing desired haplotypes of reduced zygosity, antigenically compatible with multiple patient populations, or nuclear transfer allowing generation of patient-specific cells. Methods described herein related to parthenogenesis, nuclear transfer, or pSC cell line generation. Also described herein are compositions of immunocompatible pSCs and cell lines generated by the aforementioned techniques.

Methods are provided of generating functional human oocytes following nuclear transfer of first polar body (PB1) genomes from metaphase II (MII) oocytes into enucleated donor MII cytoplasm (PBNT) and using mitochondrial replacement techniques to circumvent mother-to-child mtDNA disease transmission.

Disclosed is a novel means which makes it possible to steadily mass-produce knockout individuals even in large animals. The method of the present invention is a method for producing a non-human large mammal or fish (non-human animal) that produces gametes originating in a different individual, and comprises transplanting at least one pluripotent cell derived from a second non-human animal into an embryo derived from a first non-human animal, said embryo being at a cleavage stage and having a genome in which a function of nanos3 gene is inhibited, to prepare a chimeric embryo, and allowing said chimeric embryo to develop into an individual.

The present invention provides a novel oocyte maturation medium or/and embryo culture medium with a chemically defined supplement to produce matured oocytes at high efficiency. The inventive medium or supplement comprises three growth factors, namely, fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF-1) in a synergistic combination. Methods for oocyte and embryo culture are also provided.

The present invention provides methods and compostions to improve the efficiency of somatic cell nuclear transfer (SCNT) and the consequent production of nuclear transfer ESC (ntESC) and transgenic cells and/or non-human animals. More specifically, the present invention relates to the discovery that trimethylation of Histone H3-Lysine 9 (H3K9me3) in reprogramming resistant regions (RRRs) in the nuclear genetic material of donor somatic cells prevents efficient somatic cell nuclear reprogramming or SCNT. The present invention provide methods and compositions to decrease H3K9me3 in methods to improve efficacy of SCNT by exogenous or overexpression of the demethylase Kdm4 family and/or inhibiting methylation of H3K9me3 by inhibiting the histone methyltransferases Suv39h1 and/or Suv39h2.