163 results about "Nuclear transfer" patented technology

An improved method of nuclear transfer involving the transplantation of donor differentiated cell nuclei into enucleated oocytes of the same species as the donor cell is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring, and for production of isogenic CICM cells, including human isogenic embryonic or stem cells. Production of genetically engineered or transgenic mammalian embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.

This invention provides a method for deriving precursors to pluripotent non-embryonic stem (P-PNES) and pluripotent non-embryonic stem (PNES) cell lines. The present invention involves nuclear transfer of genetic material from a somatic cell into an enucleated, zona pellucida free human ooplastoid having a reduced amount of total cytoplasm. The present invention provides a new source for obtaining human and other animal pluripotent stem cells. The source utilizes as starting materials an oocyte and a somatic cell as the starting materials but does not require the use, creation and/or destruction of embryos or fetal tissue and does not in any way involve creating a cloned being. The oocyte never becomes fertilized and never develops into an embryo. Rather, portions of the oocyte cytoplasm are extracted and combined with the nuclear material of individual mature somatic cells in a manner that precludes embryo formation. Murine, bovine, and human examples of the procedure are demonstrated. Subsequently, the newly constructed P-PNES cells are cultured in vitro and give rise to PNES cells and cell colonies. Methods are described for culturing the P-PNES cells to yield purified PNES cells which have the ability to differentiate into cells derived from mesoderm, endoderm, and ectoderm germ layers. Methods are described for maintaining and proliferating PNES cells in culture in an undifferentiated state. Methods and results are described for analysis and validation of pluripotency of PNES cells including cell morphology, cell surface markers, pluripotent tumor development in SCID mouse, karyotyping, immortality in in vitro culture.

The present invention relates to nuclear methods and embryos developed therefrom. In particular, the present invention relates to a method of nuclear comprising the step of transferring a somatic cell nuclei into a zona pellucida-free, enucleated oocyte.

An improved method of nuclear transfer involving the transplantation of donor differentiated pig cell nuclei into enucleated pig oocytes is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring. Production of genetically engineered or transgenic pig embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.

A method for treating cells and/or nuclear transfer units and/or stem cells in culture with such compounds, individually or in combinations, is described. The method results in a globally hypomethylated genome and a restoration of cell differentiation and/or developmental potential, or potentiality. In addition, a method for the in vitro production of reprogrammed cells which have had differentiation potential (totipotential, pluripotential, or multipotential) restored by demethylating the genome is described.

An improved method of nuclear transfer involving the transplantation of donor cell nuclei into enucleated oocytes of a species different from the donor cell is provided. The resultant nuclear transfer units are useful for the production of isogenic embryonic stem cells, in particular human isogenic embryonic or stem cells. These embryonic or stem-like cells are useful for producing desired differentiated cells and for introduction, removal or modification, of desired genes, e.g., at specific sites of the genome of such cells by homologous recombination. These cells, which may contain a heterologous gene, are especially useful in cell transplantation therapies and for in vitro study of cell differentiation.

Methods for propagating haploid genomes of male or female origina and genetic screening and modification thereof are provided. These haploid genomes may be used to produce haploid embryos, and embryonic stem-like cells and differentiated cells. Also, these haploid genomes and cells containing, may be used as nuclear transfer donors to produce diploid nuclear transfer units. These diploid NT units e.g., human NT units, may be used to obtain pluripotent cells and differentiated cells and tissues.

The invention provides application of a Trim63 gene in preparation of a high muscle content and hypertrophic cardiomyopathy model cloned pig. Somatic cells of the Trim63 gene of a modified pig are utilized as nuclear transfer donor cells, oocytes are utilized as nuclear transfer recipient cells, and a cloned embryo is obtained through the somatic cell nuclear transfer technology. The cloned embryo is transferred into a pig uterus to gestate to obtain the Trim63 gene modified high muscle content and hypertrophic cardiomyopathy model cloned pig, artificial intervention methods, such as operation and so on for the cloned pig are not needed any more, and the efficiency of establishment of a disease model is improved. According to the high muscle content and hypertrophic cardiomyopathy model cloned pig, paired Cas9n targeting vectors are utilized for the first time to perform gene editing for large animals. The method is low in cost, sharply shortens the time for obtaining a homozygote pig and lays a foundation for gene function research and the disease model establishment for the large animals by utilizing the CRISPR/Cas9 technology.

The invention provides a production method for Fbxo40 gene knockout pigs. The production method comprises the steps that a CRISPR-Cas9 targeting vector and a PGK-Neo resistant gene are jointly transfected into a porcine embryonic fibroblast to obtain a G418-resistant positive monoclonal cell, an Fbxo40 gene in the positive monoclonal cell generates insertion or deletion mutation, a reading frame generates frame shift and stops in advance, then the obtained cell is cloned to serve as a donor cell for nuclear transfer, and an oocyte serves as a receptor oocyte for nuclear transfer; cloned embryos are obtained through a somatic cell nuclear transfer technique, the high-quality cloned embryos are transferred into the fallopian tube of an oestrous sow, and the Fbxo40 gene knockout pigs are obtained through whole-period development. According to the production method, the Fbxo40 knockout pigs are efficiently obtained through a CRISPR-Cas9 gene editing technique at the low cost, and animal models are supplied to research of muscle development and diseases related to the muscles.

The present invention includes compositions, methods and kits for non-nuclear transfer reprogramming an adult differentiated cell obtained from an adult tissue into an ES-like cell. The reprogrammed cell can be converted into an ES-like cell which can be re- or trans-differentiated into various differentiated cell types. The present invention further relates to identification of a novel signaling pathway, and components thereof, which effect reprogramming of a cell. The present invention further comprises compositions, methods and kits for regulating the mammalian cell cycle and cellular proliferation, as well as for treating diseases and for identifying components that affect the cell cycle, and reprogram cells, among other things.

The invention provides a method for finishing down producing goat VEGF gene fixed-point knock-in mediated by a CRISPR/Cas9 system. The method comprises the following steps: establishing a gRNA expression vector and a VEGF homologous recombination vector on the basis of the CRISPR/Cas9 system according to a CCR5 gene sequence of down producing goats; then jointly transferring three optimized vectors into fibroblasts of down producing goat fetuses to obtain VEGF gene fixed-point knocked-in positive cells; and preparing VEGF gene fixed-point integrated down producing goats by using a somatic nuclear transfer technology. The established targeting vector based on the CRISPR/Cas9 system provides a simple, rapid and safe path for fixed-point knock-in of goat VEGF genes. The method does not involve any screening marker genes in a cell line screening process, therefore, the safety of transgenic animals is greatly improved, and the method has high value on research of genetic breeding and gene functions of down producing goats.

This invention relates to methods for making immune compatible tissues and cells for the purpose of transplantation and tissue engineering, using the techniques of nuclear transfer and cloning. Also encompassed are methods for determining the effect on immune compatibility of expressed transgenes and other genetic manipulations of the engineered cells and tissues.

Provided is a method for the production of transgenic animals, especially pigs, by the use of nuclear transfer from genetically modified or other embryonic stem cells to either enucleated oocytes which were matured in vivo or in vitro and activated or to enucleated zygotes.

Methods for making human ES cells and human differentiated cells and tissues for transplantation are described, whereby the cells and tissues are created following somatic cell nuclear transfer. The nuclear transfer donor is genetically modified prior to nuclear transfer such that cells of at least one developmental lineage are de-differentiated, i.e., unable to develop, thereby resolving the ethical dilemmas involved in reprogramming somatic cells back to the embryonic stage. The method concomitantly directs differentiation such that the desired cells and tissues may be more readily isolated.

The invention relates to a method of producing a protein of interest, comprising making a non-human transgenic mammal that produces said protein in its milk, obtaining said milk from the non-human transgenic mammal and purifying said protein of interest from the milk. Transgenic bovine animals were generated, which are able to produce human growth hormone in mammary glands. The method involves cloning of a genetic construct encoding hGH gene and beta casein promoter conveniently in an expression vector. It also includes transfection procedures into fetal bovine somatic cells, generally fibroblasts, and the nuclear transfer into enucleated bovine oocytes, generating thus transgenic embryos. The method also includes other procedures to generate transgenic embryos for the further expansion of the transgenic herd, such as the subcloning of transgenic female bovines, the superovulation of transgenic cows and their insemination with semen from a non-transgenic or a transgenic male bovine, and the superovulation of non-transgenic cows and their insemination with semen from a transgenic male bovine. Afterwards, transgenic embryos give rise to transgenic cattle that produce human growth hormone in huge amounts in their milk, from which the hormone is completely purified and analysed to fulfill all the requirements for the manufacture of a pure biopharmaceutical product.

The present invention provides a method of producing an animal embryo, the method comprising transferring from a nuclear donor cell which has been selected on the basis that it is histone hypomethylated at least a portion of the nuclear contents including at least the minimum chromosomal material able to support development into a suitable recipient cell.

The invention discloses a method for processing bovine somatic cell cloned embryos constructed on the basis of somatic cell nuclear transplantation, which comprises the following steps of: injecting bovine somatic cells to be transplanted into denucleated oocyte to carry out electrofusion; and an hour later after the electrofusion is completed, selecting the successfully fused bovine somatic cellcloned embryos and processing the bovine somatic cell cloned embryos for 12 hours by 1muM of Oxamflatin. By the method, the developmental rate and the quality of the bovine somatic cell cloned embryos can be obviously improved and the bovine somatic cell cloned embryos can be efficiently produced in vitro.

An improved method of nuclear transfer involving the transplantation of donor differentiated pig cell nuclei into enucleated pig oocytes is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring. Production of genetically engineered or transgenic pig embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.

The invention discloses a method for breeding transgenic livestock rich in polyunsaturated fatty acid. The method comprises the following steps of: guiding DNA molecules which have a sequence 1 and a sequence 2 in a sequence list into somatic cells of a mammal to obtain transgenic cells in which a delta-12 fatty acid dehydrogenase and an omega-3 fatty acid dehydrogenase are expressed; cloning embryos by using the transgenic cells as nuclear transfer donor cells and using isolated ovocytes as nuclear transfer acceptor cells by nuclear transfer technology; and transplanting the cloned embryos into a uterus of the livestock by a non-operation method for gestation to obtain the transgenic livestock. The method of the invention can ensure that the delta-12 fatty acid dehydrogenase and the omega-3 fatty acid dehydrogenase are stably expressed in the body of a transgenic pig and that the content of omega-3 polyunsaturated fatty acids (PUFAs) in meat of the livestock is improved obviously.

Methods are provided for producing a human embryo capable of developing to the blastocyst stage. The method includes transferring a human somatic cell genome into a mature human oocyte by nuclear transfer and activating the oocyte, without removing the oocyte genome. Pluripotent human embryonic stem cells, and methods of obtaining these, are also provided.