158 results about "Nuclear transplantation" patented technology

The invention provides a method of cloning a reproductive and respiratory syndrome resisting pig. The method comprises the following steps: transferring CRISPR / Cas9 targeting vectors and CD163 gene homologous recombination modification vectors into fibroblasts of a pig to obtain positive clone cells, wherein the seventh exon of the endogenous CD163 gene in the positive clone cells of the pig is replaced by the tenth exon of the CD163-L1 gene of human, so that the positive clone cells are incapable of mediating PRRSV invading; obtaining a clone embryo by adopting a somatic cell nucleus transfer technology by taking positive cells as nucleus transfer donor cells and oocytes as nucleus transfer recipient cells; and transferring the clone embryo into the uterus of the pig, and impregnating to obtain a cloned pig. The method provided by the invention is low in cost, the time of obtaining the homozygous pig is shortened greatly, the expression of the CD163 gene is not affected by gene editing, and a foundation is laid for the research into the gene function of large animals and the establishment of disease models on the basis of CRISPR / Cas9 mediated homologous recombination.

The invention relates to the field of genetic engineering and genetic modification, in particular to a method for obtaining an MC4R gene knockout pig by using a CRISPR / Cas9 system for editing MC4R genes through a somatic cell nuclear transfer technology. sgRNA is designed by aiming at two sites (a 6133-6152bp sequence and a 7127-7146 bp sequence in a CDS region of the MC4R gene) in the CDS region of the pig MC4R gene for the first time; in addition, the CRISPR / Cas9 system is used for simultaneously cutting the two sites; the large segment deletion of the MC4R gene is realized; in addition, the gene knockout individual pig subjected to large segment deletion is obtained; a feasible method is provided for studying the pig MC4R gene.

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.

A nuclear transfer embryo is formed by destabilizing microtubules of an oocyte, whereby essentially all endogenous chromatin collects at a second polar body during meiosis of an oocyte. The oocyte is fused with the nucleus of a donor somatic cell of the same species of said oocyte prior to cessation of extrusion of the second polar body from the oocyte, thereby forming the nuclear transfer embryo. In one embodiment, the nuclear transfer embryo is employed to impregnate an animal, such as a mammal. In another embodiment, the donor nucleus is transgenic.

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 provides a bovine oocyte in vitro maturation culture solution and a culture method. The maturation culture solution consists of 10% of fetal calf serum and 1% of ITS-G, as well as 0.075IU / mL of HMG, 1[mu]g / mL of 17[beta]-estradiol, 10ng / mL of EGF, 2.2mg / mL of bFGF and 50ng / mL of CXCL12. The culture solution, when applied to bovine oocyte in vitro maturation culture, can improve maturation quality of bovine oocyte in vitro culture, a development rate of in vitro fertilized embryos as well as a development rate of somatic cell nuclear transplantation embryos and blastocyst quality.

The invention relates to a method and equipment for femtosecond laser somatic cell nuclear transplantation, in particular to a micro-nano control method which adopts a light knife effect induced by femtosecond laser two-photon and optical tweezers gradient force induced by high photon density. A pump laser is started-up; the laser produced by the pump laser is introduced into a resonator of a femtosecond laser; when the oscillation is done, the energy of the ultrashort femtosecond laser pulse is amplified through a regenerative amplifier, and the pulse number and the repeated frequency of laser output are chosen. The emitted femtosecond laser is divided into two beams of light, one of which used for realizing the femtosecond optical tweezers, and the other of which is used for realizing the function of the femtosecond optical tweezers through a half-permeable and semi-reflecting mirror, and then the two beams of light are coupled with an optical path through another half-permeable andsemi-reflecting mirror to realize the amplification of a spot light area; then the light beams is focused on a target cell, and the harmless transplantation operation of the somatic cell nuclear is done. A switch for controlling a light valve is arranged in a software control system, which can not only realize the operation of the laser spot on the target cell, but also adjust the scanning speed and change the exposure time to meet different requirements and enhance the success rate of the somatic cell nuclear transplantation.

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.

The present invention provides for the production of homozygous primary cells that carry a specific transgenic integration of interest on both chromosomes by bypassing breeding. These cell lines can they be used for the accelerated production of homozygous transgenic animals by somatic cell nuclear transfer. The invention is thus useful in the production of transgenic ungulate animals capable of producing desired biopharmaceuticals in their milk at higher yield than a comparable heterzygote. By combining the selection techniques of the current invention with somatic cell nuclear transfer it can be applied to large animals, where there is a strong need to shorten the time to homozygosity.

The invention belongs to the technical field of animal genetic engineering and particularly relates to a method for improving pig meat quality. The method is characterized in that a peroxisome proliferator-activated receptor (PPAR) gamma gene serves as an important candidate gene for improving the meat quality, a fibroblast cell line of a 30-day-old fetus of a large white pig is established, a SwaI linearized pN1-MCK-PPAR gamma2 expression vector is shifted to the 30-day-old fetus fibroblast cell line of the large white pig through an electrotransfection method, and a PPAR gamma transgenic pig is prepared in a method of somatic nucleus transplantation. The influence of muscle tissue overexpression PPAR gamma genes on meat traits such as intramuscular fat deposition is verified in a transgenic pig, the contradiction of simultaneously selecting meat quality and meat quantity in conventional animal breeding is overcome, and the novel method is provided for cultivating lean meat pigs with good meat quality.