42 results about "Somatic Cell Nuclear Transfer Technique" patented technology

The invention provides an overexpression porcine co-stimulatory 4-1BB vector and application thereof. PCR (polymerase chain reaction) amplification is performed on a left homologous arm and a right homologous arm of an intron 1 of a rosa26 gene, a 4-1BB regulatory sequence and an OCT4 specific promoter; the left homologous arm, a 4-1BB expression cassette, LoxP locus-contained Cre and Neo expression cassettes, the right homologous arm and negative selection DTA diphtheria toxin are connected in sequence to obtain a 4-1BB homologous recombinant vector p4BOCNDR; the vector and a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) targeting vector of sgRNA (small guide ribonucleic acid) containing the intron 1 of the specific targeting porcine rosa26 gene are transferred together into a porcine fetus fibroblast; by taking a positive cell as a donor cell and an oocyte as a recipient cell, a cloned embryo is obtained through a somatic cell nuclear transfer technique; the cloned embryo is transplanted into a porcine uterus for fetation to obtain a transgenic pig integrating a 4-1BB gene at the fixed point of a first intron of the rosa26 gene and automatically deleting a marker gene.

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 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.

The invention discloses a preparation method of liver cells with low expression or no expression of PERV. The preparation method includes the following steps: (1), respectively preparing a recombinant vector containing siRNA applied to RNA interference technology, a recombinant vector containing gRNA applied to CRIPRS/Cas9 technology and a vector containing Cas9 applied to CRIPRS/Cas9 technology; (2), jointly transfecting the three vectors to porcine somatic cells, culturing the transfected porcine somatic cells, and screening and identifying to obtain monoclonal genetically-modified porcine somatic cells; (3), utilizing somatic cell nuclear transfer technology to obtain genetically-modified pig containing nuclear genes of the genetically-modified porcine somatic cells; (4), extracting liver cells of the genetically-modified pig. According to the preparation method, knockout before transcription and inhibition after transcription of PERV genes are realized, and the technical scheme is effective and feasible.

The invention provides a transgene pig for preparing an animal model for human diseases and a culturing method thereof. An inducible expression Cre recombinase expression vector pET28a-Mxl-Cre-BGHpolyA-FRT2neoR is constructed by a genetic engineering method, and can express Cre recombinase after the expression vector is induced by interferon inducer poly IC (poly I:C) so as to be used for knocking out a target gene; porcine mechanocyte capable of inducibly expressing the Cre recmbinase can be screened through technology of cell transfection and cell screening; and the pig is cloned by the technology of somatic cell nuclear transplantation. The cloned pig can be used for preparing the animal model for knocking out genes of human diseases.

The invention relates to a method for obtaining a lymphoma minipig disease model by knocking out P53 genes, and belongs to the field of medical animal disease models. The method mainly comprises the steps that TALEN target spots of the P53 genes are designed, P53 gene knockout TALEN plasmid pCAG-pP53-L and pCAG-pP53-R of the P53 genes are assembled, TALEN plasmid is transfected and screened to obtain a P53 gene knockout positive cell line, reconstructed embryos are constructed based on the somatic cell nuclear transplantation technology and transplanted into the body of a surrogate sow to continue to develop, and a piglet with the P53 genes knocked out is obtained; the function of the P53 genes is verified by detecting the expression level of RNA and protein of the P53 genes in tissue of the cloned piglet and downstream related genes, and lymphoma in the tissue of the piglet is identified through histopathology and an immunohistochemical method. The built lymphoma disease model has great significance in research and development of occurring, forming and medicine of human lymphoma, and the reliable model is provided for research and treatment of human lymphoma diseases.

The invention discloses an expression vector resisting PCV2 and a transgenic pig and construction methods thereof, belonging to the field of gene engineering. The expression vector is constructed by respectively connecting an H1 promoter and a pPB-sh2 sequence to a pUC57-H1 plasmid and then connecting a fusion fragment containing a loxP-Neo/EGFP gene expression cassette to pPB-H1-sh2. The vector is introduced into the genome of a pig, and somatic cell nuclear transfer technology is employed to produce the transgenic pig. The expression vector provided by the invention has a piggyback transposon, greatly improves transformation efficiency, changes a common plasmid series recombination transgene integration model into a single-site single-copy integration model and better simulates the internal environment of a biological gene. The constructed transgenic pig degrades PCV2mRNA through siRNA synthesized by itself, thereby fundamentally inhibiting PCV2 infection.

The invention belongs to the technical field of biology, and specifically discloses a targeting vector for pig GHR gene knock-out and applications thereof. The targeting vector is a sgRNA expression vector based on a CRISPR/Cas system; wherein the target sequences of sgRNA are arranged on the 10th exon and 3'UTR of a pig GHR gene. The provided targeting vector is applied to targeting knock-out ofpig GHR gene, and the knock-out efficiency can reach 12.7%. After the GHR gene is successfully knocked out, somatic cells can be used to establish a GHR gene knock-out pig model by a somatic cell nuclear transplanting technology, and materials are provided for the research of physiological action and molecular mechanism of GHR gene.

The invention discloses a reconstructed oocyte of a deaf model pig and a construction method of the reconstructed oocyte, and also discloses the deaf model pig and a construction method and application of the deaf model pig. According to the reconstructed oocyte of the deaf model pig, the construction method of the reconstructed oocyte and the construction method of the deaf model pig, by knockingout a pig OSBPL2 gene, the reconstructed oocyte of a deaf mini-pig is obtained, and in combination with a CRISPR/Cas9 gene editing technology, a somatic cell nuclear transfer technology and an embryotransfer technology, high efficiency and feasibility of the construction method for the genetically engineered deaf model pig are proven. Through the construction method of the deaf model pig, genotypes and deafness phenotypes of human OSBPL2 gene mutations can be precisely duplicated, the pig of which an OSBPL2 gene is knocked out has typical characteristics of human autosomal dominant hereditary hearing loss (DFNA67), therefore, a reliable big animal model can be provided for disease research of the human hereditary hearing loss, and the deaf model pig can be applied to inner-ear gene repairing, inner-ear hair cell regeneration, hearing reconstruction and the like.

The invention discloses a building method of a donor pig suitable for xenotransplantation of tissue like islet cells and skin and belongs to the technical field of animal biology. The building methodincludes: on the basis of porcine fetal fibroblast, utilizing CRISPR/Cas9 gene editing technology to knock out three genes GGTA1, CMAH and B4GalNT2 at the same time, and inserting three humanized modifying genes hCD55, hCD59 and hCD46 in a fixed point manner; combining somatic cell nuclear transfer technology to build an xenotransplantation donor base pig modified by the above six genes; on the basis of the base pig, knocking out gene B2M, and inserting three modifying genes hCD47, A20 and CTLA4-Ig in a fixed point manner at the same time; utilizing the somatic cell nuclear transfer technologyto build a islet cell and skin xenotransplantation donor pig modified by multiple genes; performing functional verifying on the donor pig to obtain the donor pig suitable for islet cell and skin xenotransplantation. A brand-new research concept and method are provided for developing the donor pig suitable for xenotransplantation of the islet cells and the skin, and clinical conversion and application of pigs serving in heterogeneous islet cell and skin transplantation are promoted.

Provided is a method for nuclear transfer. An exogenous donor nucleus is introduced into an enucleated oocyte and one or more enucleated sperm cells or one or more enucleate sperm cell fractions are introduced into the oocyte. The one or more sperm cells or one or more sperm cell fractions may be introduced into the oocyte either before, after, or simultaneously, with the donor nucleus. Also provided are cells and embryos produced by the method.

A process for preparing transgenic she-goat to secrete human histotypic plasminogen activator mutant by nuclear transplant method features that the human histotypic plasminogen activator mutant regulated by the regulator sequence of beta-casein can be expressed in the milk gland of transgenic she-goat. It includes configuring carriers pGBCN-tPAm, pGBC2-tPAm and pGBC4-tPAm, transfecting the fibroblast of milk she-goat fetus, and cloning by nuclear transplanting.

The invention discloses a method for breeding transgenic buffalos by using somatic cell nuclear transfer technology, which comprises: obtaining buffalo fetal fibroblasts (BFF) by separation from buffalo fetuses and culture, implanting a transgenic vector (pCE-EGFP-IRES-neo), which carries a neomycin resistant gene and an enhanced green fluorescent protein (EGFP) gene, into the BFF by an electroporation process, and selecting BFF into which the EGFP gene is transferred by using G418; transplanting the BFF into which the EGFP gene is transferred into denucleated oocytes of the buffalo to construct cloned embryos, treating the cloned embryos for 5 minutes with ionomycin at a concentration of 5 mu mol/L, culturing cloned embryos in 6-dimethyl-aminopurine at a concentration of 2mmol/L for 3 hours, performing chemical activation treatment, co-culturing the cloned embryos in granulose cell monolayer liquid drops for 6 to 7 days, and obtaining the transgenic cloned blastaea; selecting transgenic cloned blastaea in which the EGFP is expressed under a reversed fluorescence microscope, and transplanting the transgenic cloned blastaea into receptor buffalos; and obtaining the transgenic buffalos after full-term pregnancy. After the irradiation by an ultraviolet lamp, the EGFP marker genes are obviously expressed in the heads and limbs of the transgenic buffalo calves, and this proves the method disclosed by the invention can successfully breed transgenic cloned buffalos.

The method of preparing nucleus embryo stem cell by means of somatic nucleus transplanting technology is one method of somatic nucleus transplanting process to prepare cell. Under microphone operation instrument, nerve nucleus of adult animal is transplanted into denucleated embryo stem cell cytoplast to form embryo stem cell with completely new genome, and the new embryo stem cell is cultured to divide and proliferate to 28 pieces. Embryo stem cell and nerve cell are homologous cells and the prepared cell is nucleus transplanted embryo stem cell with the genome of donor cell. The present invention solves serial problems in somatic nucleus transplanting and can promote the development of cell engineering and tissue engineering relevant to human embryo stem cell.

The invention relates to a method for obtaining cloned pigs with modified genes through continuous cloning and belongs to the field of animal reproductive biology.According to the method, tissue fibroblasts of obtained genetically modified pigs serve as donors, donor cells are transferred into enucleated porcine oocytes through the somatic cell nuclear transfer to build a genetically modified pig reconstructed embryos, then electro-fusion and electro-activation are conducted, the donor cells are transferred into estrous surrogate sow fallopian tube expanded portions after being cultured in a pig embryo in-vitro culture solution for 3 h, and after gestation of surrogate sows last for 114 d, a large scale of genetically modified pig individuals are obtained through delivery.The method can solve the problems that in the process that genetically modified individuals are obtained through the gene editing technology, target genetically modified individuals are low in efficiency, and difference between the target genetically modified individuals is large, so that genetically modified cloned pig individuals stable in target gene expression and phenotype can be efficiently produced on a large scale.

The invention provides a transgenic miniature pig for preparing a human hyperlipemia disease animal model and a preparation method thereof. A human apolipoprotein CIII expression vector pcDNA-CIII is constructed and expressed by using a gene engineering method; a miniature pig fibroblast capable of expressing the human apolipoprotein CIII is screened out by using cell transfection and cell screening technologies; and the miniature pig is cloned by using a somatic cell nuclear transfer technology. The cloned miniature pig can be used for research into pathogenesis of human hyperlipemia related diseases, and screening and preclinical pretesting of medicine intervention and treatment of related diseases.

The invention relates to a method for producing a parthenogenetic cloned pig by means of continuous cloning technology, which belongs to the field of reproductive biology. The method provided by the invention comprises the following steps of: the degreasing of a porcine oocyte, parthenogenetic activation, the separation of a fibroblast, and continuous somatic nuclear transfer; and specifically, the method comprises the following steps of: carrying out the parthenogenetic activation on the degreased mature porcine oocyte to obtain a parthenogenetic embryo, transplanting the parthenogenetic embryo into the body of a surrogate sow to obtain a 25-29 days old living parthenogenetic fetus, establishing a porcine parthenogenetic fetus fibroblast line which is used as a preliminary donor cell, and carrying out continuous cloning by means of somatic cell nuclear transfer technique to obtain a porcine parthenogenetic individual. By adopting the method provided by the invention, the condition that the in-vivo development stops when the porcine parthenogenetic fetus grows for 30 days is overcome, the in-vivo development time of the porcine parthenogenetic fetus is successfully prolonged, and therefore a living parthenogenetic cloned pig can be successfully obtained finally.

The invention discloses a preparation and in-vitro culture method of interspecies-cloned yak embryos. According to the method, yak-cattle interspecies-cloned embryos are constructed by using yak mammary glandular cells as donor cells and cattle or milch cow enucleated oocytes as receptor cytoplasts by a somatic nucleus transplantation technique; and an electrical activation-chemical activation-reelectrical activation mode and a self-made sequential culture system are utilized to acquire the high-efficiency high-activity yak-cattle interspecies-cloned embryos. The method disclosed by the invention can increase the yak breeding speed, shorten the yak breeding cycle, lower the yak production cost, enhance the breeding efficiency, reduce the damage level of the grassland to some extent, and effectively protect the species resource of the yak. Besides, the invention has important meanings for promoting research of interspecies-cloned yak, transgenic cloned yak embryos and nucleo-cytoplasmic interaction.

The invention discloses a preparation method of an anti-African swine fever cloned pig and belongs to the field of animal genetic engineering and genetic modification. A CRISPR/Cas9 gene knock-in technology is used, a CRISPR/Cas9 targeting vector and a pig Rosa26 gene donor vector are jointly transferred into a pig fibroblast to obtain a positive cell clone, a first intron of the pig Rosa26 gene in the positive clone is knocked in a gene capable of expressing sgRNA-Cas9 of a specific targeted knockout virus pE248R gene, such that the pE248R gene is knocked out in a targeted manner when viruses are propagated in vivo and a pig has ASFV resistance; a cloned embryo is obtained through a somatic cell nuclear transfer technology; and the cloned embryo are transferred into a pig uterus for pregnancy to obtain a cloned pig. The sgRNA-Cas9 system is knocked into a pig body for the first time and stably expressed to obtain ASFV resistance, such that a foundation is laid for breeding research on African swine fever resistance.