164 results about "Clone cell" 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 discloses a recombinant vector for the knock-in of a human Huntington gene, a construction method of the recombinant vector and application of the recombinant vector in the constructionof a model pig. According to the recombinant vector, a human mutated Huntington exon gene is knocked in a fixed point manner for the first time, a virulence gene is knocked in by virtue of a CRISPR / Cas9 technique for the first time, and a donor vector is optimized by optimizing sfRNA, so that the probability that the gene is knocked into a positive cloned cell is increased; and by combining with apig cell nucleus transplantation technique, the probability that a directly obtained positive cell is knocked into the pig is increased, and a small human Huntington gene knock-in pig is obtained, thereby proving the efficient feasibility of the method for constructing gene modified pigs. The constructed Huntington gene knock-in model pig has behavioral characteristics such as respiratory disturbance and dyskinesia similar to human Huntington diseases, and stable heritable passage can be realized, so that a reliable model is provided for the research of the human Huntington diseases; and thenumber can be guaranteed so as to realize drug screening, gene treatment, stem cell treatment and the like, and the model can be a good human disease model.

The invention discloses a method for obtaining transgenic bovine fetal fibroblast by using Cas9 cutting nuclease-mediated Ipr1 fixed point insertion. The bovine fetal fibroblast is cotransfected by adonor vector and a CRISPR / Cas0 expression vector for a target locus through an electroporation method, an MSR1 promoter in the donor vector can realize specific expression in full-time phagocytic cells, and after puromycin drug screening, the targeted positive clone cell is obtained through PCR authentication. The transgenic positive clone cell is used as a donor cell so as to obtain a transgenicclone embryo, the transgenic clone embryo is further transplanted into the uterus of a rutting acceptor calf, and finally a living Ipr1 fixed point insertion transgenic cloned calf is obtained.

Provided are methods and compositions for obtaining genome-engineered iPSCs, and derivative cells with stable and functional genome editing at selected sites. Also provided are cell populations or clonal cell lines derived from genome-engineered iPSCs, which comprise targeted integration of one or more exogenous polynucleotides, and / or in / dels in one or more selected endogenous genes.

The invention discloses a method for screening monoclonal antibodies on different antigen binding sites. The method comprises the following steps: preparing an antigen specific monoclonal antibody by an antigen immune animal with two or more antigenic determinants through a hybridoma monoclonal technology; carrying out in-vitro cell culture on positive clone cell strains to obtain cell culture liquid supernatant containing the antigen specific monoclonal antibody; adding the hybridoma cell culture liquid supernatant into a solution of rubber latex grains which are covalently cross-linked with protein A or G in a two-and-two combined manner; and incubating and adding antigens to obtain a monoclonal antibody set with the increased light absorbance, namely obtain the monoclonal antibody with the different antigen binding sites.

The current invention provides a modification procedure that reduces errors in integrated circuits due to via shorts while at the same time avoiding the unnesting of the layout design and thereby permitting verification of the layout design by LVS testing tools. The current invention identifies if potentially shorting vias have electrically redundant paths and, if so, creates cloned cells of the original cell but void of the potentially shorting vias. The cloned cell is electrically comparable to the original cell. In addition, each instantiation of the original cell in the shapes data base is replaced with the cloned cell when electrical redundancy is present. Also, the number of vias removed can be minimized or maximized while, at the same time, all via electrical shorts are removed, depending on the design requirements.

The invention relates to a hybridoma cell strain secreting a fluridone monoclonal antibody. The hybridoma cell strain is named as a monoclonal cell strain LKS, and is preserved in China General Microbiological Culture Collection Center (CGMCC) on May 13, 2021, the preservation address is No. 3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 22324. The hybridoma cell strain disclosed by the invention can efficiently and stably secrete the fluridone monoclonal antibody and has relatively good sensitivity and specificity when being applied to immunoassay detection of fluridone, the IC50 value is 0.86 ng / mL, and the cross-over rate of fluridone analogues is less than 10%.

The invention discloses a method for constructing a CHO cell strain for stably and efficiently expressing a human serum albumin and an interleukin II fusion protein. The CHO monoclonal cell strain for stably and efficiently expressing the human-derived recombinant protein can be obtained by electrically transferring a plasmid with the human serum albumin and the interleukin II fusion gene into the CHO cell. The monoclonal cell strain obtained by the invention is capable of secretory expression of the human serum albumin and the interleukin II fusion protein, the protein expression quantity is high, and the fusion protein can be obtained through the separation and purification; the in vitro biological activities of two fusion proteins are higher than the mol monomer 1L-2 activity. The CHO cell strain can be widely applied to the medicines for treating various diseases such as tumor, hepatitis, pneumonia and immunodeficiency disease.

The invention discloses a method for identifying sensitivity of an antibody as well as quality of a clone cell strain in the preparation process of the antibody. The method comprises the following steps: acquiring a solid-phase carrier, cells and the antibody, adsorbing the antibody on the solid-phase carrier, incubating the cells and the antibody adsorbed on the solid-phase carrier, keeping cells that are combined with the antibody, dyeing and counting the cells combined with the antibody, and identifying the sensitivity of the antibody and the quality of the clone cell strain according to the cell counting result. The invention further provides a kit applicable to the method. The method intuitively reflects the level of combination of the antibody and a native conformation antigen, and the identifying method is simple, quick and intuitive.

The invention provides a highly classical swine fever virus infective swine testicular clone cell strain ST-B2, which has a preservation number of CCTCC NO.C2011101, and a preparation method of the swine testicular clone cell. The method comprises the steps of: 1) subjecting a swine testicular cell to limited dilution, conducting cell cloning and subcell cloning, thus obtaining a subcellular clone strain; and 2) selecting a subclone strain of the swine testicular cell with a highest classical swine fever virus infection rate, i.e. the highly infective swine testicular clone cell of the classical swine fever virus. The invention also provides a method for preparation of a high titer classical swine fever virus solution and a classical swine fever live vaccine from the swine testicular clone cell. The swine testicular clone cell provided in the invention has a high classical swine fever virus infection rate, and the classical swine fever vaccine produced from the swine testicular cell line ST clone cell strain ST-B2 by a virus-carrying and virus transmission technique has high virus titer, hard exposure to BVDV (bovine viral diarrhea virus) pollution and good pureness. By the virus-carrying and virus transmission technique, a resurgent cell can undergo continuous passage to at least 15 generations. The cell resurrection and virus inoculation times can be reduced, the production process is simplified, and the production efficiency is improved.