29 results about "Clone cell" patented technology

The invention discloses an anti-phenacetin monoclonal antibody hybridoma cell strain AD, a preparation method and application thereof, and relates to the technical field of food safety immunodetection. The monoclonal antibody hybridoma cell strain is named monoclonal cell strain AD and the number CGMCC19681. The Phe-BA obtained by the hydrolysis of the reaction product of the phenacetin metabolite acetaminophen and ethyl 4-bromobutyrate is used as the hapten, and the hapten is coupled with the carrier protein to prepare the immunogen Phe-BA-BSA. After the mice were immunized with the immunogen Phe-BA-BSA, they were fused with myeloma cells by PEG method, screened by indirect competitive enzyme-linked immunosorbent assay and subcloned five times to obtain hybridoma cell lines. The monoclonal antibody secreted by the cell line can be made into a phenacetin detection kit, which has good affinity and detection sensitivity for phenacetin, and can be used for immunodetection of phenacetin residues in food.

The present disclosure relates to novel Spodoptera frugiperda cell lines free of Sf9 rhabdovirus, methods for making and methods for using the cell lines. In one aspect, the method includes single cell isolation to obtain a cloned cell line and to identify deletion of Sf9 rhabdovirus. In one aspect, the disclosure provides novel cell lines and kits comprising the cell lines. In one aspect, the disclosure provides a method for producing a biological product using the cell line.

The application discloses a kind of immortalization human umbilical cord mesenchymal stem cell NOTCH1-LNR overexpression stable cell line construction method and its application in liver differentiation, it is related to immortalized cell technical field.The application first connects target gene to vector, and the slow virus plasmid containing target gene is constructed, the slow virus plasmid containing target gene is introduced into 293T cell, and high titer slow virus containing target gene is generated;Immortalized human umbilical cord mesenchymal stem cell is infected with slow virus, and mixed clone cell strain is screened, and the immortalized human umbilical cord mesenchymal stem cell NOTCH1-LNR overexpression stable cell line is obtained.The application proves that the differentiation efficiency of immortalized human umbilical cord mesenchymal stem cell NOTCH1-LNR overexpression can effectively improve liver-like cell to glycogen staining and Western blotting experiment.This research result is expected to provide new technical thought for the next step development to improve the yield of MSCs liver differentiation.

The invention provides a method for rapid cloning of T-cell receptors (TCRs) (e.g., paired αβ and γδ TCR chains) and B-cell receptors (BCRs) (e.g., paired IgH or IgK or Igλ) from single cells by CDR3 substitution using single cell PCR products and Gibson Assembly techniques and a pre-generated TCR (or BCR) library in an expression vector.

The invention provides an immortalized livestock precursor fat cell line and a preparation method thereof, and belongs to the technical field of biology. The invention provides a preparation method of an immortalized livestock precursor adipocyte line, which takes adipose tissues of livestock larvae as a cell source, the cell source contains a small amount of mature adipocytes, but a main cell population is precursor adipocytes, so that the purity of a starting material for subsequent immortalization is ensured; after immortalized induction, the monoclonal cell strain is screened by using a limited dilution method, the immortalized monoclonal cell strain still has the capability of being differentiated into mature adipocytes and forming lipid droplets after induced differentiation treatment, and the immortalized cell line can keep normal cell morphology and overcome replicative senescence. The immortalized cell strain disclosed by the invention can be efficiently differentiated into fat cells with mature functions after being induced, and has normal lipid synthesis and storage capabilities.

The present application relates to a CRISPR-mediated immortalized sheep fetal skin fibroblast cell line in the field of variation or genetic engineering and application thereof.The construction of the CRISPR-mediated immortalized sheep fetal skin fibroblast cell line of the present application comprises the following steps: step one, constructing a Cas9 site-directed knockout plasmid containing an FGF5 gene target sequence; step two, constructing a homologous repair plasmid Donor-2 kb HA-SV40LT containing an FGF5 gene homologous arm; step three, constructing a donor DNA containing an SV40LT protein expression frame; and step four, preparing a sheep fetal fibroblast cell line stably expressing SV40LT protein.The immortalized SFF-FGF5 / SV40LT monoclonal cell strain created by the present application can become an ideal tool cell line for genome editing researches such as sheep CRISPR library screening, multiple gene editing and mutation gene function exploration.

The invention relates to a seawater oryzias medaka stable expression cas9 cell line, a construction method and application, and belongs to the technical field of gene editing, the cell line takes seawater oryzias medaka cells as a main body, and a cas9-Blasticidin gene is inserted into a seawater oryzias medaka cell genome. The construction method of the cell line comprises the following steps: infecting a testis cell line of oryzias melastigma by lentivirus to realize stable insertion of a cas9 gene in an oryzias melastigma genome, and screening a monoclonal cell line to obviously improve the expression quantity of cas9 in the cell line. The invention also provides a method for constructing the oryzias melastigma cell line by utilizing the cell line to edit the mstnb gene of the oryzias melastigma cell, and the method for constructing the oryzias melastigma cell line. The mstnb gene is successfully mutated by transfecting sgRNAs in the cell line.

The utility model discloses a cell cloning loop culture dish, which relates to the technical field of cell culture and comprises cell culture plates with uniform specifications. The cell culture plate is characterized by comprising a bottom plate which is arranged on the bottom surface of the cell culture plate; a cell cloning ring is arranged in the cell culture plate; a top cover is arranged at the upper end of the cell culture plate; the bottom plate and the cell cloning ring are fixed through magnetic attraction and have color difference; the cell cloning ring is arranged in the culture groove of the cell culture plate; the cell cloning ring fixing device has the beneficial effects that the original cell cloning ring is more accurately and stably fixed on the cell culture plate, and meanwhile, pancreatin is not leaked to a non-target area to digest cells. It is guaranteed that all cells obtained after digestion and elution are from the same cell clone cluster, and the purpose of accurately selecting monoclonal cell colonies is achieved.

The invention discloses a method for preparing a CD163 gene editing pig based on a CRISPR (clustered regularly interspaced short palindromic repeats) / Gs12-7MAX system. The method specifically comprises two editing strategies: one is to edit a sixth intron and a seventh intron of a CD163 gene to realize accurate deletion of a seventh exon so as to obtain a porcine fibroblast monoclonal cell with homozygous deletion of the seventh exon; and 2, editing aiming at a seventh exon of the CD163 gene, and inducing the exon to generate frame-shift mutation to obtain a gene editing pig. The technical system established by the invention has the advantages of high editing efficiency, strong accuracy and simplified operation process, provides a new effective strategy for prevention and control of PRRSV (porcine reproductive and respiratory syndrome), and has important popularization and application values in creation of new materials for pig disease-resistant breeding.

This invention relates to a hybridoma cell line that secretes a monoclonal antibody against azoxystrobin and its application, belonging to the field of immunochemistry. The hybridoma cell line of this invention was deposited on November 12, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46742, and classified as a monoclonal cell line. The monoclonal antibody secreted by the invented hybridoma cell line exhibits good detection sensitivity (IC50) against azoxystrobin. 50 With a value of 0.049 ng / mL, it can be used for the detection of antimicrobial residues.

This invention relates to a hybridoma cell line that secretes a monoclonal antibody against methylphenidate and its application. The hybridoma cell line was deposited on October 31, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46223, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and is classified as a monoclonal cell line. The monoclonal antibody against methylphenidate secreted by this hybridoma cell line exhibits good detection sensitivity (IC50) for methylphenidate. 50 With a value of 1.02 ng / mL, it can be used for the detection of methylphenidate residues.

The invention discloses a method and a kit for rapidly screening gene editing positive monoclone based on qPCR (quantitative polymerase chain reaction), and belongs to the field of gene engineering. The method comprises the following steps: preparing a monoclonal sample: preparing a single-cell suspension from cells subjected to gene editing, and carrying out single-cell sorting and culture until monoclone is formed; collecting monoclonal cells, and directly cracking by using a cracking solution to prepare a qPCR template; qPCR detection: carrying out qPCR amplification and detection on the sample by using a specific primer and a probe designed aiming at a target gene editing event; and result judgment: judging whether the monoclone is gene editing positive clone or not by analyzing the Ct value of the qPCR. According to the present invention, the specific primers and the probe of the target gene editing site are designed, and the qPCR is combined to directly detect the genome variation of the monoclonal cell so as to achieve the early rapid screening;

The invention relates to the technical field of biology, in particular to a human pluripotent stem cell recurrent mutation monoclonal cell strain and application thereof, and the cell strain can provide a reference model for research on phenotypes, mechanisms and related diseases of recurrent mutation of pluripotent stem cells.

A myogenin-expressing fibroblast-like cell (MEFLC) line and a construction method and a use thereof are provided. The MEFLC line was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on Apr. 23, 2022 with an accession number of GDMCC NO: 62409. The construction method includes: 1) constructing a pCW-MYOG-T2A-Puro lentivirus; 2) infecting a human induced pluripotent stem cell (iPSC) line with the lentivirus, inducing the expression of an MYOG gene, and screening out a positive monoclonal cell line; 3) subjecting the positive monoclonal cell line to expanded cultivation, during which the expression of the MYOG gene is continuously induced; and 4) changing cultivation conditions, continuously inducing the expression of the MYOG gene, and screening out positive monoclonal cell lines to ensure the purity of MYOG-positive cells until a cell morphology changes significantly into fibroblastoid cells to obtain the MEFLC line.

The present application relates to the field of cell culture, in particular to a culture and screening method of MDCK monoclonal cell strain, and provides a culture method of MDCK monoclonal cell strain, comprising the following steps: S1, culturing pretreated cells to logarithmic phase; S2, inoculating the cells in logarithmic phase in S1 to the first culture carrier after diluting to a concentration of 1 cell / 100 μL; S3, inoculating the single cell colony in the first culture carrier in S2 to the second culture carrier after digestion, until the cell fusion degree reaches 70-80%, and obtaining the MDCK monoclonal cell strain. The present application realizes early detection without expanding the monoclonal cells to T75 culture flask, avoids the step of gradually expanding and culturing for 7-10 days in the traditional method, greatly shortens the overall screening period from the original 14-21 days, and can quickly match the demand of H3N2 virus variation for highly adaptive cell strains.

This invention discloses the preparation and application of a universal mRNA vaccine for H5N1 influenza. The mRNA vaccine contains mRNA molecule 1 and mRNA molecule 2; mRNA molecule 1 encodes the H5m protein; mRNA molecule 2 encodes the N1m protein; the H5m protein is the protein shown in SEQ ID NO:1; the N1m protein is the protein shown in SEQ ID NO:2. The universal mRNA vaccine for H5N1 influenza prepared by this invention possesses broad-spectrum protection, rapid iteration, and low-cost mass production characteristics. It can efficiently express native conformational proteins in host cells, not only covering the main variation spectrum of the H5N1 virus, but also overcoming the broad-spectrum limitations of traditional vaccines by activating cross-neutralizing antibodies and polyclonal T-cell immunity. This invention provides key technical support for addressing the continuous evolution and potential pandemic of H5N1.

The application discloses a platelet detection kit based on multi-parameter flow cytometry and a use method thereof, relates to the technical field of in-vitro diagnosis, and covers the core pathological links of thrombosis and downstream key events of thrombosis by simultaneously detecting the proportion of PNH clone cells, the platelet activation state and the proportion of platelet-leukocyte aggregates (PLAs), thereby efficiently and accurately evaluating the thrombosis risk of PNH patients. The application directly detects cell surface markers and cell-cell interaction by using multi-color flow cytometry, can early and sensitively identify abnormal activation signals, and avoids the limitations of traditional coagulation indexes (such as D-dimer) in PNH thrombosis early warning.

The disclosure belongs to the technical field of animal bioengineering. Disclosed is a method for knocking out a bovine FBXO40 gene and an application thereof. A CRISPR / Cas12i system is used to knock out the bovine FBXO40 gene. The CRISPR / Cas12i system targets exon 5 of the bovine FBXO40 gene, and a nucleotide sequence of the exon 5 of the bovine FBXO40 gene is shown in SEQ ID No. 1. In the disclosure, a method for precise knockout of the bovine FBXO40 gene using a CRISPR / Cas12i gene editing technology is provided, and stably inheritable monoclonal cell lines and embryos with gene knockouts are successfully constructed. The method can provide experimental references for studying a molecular mechanism for bovine skeletal muscle development, and can also be applied in the breeding of new cattle breeds with high-meat-yielding.

The application discloses a preparation method of cat induced pluripotent stem cells, which comprises the following steps: constructing piggyBac transposon plasmids carrying cat source OCT4, SOX2, KLF4 and c-MYC four factors in series, piggyBac transposon plasmids carrying cat source or pig source NANOG and LIN28 two factors in series and piggyBac transposon plasmids carrying monkey kidney virus SV40LT plasmids, and transfecting the plasmids into cat fetal fibroblasts; picking up cloned cells with embryonic stem cell morphologies, and carrying out subculture of cell clones meeting the characteristics of embryonic stem cells through identification and screening, so that the cat induced pluripotent stem cells are obtained. The preparation method of the cat induced pluripotent stem cells is safe, efficient and has good differentiation potential, lays a scientific foundation for researching in-vitro culture conditions of feline ESCs and establishment of feline iPSCs, and lays a scientific foundation for further cultivating cloned cats and gene modified cats and researching in related disease mechanisms, screening drugs and regeneration and repair.

The invention discloses a cell strain A375-H for constructing a melanoma liver metastasis model as well as a construction method and application of the cell strain A375-H, and relates to the technical field of biology, and the preservation number of the cell strain A375-H is CGMCC No.46344. The cell strain is classified and named as an A375 high metastatic melanoma cell strain. The A375-H cell strain with high metastatic property and liver metastatic potential is efficiently and conveniently obtained by adopting a continuous 10-round Transwell screening combined monoclonal cell selection method. The A375-H cell strain has high liver metastasis invasion potential, and a unique seed cell model is provided for researching the liver metastasis mechanism of melanoma.

The invention discloses an immortalized bovine muscle-derived stem cell line as well as a construction method and application thereof, and belongs to the technical field of stem cells and biological cell lines. The bovine muscle-derived stem cell line is characterized in that telomerase reverse transcriptase (TERT) and cyclin dependent kinase (CDK) genes are overexpressed in a mode of lentivirus packaging and infection, a cell strain which is single in source and stably expresses a target gene is obtained through monoclonal screening, gene verification and functional verification, and the verification shows that the cell line overexpresses the CDK4 and TERT genes, so that the cell line can be used for preparing the bovine muscle-derived stem cell line with the stable expression of the target gene. The monoclonal cell strain can stably proliferate and maintain good differentiation characteristics. The immortalized bovine muscle-derived stem cell line is prepared, and a good seed cell resource is reserved for commercialized production of cultured meat.

The invention relates to the technical field of biological medicine, and discloses a construction method and application of a CAR-M cell based on an ICAM-1 targeting ligand gene, a transfer plasmid containing the ICAM-1 targeting ligand gene, a VSV-G envelope plasmid and a lentivirus packaging auxiliary vector are transfected to an HEK293T packaging cell, after transfection is finished, a clear virus solution is obtained through collection and culture, and after concentration, the CAR-M cell is obtained. The preparation method comprises the following steps of: adding the mCherry-ICAM-1 lentivirus suspension into a culture medium; tHP-1 cells are transduced by using the virus, and then the transduced THP-1 cells are sequentially subjected to antibiotic screening and monoclonal cell screening, so that CAR-M cells are obtained. According to the invention, the ICAM-1 extracellular domain is integrated into the extracellular antigen binding region of the CAR, so that the CAR-M cell is successfully constructed, and the macrophage is endowed with the capability of directionally recognizing and eliminating iRBCs.

The invention relates to systems comprising at least a cell culturing device and a cell sorting device, and optionally a direct connection from the cell culturing device to the cell sorting device, and a direct connection from the cell sorting device to the cell culturing device. The invention further relates to methods for developing and / or optimizing a cell culture process, comprising at least the steps of growing a cell culture of a polyclonal population of cells in a cell culturing device and selecting cells in the cell sorting device, and optionally transferring cells from the cell culturing device into a cell sorting device by direct connection(s) from the cell culturing device to the cell sorting device, and recirculating selected cells from the cell sorting device back to the cell culturing device by direct connection(s) from the cell sorting device to the cell culturing device.

The invention belongs to the technical field of ST cell full suspension culture, and particularly relates to a rapid domestication method suitable for serum-free suspension culture of ST cells, which is characterized by comprising the following steps: (1) gradient domestication stage: according to a conventional gradient domestication method, reducing the concentration of adherent ST cell culture serum to 2%; (2) low-serum monoclonal screening: screening monoclonal cell strains with growth advantages in a low-serum culture medium through a limited dilution method, and carrying out enlarged culture; and (3) serum-free suspension adaptive domestication: digesting the amplified and cultured monoclonal cells, and transferring the digested monoclonal cells into a shake flask for serum-free suspension adaptive domestication. The invention provides an efficient and low-cost ST cell serum-free suspension domestication method, which can completely eliminate adherent dependence, improve cell density to 8.0 * 10 < 6 > cells / ml, ensure that the motility rate is greater than 93% after continuous passage of 160 generations, and verify genetic stability and cryopreservation adaptability.

The invention provides a construction method of a monoclonal cell strain for knocking out a Tan sheep FABP4 gene, which comprises the following steps: knocking out the Tan sheep FABP4 gene by using a CRISPR / Cas9 system, targeting a first exon of the Tan sheep FABP4 gene by using the CRISPR / Cas9 system, and showing a nucleotide sequence of the first exon of the Tan sheep FABP4 gene as SEQ ID NO.1; sgRNA in the CRISPR / Cas9 system targets the nucleotides at the 2-8 sites at the 5'end in the sequence SEQ ID NO.1, and the nucleotide sequence of the sgRNA is as shown in SEQ ID NO.2; the method comprises the following steps: constructing a Lentiti-CRISPR V2-Puro-OaFABP4-sgRNA recombinant plasmid, and carrying out lentivirus packaging, drug screening and monoclonal screening, so as to obtain the monoclonal cell strain of which the Tan sheep FABP4 gene is knocked out. The invention also provides application of the monoclonal cell strain, and the monoclonal cell strain is used for constructing a cell model of a Tan sheep precursor fat cell differentiation regulation mechanism. According to the invention, the Tan sheep FABP4 gene is knocked out by using a CRISPR / Cas9 system, so that the research on the function and regulation mechanism of the FABP4 gene in Tan sheep precursor fat cell differentiation is facilitated, and a new molecular target is provided for controlling excessive fat accumulation.

The invention provides a preparation method and application of a multi-type CAR-immune cell mixed product containing CAR-macrophages and T cells, and relates to the technical field of biology. The preparation method of the mixed product containing the CAR-macrophages and the T cells, provided by the invention, comprises the following steps: culturing peripheral blood mononuclear cells obtained by separation in a culture medium, introducing a gene for coding a chimeric antigen receptor into the macrophages, and continuously culturing to obtain the multi-type CAR-immune cell mixed product containing the CAR-macrophages and the T cells. According to the preparation method, the mixed multi-type CAR-immune cell (CAR-mix) containing CAR-macrophages and T cells is prepared by adopting a one-step method, the cell yield is high, the proportion of the CAR-macrophages to the T cells in the prepared immune cell is proper, the synergistic effect of the CAR-macrophages and the T cells can be fully exerted, the generation of polyclonal T cells is stimulated, and the CAR-mix is used for preparing the CAR-macrophages and the T cells. A stronger solid tumor resisting function is realized.

The application discloses a cell strain A375-H for constructing a melanoma liver metastasis model and a construction method and application thereof, relates to the technical field of biology, and has a preservation number of CGMCC No.46344; and a classification name of A375 high-metastasis melanoma cell strain. A375-H cell strain with high metastasis and liver metastasis potential is efficiently and conveniently obtained by using a continuous 10-round Transwell screening combined with a single clone cell picking method. The A375-H cell strain has high liver metastasis potential, and provides a unique "seed cell" model for studying a liver metastasis mechanism of melanoma.

The invention provides a high-yield single cell screening method and system, a terminal and a medium, single cells are screened based on cell images in nozzles of a printing chip after a cell solution is applied, a target cell sample is determined according to a protein yield detection result detected after the screened single cells are cultured, and a single cell recognition model is constructed; and based on the single cell identification model, determining screened high-yield single cells according to an input cell image in each nozzle of the printing chip after the cell solution is applied. According to the method, the cells in the nozzle of the printing chip are identified through the image, and the high-yield cells are screened out, so that the construction efficiency of the monoclonal cell strain can be improved.

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.