57 results about "Clone cell" patented technology

The invention discloses a double-gene screening expression vector of a CHO monoclonal cell strain and a preparation method and application of the double-gene screening expression vector, the double-gene screening expression vector comprises a glutamyl synthetase GS screening gene and an antibiotic screening gene, the antibiotic screening gene is subjected to weakening expression treatment, that is, an IRES sequence exists in front of the antibiotic screening gene, and an IRES sequence exists in front of the antibiotic screening gene. A target expression gene and an antibiotic screening marker gene share one set of promoter and terminator, and when the double-gene screening expression vector disclosed by the invention is used for screening CHO monoclonal cell strains, the screening efficiency can be remarkably improved, the screening time is saved, and the target gene has high expression quantity.

The invention provides a construction method and application of a gene modified K562 cell line, and belongs to the technical field of biological medicines. According to the invention, two viruses CD86-4-1BBL and mbIL-21 are respectively subjected to gene transduction packaging by adopting a third-generation lentivirus four-plasmid packaging system, a K562 cell line is infected by the lentiviruses CD86-4-1BBL and mbIL-21, and a monoclonal cell line capable of stably expressing CD86, 4-1BBL and mbIL-21 proteins is obtained through screening. According to the method, cell lines with the cloning numbers of V5-E9, V10-A5, V5-D6 and V6-D7 are obtained through screening, the four cloning cell lines can serve as feeder cells for stimulating NK cell amplification after being amplified and subjected to irradiation treatment, NK cell amplification can be effectively promoted, the amplification multiple of the NK cells reaches up to 6000-8000, and large-scale preparation and production of the NK cells for treatment are met.

The invention relates to the technical field of biology, in particular to a method for cloning medicinal plant stem cells and obtaining a high-yield cell line, and the cell line with high yield of specific active substances is obtained through establishment of a suspension cell line, obtaining of single cells, cell cloning culture and screening of cloned cells.

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 invention discloses a culture method of a human 293 monoclonal cell for expressing recombinant human serum albumin. The invention provides a culture method of a human 293 monoclonal cell for expressing recombinant human serum albumin. Monoclonal cells cultured by the first culture medium are fusiform, the cell adherence is tight, the cell boundary is not clear, the cell clone formation rate is high, and the division growth of the monoclonal cells is fast; after the second culture medium is replaced, standing culture is conducted to suspension culture, and finally the recombinant human serum albumin 293 monoclonal cells capable of growing in a suspension mode are obtained. The screening of human 293 monoclonal cells expressing recombinant human serum albumin from single cell cloning to suspension single cell cloning is realized, and meanwhile, a feasible method is provided for screening and culturing the monoclonal cells. Through a batch feeding culture process, the expression quantity of the recombinant human serum albumin can reach 6.3 g / L, and commercial amplification production of the recombinant human serum albumin can be supported.

The invention relates to a 3D cell clone culture screening method and system, and the method comprises the following steps: obtaining an image of a clone cell to be selected through an inverted microscope, and calculating the position information of the clone cell to be selected in X and Y directions; obtaining the Z-axis position of an inverted microscope focusing mechanism when the clone cell needing to be selected is clearest in the inverted microscope and the Z-axis position of the inverted microscope focusing mechanism when the bottom of the culture hole is calibrated, and calculating the position information z of the clone cell needing to be selected away from the bottom of the culture hole; according to the position information, a three-axis moving mechanism is used for driving a pipettor to suck clone cells needing to be selected, and the clone cells are transferred into a clone receiving plate. The height position of the clone cell is calibrated through the focusing distance of the inverted microscope, so that the clone cell can be accurately selected, the accuracy of cell screening is ensured, and the method has very high universality, universality and use value.

An antigen (Ag) that elicits a polyclonal T cell receptor like antibody response from endogenous B cells. The Ag comprises a single chain peptide MHCI (pMHCI) complex, that directs B cell responses to the displayed peptide without reactivity to the rest of the pMHCI molecule. Also, methods of using the Ag to induce polyclonal T cell receptor like (TCRL) antibodies (Abs) and using the Ag to deliver the single chain peptide to a target cell.

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 an imaging structure for cell sorting, cell sorting equipment and a cell sorting method, and belongs to the technical field of cell culture. The imaging structure comprises an illumination light source, the illumination light source is configured to be arranged at a preset height position, the illumination light source defines a channel for the pipette to pass through, and the pipette can penetrate through the channel to move in the direction close to or away from the container; the imaging module is arranged opposite to the illumination light source, and the imaging module is used for imaging a target position of the container from the lower part of the container; wherein an illumination light beam emitted by the illumination light source at least can illuminate a target position, and the pipette can move to the target position of the container to suck and / or spit out target cells according to an output result of the imaging module. According to the application, the position of the clone cell can be accurately identified, and the accuracy of clone cell selection is improved.

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 invention relates to a CRISPR (clustered regularly interspaced short palindromic repeats)-mediated immortalized sheep fetal skin fibroblast line and application thereof in the field of variation or genetic engineering. The construction of the CRISPR-mediated immortalized sheep fetal skin fibroblast line comprises the following steps: step 1, constructing a Cas9 site-directed knockout plasmid containing an FGF5 gene target sequence; step 2, constructing a homologous repair plasmid Donor-2 kb HA-SV40LT containing a homologous arm of the FGF5 gene; step 3, constructing a donor DNA (Deoxyribose Nucleic Acid) containing an SV40LT protein expression cassette; and step 4, preparing the sheep fetal fibroblast line capable of stably expressing the SV40LT protein at a fixed point. The immortalized SFF-FGF5 / SV40LT monoclonal cell strain created by the invention can become an ideal tool cell line for genome editing research such as sheep CRISPR library screening, multiple gene editing, mutant gene function exploration and the like.

There are provided methods for detecting and classifying a B-cell malignancy selected from the group consisting of a B-cell lymphoma, a B-cell leukemia, and a plasma cell dyscrasia in a subject. The methods employ a tumor cell profiling algorithm to analyze single-cell RNA sequencing data obtained from a sample of, e.g., the subject's blood or bone marrow, enriched for a cell type comprising tumoral cells using a cell surface marker. This algorithm identifies one or more one or more expanded clonal cell populations and categorizes each of the one or more expanded clonal cell populations as malignant or pre-malignant tumor cell population if expression of one or more malignancy markers is detected. The one or more malignant or pre-malignant tumor cell population(s) can be analyzed using a statistical model or machine learning algorithm to classify the B-cell malignancy.

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.

The application discloses a method for simultaneously producing multiple recombinant adeno-associated viruses, and belongs to the field of gene therapy. The method comprises the following steps: stably integrating multiple target genes into a host cell genome or stably integrating multiple serotype capsid protein Cap genes into the host cell genome; screening a single clone cell which stably expresses the target genes or the serotype capsid protein Cap genes; co-transfecting a transfer plasmid containing AAV inverted terminal repeat sequences, a Rep gene plasmid and an adenovirus auxiliary gene plasmid into the single clone cell which stably expresses the target genes or the serotype capsid protein Cap genes; and harvesting and purifying recombinant AAV viruses to obtain an AAV virus mixture with a specific ratio of target genes or a hybrid AAV virus with a specific ratio of capsid proteins.

The present invention relates to a method of selecting a cloned cell line for the production of a therapeutic protein, the method comprising the steps of: measuring, for a plurality of cloned cell lines, the product concentration of each cloned cell line; determining product concentration atlas data for each clone cell line based on the product concentration, inputting the product concentration atlas data into a learning model comprising a modeling framework, where the modeling framework comprises multivariate potential variable modeling, a multidirectional analysis structure and an evolution model structure; generating an output indicative of production stability of each cloned cell line using the learning model; and selecting a cloned cell line for the production of a therapeutic protein product based on the output. The invention also relates to a system for determining the production stability of a cloned cell line or selecting a cloned cell line.

The invention relates to a myogenic cell rapid proliferation gene knock-in bovine primary cell and a preparation method thereof, and belongs to the technical field of gene editing and biology. Specific sgRNA is designed aiming at a bovine genome safety site H11, and directional insertion of a target gene is realized by taking a gene containing a homologous arm and a muscle-promoting cell proliferation gene Ufc1 and a gene Ufl1 as a donor template through Cas9 protein mediated DNA double-strand cutting. The method has the advantages that the cutting efficiency of the safety site H11 is improved to 90% or above, so that the obtaining rate of the positive monoclonal cell strain can be remarkably improved; the ssDNA is used as a donor fragment to be introduced into bovine fibroblasts, the survival rate of electrotransfected cells is two times that of double-stranded plasmid DNA, and meanwhile, the transfection efficiency is improved by more than four times. The cell proliferation efficiency of the Ufc1 gene knock-in cell strain is improved by 1.6 times compared with that of a wild type, and the Ufc1 gene knock-in cell strain is applied to cattle and provides important technical support for genetic improvement of muscle tissue engineering and animal husbandry.

The application discloses an immortalized human hair papilla cell line and application thereof, and belongs to the technical field of immortalized cells, and particularly relates to a preparation method of an immortalized human hair papilla cell line; the method comprises the following steps: infecting human hair papilla cells by using an SV40 LT overexpression vector combined with a dyeing aid, treating the human hair papilla cells by using a puromycin screening solution, picking single clone cells by using a limited dilution method, and expanding the culture of the single clone cells to obtain the immortalized human hair papilla cell line; the dyeing aid comprises polybrene; compared with human hair papilla cells, the immortalized human hair papilla cell line prepared by the preparation method has higher cell activity and cell proliferation capacity, and can form 3D cell balls when cultured in vitro in a 3D mode, and the immortalized human hair papilla cell line retains the ability of inducing hair follicle formation, and can be applied to the fields of hair follicle tissue engineering, 3D cell culture or hair follicle organoid construction and the like.

The invention provides a CD4 + T monoclonal cell culture medium, a culture method and application of the CD4 + T monoclonal cell culture medium. The culture medium comprises N-acetylcysteine and Trolox. According to the invention, the long-standing technical problem that natural CD4 + T cells are difficult to directly and stably form a monoclonal line is successfully solved. Through a density gradient dilution method and a culture medium formula, the formation of a monoclonal cell line with uniform and stable genetic background by efficient amplification starting from a single CD4 + T cell is realized for the first time. The CD4 + T cell is high in monoclonal formation efficiency and good in monoclonal growth state, and the preparation efficiency of the gene editing cell strain is greatly improved. According to the invention, a cell line with hCCR5-delta 32 CD4 + T cell point mutation is successfully constructed by using a monoclonal cell culture method, and important scientific research reference data is provided for treatment of diseases related to the CCR5 gene.

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.