59 results about "Cell Clone" patented technology

The embodiment of the invention provides an end-to-end B cell clone pedigree forest construction method and related equipment, and can be applied to the technical field of data processing. According to the method, a plurality of obtained receptor sequencing sequences are subjected to germline comparison identification to obtain a first test Fv sequence corresponding to each receptor sequencing sequence, and a germline Fv sequence corresponding to each receptor sequencing sequence is generated; performing integrity filtering on the first test Fv sequence, performing clone type division to obtain a plurality of first clone type sets, constructing corresponding first evolutionary trees to form a first pedigree forest on the basis of a second clone type set contract type conversion probability, and performing node optimization on all the first evolutionary trees to obtain a second pedigree forest; and after it is determined that the homotype category conversion probability after updating based on all the second evolutionary trees meets the preset requirement, visualization processing is performed on all the second evolutionary trees, so that the systematic cognition efficiency of related personnel on the adaptive immune response mechanism can be improved.

The invention discloses an SOD2 knockout cell line and application of the SOD2 knockout cell line in anti-poxvirus, and relates to the technical field of antiviral research. According to the invention, sgRNA of targeted superoxide dismutase 2 (SOD2) is designed, the sequence of the sgRNA is shown as SEQ ID NO.1-SEQ ID NO.2, then a stable SOD2 knockout human-derived non-small cell lung cancer A549 cell line is established by combining CRISPR / Cas9 gene editing with a lentiviral vector delivery technology, single-cell cloning is obtained through multiple rounds of puromycin screening, and the SOD2 gene knockout A549 cell strain is successfully constructed. Vaccinia virus Tian Tan strain infection shows that the number of plaques of SOD2 knockout cells is obviously greater than that of the plaques (about 2.3 times) of normal cell infection, and the plaques are relatively large, so that fine SOD2 has the effect of limiting intercellular transmission of poxvirus. The invention lays a foundation for research and preparation of antiviral drugs.

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 is applicable to the technical field of biotechnology and cell biology, and provides a method for quickly constructing an IgG antibody high-expression stably-transfected CHO cell strain, which comprises the following steps: S1, constructing antibody plasmids; s2, carrying out cell resuscitation, passage and plasmid transfection; s3, screening positive cells; s4, preparing, namely screening high-expression stably transfected single cell clones; s5, cell feeding batch culture; s6, verifying the stability of the cell strain expressed by the antibody, according to the method provided by the invention, after the plasmid vector enters the cell through electrotransfection and standing culture is carried out for 48 hours, shaking culture is carried out, the cell recovery time can be shortened, the recovery speed is higher than that of traditional standing culture, and the cell state is better than that of a standing state; according to the method, high-density cells are inoculated during cell fed-batch culture, and under the high-density inoculation, the logarithmic phase of the cells is shortened, and the cells enter a protein production phase earlier, so that higher expression quantity can be achieved, the overall production period is greatly shortened, and a large amount of cost is saved.

The invention discloses a method for cultivating triploid kelp sporophytes and identifying ploidy of induced protonema, and belongs to the field of kelp breeding. The method is based on a capillary glass needle separation technology for inducing filamentous somatic cells by kelp and a molecular biological technology for specific marking of sex of kelp gametophytes. The sex specific marker of the kelp gametophyte is used for carrying out PCR (Polymerase Chain Reaction) amplification and 1.5% agarose gel electrophoresis detection on the separated induction protonema single-cell cloning line, so that the genetic sex of the kelp induction protonema can be accurately identified; and the ploidy of the induced protonema of a part of varieties (lines) is directly determined from the molecular level. The method for synchronously detecting the genetic sex and ploidy of the induced protonema breaks through the bottlenecks of chromosome counting and flow cytometry operation, can be completed only through one-time PCR amplification, and is simple, convenient, rapid, short in time consumption, capable of completing detection of a plurality of samples at the same time and low in cost.

The invention discloses a cell-free cloning reaction system, a kit and application of the cell-free cloning reaction system. The system comprises a cell lysis solution, an inserted DNA fragment and a linear vector, the cell lysis solution is obtained from Escherichia coli which is subjected to induced expression and then subjected to lysis, and a lambda Red system and a ligase system are expressed in the Escherichia coli; the lambda Red system comprises Exo protein, Beta protein and Gam protein; and the ligase system comprises DNA ligase. The optimized lambda Red system and the efficiently expressed DNA ligase system can complete efficient splicing of multi-fragment DNA within 15 minutes at 37 DEG C, still maintain a high positive rate in a multi-fragment assembly system, show an effect superior to Gibson cloning, and expand the applicability of the Gibson clone in complex pathway synthesis and gene line reconstruction.

The invention relates to the technical field of gene engineering, in particular to SgRNA for targeted knockout of an MINOS1 gene and application of the SgRNA. According to the invention, a CRISPR / Cas9 gene knockout technology is adopted, a LentiCRISPR-V2 vector is constructed by using a CRISPR / Cas9 system, the LentiCRISPR-V2 vector is co-transferred into a porcine small intestine epithelial cell to obtain a positive cell clone, an MINOS1 gene in the positive cell clone is not expressed, the cell death rate under a hypoxia condition can be obviously reduced, and the positive cell clone has hypoxia tolerance. By regulating and controlling MINOS1 expression, the adaptive capacity of an organism to a low-oxygen environment can be enhanced, and a new thought is provided for preventing and relieving altitude stress and improving physiological tolerance in an extreme environment; besides, the hypoxia characteristic of the tumor microenvironment is a key factor causing chemoradiotherapy drug resistance, and the MINOS1 knockout related strategy can reverse the hypoxia tolerance phenotype of tumor cells, so that the sensitivity of the tumor cells to treatment is enhanced, and an innovative direction is provided for overcoming tumor drug resistance.

The application discloses a gene editing system for constructing a three-gene (GHR gene, IGF1 gene and IGF2 gene) combined mutation small pig nuclear transfer donor cell and application thereof. The application provides a kit comprising GHR-E5-gRNA2 shown in SEQ ID NO: 36, GHR-E5-gRNA3 shown in SEQ ID NO: 37, IGF1-E4-gRNA1 shown in SEQ ID NO: 38, IGF1-E4-gRNA2 shown in SEQ ID NO: 39, IGF2-E4-gRNA2 shown in SEQ ID NO: 40, IGF2-E4-gRNA7 shown in SEQ ID NO: 41 and NCN protein. The NCN protein is a Cas9 protein or a fusion protein with the Cas9 protein. The application adopts CRISPR / Cas9 technology combined with double gRNA editing to perform combined knockout of the GHR gene, the IGF1 gene and the IGF2 gene, and obtains a single cell clone with three-gene combined knockout, thereby laying a foundation for breeding small pigs and growth and development disorder or retardation model pigs through somatic cell nuclear transfer animal cloning technology in the later stage.

The application discloses a monoclonal antibody specifically combined with PGLYRP1 and a preparation method and application thereof. The monoclonal antibody capable of accurately recognizing PGLYRP1 is constructed, and the monoclonal antibody can stably recognize the conservative domain of PGLYRP1. The monoclonal antibody can significantly reduce cross-reaction and improve the accuracy of experimental results, and is particularly suitable for signal detection in complex tissues. In addition, the monoclonal antibody is derived from a stable cell clone, can be cultured and amplified for a long time, and ensures the quality stability of the product, so that the problems of large batch-to-batch difference and non-reproducibility of polyclonal antibodies are overcome.

Disclosed is an efficient and non-genetically modified iPSC-induced, industrialized single clone selection platform, and a use. The platform can efficiently perform reprogramming, and only requires the use of a minimal number of reprogramming factors (OCT4, SOX2, E6, E7). During the single clone separation stage of the present invention, SSEA4 / TRA-1-60 is used as a screening marker, and a large number of single cell clones are obtained by means of flow cytometry. The platform described in the present invention has advantages such as high reprogramming efficiency, high safety, easy operation, and large-scale production.

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.

This invention discloses an APLN monoclonal antibody, its preparation method, and its applications. The APLN monoclonal antibody includes a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is shown in SEQ ID No:1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:2. The APLN monoclonal antibody is obtained by immunizing mice with APLN protein, followed by single-cell B-cell sorting and single-cell PCR. Utilizing advanced single-cell B-cell cloning technology combined with single-cell PCR, a novel APLN monoclonal antibody has been developed. The obtained antibody sequence is the result of natural affinity maturation screening by the in vivo immune system, requiring no additional humanization or in vitro affinity maturation steps. It retains the natural high affinity and specificity, and can directly obtain all-natural antibodies with high affinity and high specificity.

The invention relates to the technical field of molecular immunology and biology, in particular to a sicca syndrome model mouse B cell immune repertoire sequencing and subpopulation analysis method. The method comprises the following steps: S1, sample preparation: preparing a single-cell suspension from a target autoimmune disease animal model and a preset tissue of a control animal, and carrying out B cell enrichment treatment on the single-cell suspension to obtain a B cell sample; s2, multi-dimensional data parallel analysis: analyzing the B cell sample, and performing parallel construction of the multi-dimensional data; and S3, integrated correlation analysis: carrying out correlation analysis on the immune group library data, the cell subset data and the signal channel data obtained in the step S2 to establish a correlation relationship among specific B cell cloning characteristics, specific B cell subset changes and specific signal channel activity, and carrying out multilevel analysis and generating a B cell immune response mechanism of the autoimmune disease. The efficiency and accuracy of disease mechanism research and drug action mechanism evaluation are remarkably improved.

This application provides an end-to-end method and related equipment for constructing a B-cell clonal lineage forest, applicable to the field of data processing technology. This application identifies the first test Fv sequence corresponding to each receptor sequencing sequence by performing germline alignment on multiple acquired receptor sequencing sequences, and generates a germline Fv sequence corresponding to each receptor sequencing sequence. Then, the first test Fv sequences are filtered for integrity and further classified into several first clonal type sets. Based on these second clonal type sets and the same-type class conversion probability, corresponding first phylogenetic trees are constructed to form a first lineage forest. Node optimization is performed on all first phylogenetic trees. After confirming that the same-type class conversion probability meets preset requirements after updating all second phylogenetic trees, all second phylogenetic trees are visualized, thereby improving the efficiency of relevant personnel's systematic understanding of adaptive immune response mechanisms.

The present disclosure relates to methods of producing an enveloped virus in a suspension cell culture, the method comprising culturing a suspension cell line expressing a tetracycline-suppressible gene expression system in a cell culture medium. The present disclosure also relates to stable producer cell clones capable of producing an enveloped virus in a suspension cell culture.

The application discloses a high-precision and high-coverage pedigree tree tracking method, and relates to the technical field of cell pedigree tracking, and specifically comprises the following steps: selecting single-direction RNA1, single-direction RNA2, single-direction RNA3 and single-direction RNA4 which are completely different in sequence as backups, and selecting single-cell clone amplification to prepare a pedigree barcode with 13 editing sites as a backup; and adopting a cell pedigree barcode technology to match the single-direction RNA1, the single-direction RNA2, the single-direction RNA3 and the single-direction RNA4 with the 13 editing sites of the pedigree barcode respectively. The high-precision and high-coverage pedigree tree tracking method increases the average expression amount of the pedigree barcode in all cells, reduces the speed of consumption of the editing sites, increases the traceable time, simultaneously reduces the proportion of cross-site deletion mutations, and obtains a high proportion of single leaves of a cell pedigree tree, so that a pedigree tree determination method with a single-cell level resolution is realized, and the mortality of cells in clone amplification and the loss rate in cell digestion are reduced.

This invention discloses a gene editing system for constructing porcine nuclear transplantation donor cells for ALS models with SOD1 gene mutations and its applications. This invention also provides a method for preparing recombinant cells: SOD1- g RNA1 (target sequence binding region as shown in nucleotides 3-22 of SEQ ID NO: 18), SOD1- g RNA6 (target sequence binding region as shown in nucleotides 3-22 of SEQ ID NO: 19), SOD1-mutant-ss160 (shown in SEQ ID NO: 20), and NCN protein (Cas9 protein or a fusion protein containing Cas9 protein) were co-transfected into porcine cells to obtain recombinant cells. This invention utilizes CRISPR / Cas9 technology combined with ssODN homologous recombination technology to perform point mutation gene editing of the SOD1 gene, mimicking the natural pathogenesis and genetic characteristics of ALS, and obtaining single-cell clones with precise point mutations in the SOD1 gene. This lays the foundation for later breeding of ALS disease model pigs using somatic cell nuclear transfer animal cloning technology.

The invention provides an umbilical cord matrigel stem cell culture medium as well as a preparation method and application thereof, and belongs to the technical field of stem cells. Comprising the following raw materials: polyethylene glycol, cane sugar, an exosome composition, a cell factor composition, globulin, tween-80 and a PBS buffer solution. The culture medium can promote the growth of cell mitosis and mesoderm cells, the cells are not prone to deformation in in-vitro culture, the cell clone formation rate retention rate after multiple passage is high, the cells can still keep the characteristics of primary cells after being gradually differentiated, and the culture medium has wide application prospects.

The invention discloses an identification method of a single cell ecDNA cloning trajectory. Potential tumor markers in single cell ATAC data are identified based on ecDNA cloning characteristics. The cancer marker is recognized through the ecDNA, tumor cells can be sensitively recognized, and early diagnosis can be carried out; the copy number, the structure and the distribution of the ecDNA often have great heterogeneity in tumor cells. Different types of ecDNA clones may be carried among different tumor cell populations, so that the evolution trajectory of tumors and the diversity of the cell populations can be disclosed; according to the present invention, the gene structure change of the tumor cell population in different stages can be revealed by researching the clone trajectory of the ecDNA so as to provide the potential marker for the early screening and monitoring of cancers.

The present disclosure relates to the technical field of cell culture. It provides a method that facilitates the selection of single cell clones or cell pools for the manufacture of antigen binding proteins having two to four different antibody chains. The method utilizes ddPCR to quantify the transcript level.

The utility model relates to the related technical field of single cell picking devices, in particular to a single cell clone picking device, which is used for picking single cells in a cell culture dish, the cell picking culture dish is positioned in a picking area of an inverted microscope objective table, and the single cell clone picking device is used for picking the single cells in the cell culture dish. Microscope collecting lenses with fixed positions are arranged above the picking area at intervals, and microscope collecting lenses are arranged above the picking area of the inverted microscope objective table at intervals; cells at the bottom of the culture dish are scraped off through the cell suction tube opening, the cells are sucked and stored in the cell suction tube by adjusting the position of the rubber plug through the suction adjusting assembly, the sucked cells can be pushed into a new culture dish containing a culture medium, and picking and transferring of single cell clones are achieved. The device is easy to assemble and disassemble, and is suitable for most inverted microscopes, and the cell suction tube opening covers single cell cloning, so that other parenchyma cells cannot be doped in the picking process, and the unicity of single cell cloning is ensured.

The present discloses provides the use of tributyrin as an additive for embryonic development culture medium in vitro, in the present disclosure, the tributyrin is applied as an additive to the development culture medium of mouse embryos in vitro for the first time, the tributyrin can significantly increase the rate of blastocyst, and reduce the ROS content in the embryos, improve the mitochondrial membrane potential in the embryos, increase the ATP level and the expression of antioxidant genes in the embryos, improve the DNA methylation and histone modification level in the embryos, promote the embryos development in vitro; In addition, tributyrin, as natural antioxidant and apparent drug, which is safe, non-toxic and side effects; the tributyrin provides strong support for the efficient embryos development of human assisted reproductive technology, mammalian fertilization embryos, parthenogenetic embryos and somatic cell cloned embryos and other embryo engineering technologies in vitro.

The application discloses a gene editing system for constructing a GP130 gene mutation gastric cancer model pig nuclear transfer donor cell and application thereof. The gene editing system comprises a high-efficiency Cas9 protein prepared according to the method of the application, a high-efficiency target gRNA for the GP130 gene screened, and a single-chain Donor DNA containing a GP130 mutation site, and the optimal use amount ratio of the components of the system is optimized, and finally the single-cell clone ratio of the target site point mutation is 22.5%, which is much higher than the conventional point mutation efficiency (<5%).

This invention discloses an siRNA for inhibiting MCM7, a composition thereof, and its application. The siRNA designed and validated in this invention can efficiently inhibit MCM7 gene expression, thereby inhibiting DNA synthesis, cell proliferation, and cell clone generation in cancer cells, achieving the purpose of preventing and treating tumors. This invention provides a new target and candidate drug for cancer prevention or treatment.

This invention discloses a CRISPR system for constructing pig nuclear transfer donor cells for cataract models with YAP1 gene mutations and its applications. The invention provides the application of YAP1-gRNA2 (SEQ ID NO: 18), YAP1-gRNA3 (SEQ ID NO: 19), and NCN protein in a preparation kit; the kit is used for: preparing recombinant cells; preparing pig cataract models; preparing cataract cell models, cataract tissue models, or cataract organ models. This invention uses CRISPR / Cas9 technology combined with dual gRNA editing to knock out the YAP1 gene, simulating the natural pathogenesis and genetic characteristics of cataracts, and obtaining single-cell clones with the YAP1 gene knockout, laying the foundation for later breeding pig cataract models through somatic cell nuclear transfer animal cloning technology. This invention has significant application value for the development of cataract drugs and elucidating the pathogenesis of this disease.

This invention discloses a method for intelligently recognizing T-cell antigens at the atomic level, comprising the following steps: representing antigens, HLA, and TCR as a graph with atoms as nodes and chemical bonds as edges; constructing an atomic-level intelligent prediction model for antigen-HLA binding to screen antigens that can be presented by HLA; constructing an atomic-level intelligent prediction model for antigen-TCR interaction to screen antigens that can be recognized by TCR; calculating the immunogenicity of antigens based on T-cell clone frequencies and identifying highly immunogenic antigens, thus forming an intelligent T-cell antigen recognition method. This invention overcomes the problem of existing technologies that only recognize T-cell antigens at the sequence or residue level, resulting in most candidate antigens failing to elicit an immune response, and can accurately screen for immunogenic neotumor antigens at the atomic level.

The present disclosure generally relates to the manufacturing of gene therapy products, and specifically to methods of producing an enveloped virus from a stable producer cell clone or a stable producer cell pool overexpressing vesicular stomatitis virus G (VSV-G) in suspension cell culture.

The present invention provides a genetically modified cell line producing a recombinant glycoprotein having N-glycans with terminal mannose and a method for producing the recombinant glycoprotein or a method for generating the cell, clone or cell line. The cell line comprises an insertion of less than 600 bp in the coding region of a chromosomal sequence encoding MGAT1.

The present invention addresses the problem of providing a cultured cell line that has a constant susceptibility to infection by human sapovirus as well as providing measures for establishing said cultured cell line, and of contributing to research on human sapovirus infections and to the development of therapeutic and preventive measures; the inventors of the present invention achieved the present invention after finding that, by performing transformation using a Cas9 gene on Caco-2, which is an easily obtainable commercial cell line derived from human colon cancer, it is possible to acquire, as clones, cells having constant susceptibility to infection by human sapovirus, that a dramatically improved propagation efficiency of human sapovirus is observed in this human sapovirus infection-susceptible cell line, and that propagation by using a subculture is also possible.

The application discloses a monoclonal antibody targeting PGLYRP1 and a preparation method and application thereof. The monoclonal antibody capable of accurately recognizing PGLYRP1 is constructed, and the monoclonal antibody can stably recognize the conserved domain of PGLYRP1. The monoclonal antibody can significantly reduce cross-reaction and improve the accuracy of experimental results, and is particularly suitable for signal detection in complex tissues. In addition, the monoclonal antibody is derived from a stable cell clone, can be cultured and amplified for a long time, ensures the quality stability of the product, and overcomes the problems of large batch-to-batch difference of polyclonal antibodies and non-reproducibility.