30 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 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 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.

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 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 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.

The present application relates to the technical field of biology, in particular to a method for obtaining a medicinal plant stem cell clone and a high-yield cell line, which adopts the establishment of a suspension cell line, the acquisition of single cells, cell clone culture and the screening of clone cells to obtain a cell line with high yield of specific active substances.

A method for cell culture expansion and maintenance, comprising seeding, in a first cell culture cassette, a plurality of cell colonies adhered to a first surface of the first cell culture cassette; capturing, by an optical engine, time-series images of the plurality of cell colonies as the plurality of cell colonies expand; determining, by a platform manager, a subset of cell colonies to remove from the first surface based on the time-series images; removing, by the optical engine, the subset of cell colonies from the first surface; washing, by a fluid management system, the removed subset of cell colonies from the first cell culture cassette; repeating steps for one or more iterations; harvesting a set of remaining cell colonies from the first cell culture cassette; seeding the set of remaining cell colonies into a second cell culture cassette; repeating steps for additional iterations in the second cell culture cassette.

The invention discloses sgRNA for accurately editing a PSEN1 p.M146V site of a tree shrew and application of the sgRNA, and belongs to the technical field of gene editing. The nucleotide sequence of the sgRNA designed by the invention is shown as SEQ ID NO.1. The sgRNA can guide a base editor to be precisely positioned to a PSEN1 p.M146V site, a spermatogonial stem cell line for precisely editing the PSEN1 p.M146V site of the tree shrew is obtained, the precisely edited spermatogonial stem cell line is obtained through single cell cloning, screening and enrichment, and the stably inherited PSEN1 p.M146V gene is obtained through reproductive line transmission to edit the tree shrew. The sgRNA provided by the invention is accurate in design and high in editing efficiency, and the editing efficiency of the obtained tree shrew spermatogonial stem cells is as high as 100%. The spermatogonial stem cell line for precisely editing the tree shrew PSEN1 p.M146V site and the stably inherited PSEN1 p.M146V gene editing tree shrew are obtained for the first time, a research basis is provided for researching the degenerative phenotype, pathological characteristics and molecular mechanism of the Alzheimer's disease caused by the mutation, and an important experimental platform is provided for screening related drugs.

The invention relates to a duck induced pluripotent stem cell differentiation method which comprises the following steps: (1) establishment of duck iPS cells: separating and culturing adult cells, transfecting transcription factor plasmids, and screening and amplifying iPS cell clone; (2) differentiating the duck iPS cells into PGC cells: preparing an induced differentiation culture medium, and performing differentiation induction on the iPS cells; and (3) cryopreservation and resuscitation of the PGC cells: preparing a cryoprotectant, carrying out cryopreservation on the PGC cells, and resuscitating the PGC cells. According to the method, differentiation of duck iPS cells and preservation and recovery of PGC cells are successfully achieved, genetic resources of endangered duck species can be effectively preserved, population rejuvenation and ecological restoration of the endangered duck species are promoted, and important technical support is provided for global biodiversity protection.

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 belongs to the technical field of cell clone acquisition, and discloses a needle head, a teasing needle and an adherent cell clone acquisition method.The needle head comprises a needle head body and a supporting column, the needle head body is provided with a suction channel, and the suction channel penetrates through the first end face, in the height direction, of the needle head body; the multiple supporting columns are arranged on the outer surface of the needle head body at intervals in the circumferential direction of the needle head body, and the supporting columns protrude out of the first end face; the adherent cell clone obtaining method comprises the steps that when cell clone obtaining is conducted, the needle body is placed in a culture solution of a culture container, all the supporting columns abut against the inner wall of the culture container, a flow field is more stable, then cell clone is sucked through the suction channel, and therefore when the cell clone is sucked, the cell clone is separated from the culture solution, and the adherent cell clone is obtained. The distance between the first end face and cell clone does not need to be noticed; meanwhile, the needle head can also form a relatively stable flow field on the surface, so that the acquisition efficiency of adherent cell clone is improved.

The application discloses a rhabdovirus-free Sf9 cell screening method of double pretreated conditioned medium, relates to the technical field of biology, and obtains the rhabdovirus-free Sf9 cell by double pretreatment of the conditioned medium through cross-flow filtration and beta-propiolactone inactivation and hydrolysis, and combination of single cell cloning screening. The method realizes efficient virus interception and high-activity retention of nutritional factors, the single cell cloning formation rate is greater than 40%, and is suitable for virus-free cell bank construction and biological product production.

Disclosed herein are compositions, culture systems, and methods for stable clonal expansion of renal unit progenitor cells (NPCs) and generation of renal unit organoids. The compositions and culture systems allow for high purity and clone scalable NPC populations, which enable genomic range screening and rapid, efficient and scalable organ-like models of kidney disease.

The application provides a multi-omics joint detection system for prostate cancer recurrence risk assessment, the application synchronously acquires genomic, transcriptomic, epiproteomic and metabolomic data of different regions of a tumor through spatially resolved in situ capture technology, and integrates multi-dimensional information such as circulating tumor DNA epigenetic memory, urological microbiome-host interaction, single-cell clone evolution and tumor microenvironment three-dimensional topology. The system uses a dynamic Bayesian fusion engine to perform probabilistic risk calculation, combines digital twin technology to simulate treatment response, and realizes model adaptive updating through longitudinal follow-up data. The output result has high interpretability and can directly show key driving factors and their clinical interventional properties. The system breaks through the limitations of traditional static and single-omics models, significantly improves the prediction accuracy of recurrence, especially in low-risk populations, and provides intelligent support for individualized auxiliary treatment decisions.

The application provides a small molecule composition for partial reprogramming of pig somatic cells, a partially reprogrammed nuclear donor cell and application thereof, and belongs to the field of animal biotechnology and genetic engineering. The small molecule composition comprises CHIR99021, Repsox-616452, TTNPB and SAG. The small molecule composition provided by the application can safely and significantly reverse the epigenetic and functional aging state of pig aging somatic cells edited by "3+X", restore the nucleo-cytoplasm compartmentalization integrity, and improve the cell cloning expansion capacity by more than 200%. The early abortion rate can be reduced from more than 70% to less than 10% by using the obtained partially reprogrammed nuclear donor cell as a nuclear donor for SCNT, reaching the industry limit level. The application provides an efficient key technology for the large-scale and economic production of "gold standard" donor pigs.

This invention discloses a method for gene transfection of fibroblasts. The method includes: obtaining a fibroblast suspension with a cell concentration of 1-2 cells / µL; preparing cell suspension droplets in a cell culture dish, each droplet consisting of 10±0.5µL of the fibroblast suspension; incubating the cell culture dish containing the cell suspension droplets in a microenvironment, and adding cell culture medium after cell adhesion; obtaining a plasmid solution containing a linearized target gene with a concentration of 150-200 ng / µL; aspirating the plasmid solution into an injection needle, adjusting the angle between the injection needle and the horizontal plane to 30-60°, and injecting it sequentially into each cell; performing single-cell culture after injection to obtain stably transfected transgenic cell clones. This method aims to obtain a cell line without selection markers while achieving high-efficiency and stable transfection.

The application discloses a cell-free cloning reaction system, a kit and application thereof. The system comprises a cell lysate, an inserted DNA fragment and a linear carrier. The cell lysate is obtained from induced and lysed Escherichia coli, wherein a lambda Red system and a ligase system are expressed. The lambda Red system comprises Exo protein, Beta protein and Gam protein. The ligase system comprises DNA ligase. The optimized lambda Red system and the high-efficiency DNA ligase system can complete high-efficiency splicing of multi-fragment DNA within 15 minutes at 37 DEG C, and still maintain a high positive rate in a multi-fragment assembly system, which is superior to Gibson cloning and expands the applicability in complex pathway synthesis and gene circuit reconstruction.

This invention relates to the fields of biotechnology and cell biology, and provides a method for rapidly constructing a stable CHO cell line with high IgG antibody expression. The method includes the following steps: S1. Antibody plasmid construction; S2. Cell resuscitation, passage, and plasmid transfection; S3. Positive cell screening; S4. Preparation and screening of stable single-cell clones with high expression; S5. Feed-and-carry batch culture; S6. Verification of the stability of the antibody-expressing cell line. The method provided by this invention, after the plasmid vector enters the cells via electroporation, performs shaking culture after 48 hours of static culture, which accelerates cell recovery time, resulting in faster recovery and better cell condition compared to traditional static culture. During feed-and-carry batch culture, high-density cell inoculation shortens the logarithmic growth phase, allowing cells to enter the protein production phase earlier, thereby achieving higher expression levels, significantly shortening the overall production cycle, and saving substantial costs.