25 results about "Stable cell line" patented technology

The invention provides a gene editing cell and a method for producing extremely high molecular weight hyaluronic acid by using the cell. The preparation method comprises the following steps: by taking PUC57 as a skeleton carrier, constructing a recombinant plasmid containing an HAS2 gene, and transfecting a mole NMRHas2 gene, a chicken CHICKHAS2 gene, an astronomy mouse SNMHAS2 gene, a dakalimeris mouse DMRHAS2 gene or a mole mouse BMRHAS2 gene into chicken fibroblasts through lipidosome to construct a stably transfected cell line, so that hyaluronic acid with extremely high molecular weight can be stably obtained. The method provided by the invention overcomes the defects of non-uniform molecular weight distribution of HA product sources, low molecular weight and single method in the prior art, and has important significance for developing medicines for treating osteoarthritis and novel medical equipment products.

The invention discloses a chemical defined culture medium formula for subculture of CHO-K1 host cells and high expression of an engineering cell strain product of a stable cell line constructed by taking CHO-K1 cells as hosts. The main components of the feed only comprise amino acids, inorganic salts, vitamins, trace elements and other components limited by chemical components. According to the invention, the growth density and motility of the engineering cell strain taking CHO-K1 as the host are improved, the tolerance of cells is increased, the maintenance period of the cells is prolonged, the product expression quantity of the CHO-K1 engineering cell strain is improved, and the cost of the culture medium is greatly reduced.

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 discloses a homologous recombination system, a transposition system, a fruit fly animal gene high-throughput screening method and application. The screening method comprises the following steps: firstly, separating and establishing a stable cell line from a fruit fly animal; then co-transfecting a CRISPR / Cas9 mediated homologous recombination system, and obtaining a cell line for stably expressing Cas9 protein through puromycin screening and a limited dilution method; the sgRNAs plasmid library constructed by combining a PiggyBac transposon system can realize efficient screening and functional analysis of a target gene in the Cas9 cell line. The high-throughput screening method provided by the invention is widely applicable to gene function research and target gene screening, is simple in preparation process and convenient to operate, has good universality, and provides effective technical support and solution for related fields.

This useful solution, in the field of biotechnology and regenerative medicine, relates to a process for enhancing the efficiency of induced pluripotent stem cell (iPSC) colony formation from human peripheral blood mononuclear cells (PBMCs). The solution aims to overcome the low reprogramming efficiency, unstable colony formation, and limited iPSC cloning rates commonly encountered in PBMC iPSC generation processes. The technical essence of the solution lies in the synchronized optimization of the reprogramming process, including pre-activating PBMCs with an appropriate cytokine combination at predetermined times before reprogramming, using a non-integrated Sendai virus system under optimized transformation conditions, switching culture media at defined intervals to promote cell state transitions, and applying a selection, enrichment, and stabilization process for iPSC cloning in the initial passages after colony formation.The process also incorporates cell line quality control steps through assessment of residual Sendai virus RNA removal and mycoplasma contamination testing during line maintenance. Experimental results show that the proposed process increases the number of iPSC colonies formed, improves the rate of establishing stable iPSC lines, and enhances cell homogeneity and pluripotency maintenance. The solution can be applied in basic research, pathogenesis modeling, drug screening, regenerative medicine, and studies using induced pluripotent stem cells derived from human peripheral blood cells.

Described herein are polynucleotide constructs for inducible production of rAAV virions. Also provided are stable cell lines for inducible expression of rAAV virions that include a payload polynucleotide.

The invention relates to a Rosa26 site-targeted Vero cell line as well as a construction method and application thereof, and belongs to the technical field of gene engineering. The invention provides a Vero cell line targeting a Rosa26 site. The Vero cell line comprises an attP sequence integrated into the Rosa26 site of a genome of the Vero cell line. Researches show that by co-transfecting donor plasmids expressing exogenous genes and attB sites and Bxb1 expression plasmids to the Vero cell line, the exogenous genes can be rapidly integrated to a genome of the Vero cell line, and then the Vero stably transfected cell line capable of stably expressing the exogenous genes for a long time is constructed. According to the Vero cell line provided by the invention, the construction time of the Vero stable transfection cell line for expressing an exogenous gene is greatly shortened, and based on insertion of the Rosa26 site, the Vero cell line provided by the invention can maintain the stability and consistency of a genome for a long time, so that long-term stable expression of the exogenous gene is ensured.

The invention relates to the field of biomedical research, and provides a lung cancer cell strain (H1299) capable of stably and low-expressing Integrin alpha6 protein and a construction method of the lung cancer cell strain (H1299). The preparation method comprises the following steps: designing and synthesizing an shRNA (short hairpin Ribonucleic Acid) sequence for specifically knocking down gene expression aiming at a human Integrin alpha6 gene, forming a double-chain fragment through annealing, and inserting the shRNA sequence into a pLKO.1-puro lentiviral vector by adopting a connection independent cloning (LIC) method, so as to construct a pLKO.1-ITG alpha6-shRNA recombinant plasmid. Furthermore, the recombinant plasmid and lentivirus packaging plasmids psPAX2 and pVSVG are co-transfected to an HEK293T cell by utilizing a lentivirus packaging system, and the lentivirus is produced by packaging. And infecting a target lung cancer cell strain H1299 by using the obtained lentivirus, and then carrying out puromycin resistance screening and continuous subculture to finally obtain the stable and continuous low-expression Integrin alpha6 protein H1299 cell strain. A fluorescence microscope, a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) and an immunoblotting technology are used for verifying that the gene silencing efficiency of the Integrin alpha6 in the cell strain can reach more than 80%. The stable cell strain constructed by the invention provides a reliable and efficient tool cell model for researching the molecular mechanism of the Integrin alpha6 in the processes of occurrence and development, invasion and metastasis, energy metabolism, immune escape and the like of lung cancer, and also provides a new experimental material for screening antitumor drugs targeting the Integrin alpha6.

The invention provides a low-autophagy Vero cell line based on a DDIT3 inhibition gene as well as a construction method and application of the low-autophagy Vero cell line. The cell line stably knocks down DDIT3 expression through lentivirus-mediated shRNA, the autophagy level under the condition of nutrition deficiency is remarkably reduced, the cell proliferation capacity is enhanced, and the yield of influenza A H1N1 viruses is remarkably increased. The construction method comprises the following steps: cloning shRNA (short hairpin ribonucleic acid) to a GV493 lentiviral vector, co-transfecting 293T cell packaging virus with psPAX2 / pMD2. G helper plasmid, infecting Vero cells, and screening through puromycin to obtain a stable cell line. The cell line solves the problem of growth stagnation caused by over-activation of autophagy in serum-free suspension culture of traditional Vero cells, and is suitable for large-scale vaccine production.

The invention discloses a PINK1 inducible expression cell line of a porcine umbilical vein endothelial cell and a construction method and application thereof, a stable cell line with controllable PINK1 expression level is constructed by introducing a porcine PINK1 gene into the porcine umbilical vein endothelial cell and combining with an inducible expression system taking doxycycline as an inducer. The cell line is in a low-expression or non-expression state under the condition that an inducer is not added, and dose-dependent induced expression can be realized after doxycycline is added. Experimental results show that the induced expression of PINK1 can regulate and control the expression of angiogenesis related genes, activate mitochondrial autophagy related pathways under oxidative stress conditions, and influence the expression of cell proliferation and apoptosis related proteins at the same time. The cell line is clear in construction method and good in stability, can be used as an in-vitro model for researching angiogenesis, oxidative stress and related regulation and control mechanisms of porcine vascular endothelial cells, and has good practical value.

This invention provides a method for producing engineered virus-like particles using stable cell lines, and the engineered virus-like particles thereof. The method comprises the following steps: constructing a stable BaEV retrovirus packaging cell line; transfecting the BaEV retrovirus packaging cell line obtained in step 1 with a Gag-Cas9 plasmid; constructing a self-inactivating delivery plasmid for a retrovirus packaging system; and selecting a stable cell line capable of producing engineered virus-like particles. This allows for the production of engineered virus-like particles through stable cell lines. This invention innovatively develops a method for preparing eVLP stable cell lines. This method overcomes existing production bottlenecks, providing a stable, efficient, and economical gene-editing vector solution for research and clinical applications, and promoting further development in the field of gene therapy.

The present application provides a method for batch preparation of immune cell therapy products without electric conversion, the CD7 gene of the immune cells is knocked out and a chimeric antigen receptor targeting CD7 is expressed at the same time, the method comprises producing engineered viroplasm by a stable cell line, knocking out the CD7 gene in the immune cells by using the engineered viroplasm, and transducing the immune cells by using a CD7 CAR retrovirus vector, thereby obtaining the immune cell therapy product. The present application aims at the difficulties in the current CAR-T cell preparation, such as high cost, poor popularity, and high risk of failure in the application of CRISPR electric conversion technology, introduces eVLP technology for efficient and convenient gene editing, and ensures safety, saves cost, reduces operation complexity, and greatly improves the success rate of immune cell preparation. Based on clinical needs, the present application uses a CD7 eVLP vector combined with a CD7 CAR retrovirus to construct a CD7 KO+CD7 CAR T cell therapy product.

The invention discloses a Grin2b stably transfected cell strain, a construction method and application thereof and a method for screening pollutant high-throughput neurotoxicity or learning and memory disorders, and belongs to the technical field of biologication.The construction method comprises the following steps that 1, cells are cultured; (2) constructing a plasmid vector, wherein the plasmid vector comprises a Grin2b coding sequence; (3) virus coating and virus collection; (4) testing the titer of the lentivirus; (5) stable plant screening; and (6) carrying out stable plant transformation function verification. The Grin2b stably transfected cell strain based on the Grin2b related ion channel constructed by the invention provides a brand new technical platform for high-throughput neurotoxicity recognition, learning memory screening and mechanism research of organic pollutants and degradation products thereof.

On the basis of a Galpha s-cAMP-PKA signal channel, which plays a normal physiological role in vivo, of thyroid stimulating hormone and the regulation and control effect of the thyroid stimulating hormone on thyroid hormone synthesis, a normal human thyroid follicular epithelial cell line Nthy-ori 3-1 is transformed into a TSHR overexpressed stable cell line for the first time, the expression quantity of TSHR in a cell membrane is obviously increased, and the TSHR expression level is improved; the response sensitivity of the Nthy-ori 3-1 cell to a thyroid stimulating hormone drug is obviously improved; by detecting the synthesis level of cAMP and thyroid hormone before and after the stimulation of the thyroid stimulating hormone, it is verified that the TSHR overexpression Nthy-ori 3-1 cell line established in the research has great superiority for determining the biological activity of the human thyroid stimulating hormone.

This invention relates to a drug screening method for diseases closely related to label-free G protein-coupled receptors (GPCRs), specifically to a label-free cell screening model for the GPR183 receptor and the application of drugs prepared from it. The label-free cell screening model is a stable cell line CHO-K1-GPR183 that highly expresses the GPR183 receptor. The GPR183 receptor agonist compound N-demethylnuciferine was obtained through screening using the GPR183 cell screening model established in this invention. Current research indicates that the GPR183 receptor is closely related to the occurrence and development of neuroinflammatory / autoimmune diseases such as multiple sclerosis, colitis, type 1 diabetes, and arthritis, as well as cancer and metabolic diseases, thus providing potential lead compounds for drug development in these diseases.

To provide an inducible promoter for viral vector production.SOLUTION: Aspects described herein relate to a stable cell line for recombinant viral vector (e.g., recombinant adeno-associated viral vector) production comprising at least one inducible promoter operably linked to a heterologous gene encoding a toxic protein. Further provided herein are methods for generating stable cell lines and methods for viral vector production. The present invention relates to cell lines for the rapid and scalable production of viral vectors, such as adeno-associated virus (AAV).SELECTED DRAWING: None

This invention discloses a viral vector targeting KIF4A to inhibit angiogenesis, its preparation method, and its applications. The viral vector contains three shRNA gene fragments targeting the KIF4 protein-coding region, as shown in SEQ ID NO.1-SEQ ID NO.3. Using a lentiviral packaging system, a stable KIF4A knockdown cell line can be constructed, effectively inhibiting endothelial cell proliferation and angiogenesis, and also inhibiting the paracrine promotion of angiogenesis by glioma cells, providing a new strategy for selecting targets for anti-angiogenic tumor therapy.

Aspects described herein relate to stable cell lines for the production of recombinant viral vectors, such as recombinant adeno-associated viral vectors, comprising at least one inducible promoter operably linked to a heterologous gene encoding a virulence protein. Further provided herein are methods for making stable cell lines and methods for viral vector production.

This invention provides enzymes that promote the stable and / or intracellularly abundant synthesis of luciferin from precursor compounds, and the reduction of oxyluciferin to luciferin, as well as uses thereof. [Solution] A method for preparing compounds identical to fungal luciferin and preluciferin in vitro or in vivo is provided; an expression cassette comprising nucleic acids, vectors, and regulatory elements necessary for nucleic acid expression in selected host cells; further, a combination of nucleic acids for obtaining cells, stable cell lines, transgenic organisms (e.g., plants, animals, fungi, or microorganisms), autoluminescent cells, cell lines, or transgenic organisms containing nucleic acids, vectors, or expression cassettes; a combination of proteins for producing luciferin or its precursors from simpler compounds; and a kit comprising nucleic acids, vectors, or expression cassettes for producing luminescent cells, cell lines, or transgenic organisms.

Method of generating a cell for producing a viral vector, the method comprising the following steps: providing a cell; targeted insertion into a first genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell; and targeted insertion into a second genomic safe harbour site of the cell of a nucleic acid molecule comprising a nucleotide sequence encoding one or more viral vector proteins; wherein the first and second genomic safe harbour sites are different. A cell with a modified genome for producing a viral vector comprising: a nucleotide sequence encoding a protein capable of exporting a viral vector transcript from a nucleus of the cell inserted into a first genomic safe harbour site of the cell; and a nucleotide sequence encoding one or more viral vector proteins inserted into a second genomic safe harbour site of the cell; wherein the first and second genomic safe harbour sites are different. Uses of the produced viral vectors and the cell, for example as a medicament or in the production thereof.

The invention relates to the technical field of gene therapy, in particular to a method for knocking out a Bax gene on the basis of an HEK293 cell line by utilizing a CRISPR / Cas9 technology, constructing a stable cell line expressed by foreign protein and improving the AAV packaging efficiency. The invention discloses a Bax2.6 cell strain. A pro-apoptosis factor Bax gene is knocked out from an HEK293T cell. Compared with a wild type HEK293T cell, the Bax2.6 cell strain provided by the invention shows higher cell activity under various stress conditions, the cell survival rate of the Bax2.6 cell strain is improved by at least 20% under the treatment conditions of small molecule inhibitors ZV-FMK, Nec-1, puromycin (Puro), cis-platinum (CDDP) and rotenone, and the Bax2.6 cell strain shows excellent environmental tolerance and production robustness. When the Bax2.6 cell strain provided by the invention is used for transient transfection and packaging of an adeno-associated virus (AAV) vector, the copy number of the obtained AAV virus can be up to 2 * 10 copies / mL, and the AAV packaging efficiency is improved by more than 10 times compared with that of a conventional HEK293T cell line.

  42      Abstract  The present invention is directed to nucleic acid molecules which encode novel luciferases, functional fragments thereof, homologs and mutants, as well as to proteins encoded by said nucleic acids. The nucleic acid molecules of interest are isolated from fungi or obtained by genetic engineering methods. Also, host cells, stable cell lines and transgenic organisms comprising said nucleic acid molecules are provided. In addition, antibodies specific to the proteins of the present invention are provided. Said proteins and nucleic acids are used in many applications and methods, in particular, in labelling organisms, cells, cellular organelles, or proteins. Also, said protein and nucleotide compositions are used in methods for detecting protein-protein interactions, for testing promoter activity under various conditions. Finally, provided are kits for the use of proteins and nucleic acids of the present invention in the diversity of methods and applications. Abstract The present invention is directed to nucleic acid molecules which encode novel luciferases, functional fragments thereof, homologs and mutants, as well as to proteins encoded by said nucleic acids. The nucleic acid molecules of interest are isolated from fungi or obtained by genetic engineering methods. Also, host cells, stable cell lines and transgenic organisms comprising said nucleic acid molecules are provided. In addition, antibodies specific to the proteins of the present invention are provided. Said proteins and nucleic acids are used in many applications and methods, in particular, in labelling organisms, cells, cellular organelles, or proteins. Also, said protein and nucleotide compositions are used in methods for detecting protein-protein interactions, for testing promoter activity under various conditions. Finally, provided are kits for the use of proteins and nucleic acids of the present invention in the diversity of methods and applications. 42 20 24 20 33 78 21 M ay 2 02 4 A b s t r a c t 2 0 2 4 2 0 3 3 7 8 2 1 M a y 2 0 2 4

This invention belongs to the field of molecular biology, specifically relating to the application of SND1-overexpressing cells in promoting small RNA virus replication. The invention first constructs an SND1 gene overexpression vector, transfects it into BHK-21 cells, and then selects a stable cell line. Infection of the cell line with foot-and-mouth disease virus (FMDV) and Seneca virus (SVV) revealed that the titers of FMDV and SVV, as well as the expression levels of viral RNA and proteins, were significantly higher than in wild-type cells, indicating that SND1 promotes FMDV and SVV replication. The constructed SND1-overexpressing cells can efficiently proliferate FMDV and SVV and can be used as cell lines for FMDV and Seneca virus vaccine production. This provides a new tool for FMDV and SVV mechanism research, vaccine preparation, and the development of detection reagents, and has significant scientific research and application value.

Provided are an anti-ANGPTL3 humanized monoclonal antibody stable cell line and a use thereof. An anti-ANGPTL3 humanized monoclonal antibody expression vector is introduced into a host cell by means of electrotransfection; and after multiple times of screening, a series of stable and controllable cell lines with high expression levels are finally selected. Further disclosed is an ANGPTL3 humanized monoclonal antibody on the basis of a hybridoma cell. The monoclonal antibody can be used in a reagent for detecting ANGPTL3, and can also be used in the preparation of a drug for preventing and / or treating kidney diseases or metabolic diseases.

The invention discloses an NFE2L3-multi-tag fusion stable cell line, a special tool and a construction method and application of the NFE2L3-multi-tag fusion stable cell line, and belongs to the technical field of crossing of molecular biology and cell biology. According to the invention, a CRISPR-Cas9 gene editing technology is adopted, multiple types of tags such as GFP, Flag and the like are inserted in the same frame in front of a termination codon of an NFE2L3 gene, and specific sgRNA targeting NFE2L3, a universal HDR donor vector and a construction composition are provided in a matched manner. The construction method comprises the steps of sgRNA design, donor vector construction, adaptability delivery and positive screening, and overcomes the defects of unstable expression, gene function interference and the like in the prior art. The obtained cell line can avoid the problem of insufficient NFE2L3 special antibodies, is suitable for ChIP-seq, living cell imaging and other experiments, assists downstream target gene analysis, upstream regulatory factor screening and stress condition response identification, and provides an efficient tool for NFE2L3 functional mechanism and tumor related research.