54 results about "Stable cell line" patented technology

The invention provides a cyclic compound with a structure as shown in a formula (I) or a pharmaceutically acceptable salt or a stereoisomer or a prodrug molecule of the cyclic compound, and a pharmaceutical composition and application of the cyclic compound or the pharmaceutically acceptable salt or the stereoisomer or the prodrug molecule of the cyclic compound. The compound can be used as a protein kinase inhibitor, has very strong inhibitory activity on FLT3 mutant kinase, has very strong proliferation inhibitory activity on Ba / F3-FLT3-ITD and stable strain cells containing drug-resistant point mutation, can inhibit proliferation, migration and invasion of various tumor cells, and especially can overcome the drug resistance of existing clinical drugs. The cyclic compound provided by the invention can be used for preparing medicines for preventing or treating FLT3 tyrosine kinase mediated diseases (such as hematological malignancies), and can be used for treating various hematological malignancies of human beings and other mammals. # imgabs0 #

The invention relates to the fields of epigenetic regulation and tumor treatment, and aims to evaluate that the cell distribution of the mutant is consistent with that of H3 by constructing Bas-2B (H3K9R) and Bas-2B (H3K18R) stably transfected cell strains and analyzing the cell positioning condition of the H3K9R / H3K18R mutant through an immunofluorescence technique. According to the present invention, a Western blotting technology is adopted to detect the H3K9 / H3K18 lactic acid modification level of Bas-2B (H3K9R) and Bas-2B (H3K18R) cells compared to control cells so as to verify the influence of the H3K9R / H3K18R mutant on the H3K9 / H3K18 lactic acid modification; after Bas-2B (H3K9R), Bas-2B (H3K18R) cells and control cells of the Bas-2B (H3K9R) and the Bas-2B (H3K18R) cells are subjected to nickel compound exposure treatment, the influence of the H3K9R / H3K18R mutant on the nickel compound induced lung cancer is evaluated through a soft agar cloning formation experiment. An AGS cell line stably transfecting H2BK16R is constructed, and the cell localization condition of the H2BK16R mutant is analyzed through an immunofluorescence technology. According to the present invention, a Westernbloting technology is adopted to detect the lactic acid modification level of AGS (H2BK16R) cells so as to verify the influence of the H2BK16R mutant on the H2BK16 lactic acid modification; the influence of the H2BK16R on the malignant proliferation ability of the gastric cancer is researched through a soft agar clone formation experiment.

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.

The present invention provides a method of electrotransfection-free batch preparation of an immune cell therapeutic product, the CD7 gene of the immune cell itself being knocked out and simultaneously expressing a CD7-targeted chimeric antigen receptor, comprising the production of engineered viroid particles by a stable cell line, the knockout of the CD7 gene in the immune cell using the engineered viroid particles, and the preparation of the therapeutic product of the immune cell, the preparation of the therapeutic product of the immune cell, the preparation of the therapeutic product of the immune cell, and the preparation of the therapeutic product of the immune cell. The CD7 CAR retroviral vector is used for transducing immune cells, so that the immune cell treatment product is obtained. Aiming at the difficulties of high cost, poor universality and high failure risk of CRISPR application electrotransfection technology in current CAR-T cell preparation, the eVLP technology is introduced to carry out efficient and convenient gene editing, so that the safety is guaranteed, the cost is saved, the operation complexity is reduced, and meanwhile, the success rate of immune cell preparation is greatly improved. Based on clinical requirements, a cell therapy product of CD7 KO + CD7 CAR T is jointly constructed by combining a CD7 eVLP vector with a retrovirus of CD7 CAR.

The invention provides a human diploid stably transfected cell line suitable for coxsackie virus A6 culture and application of the human diploid stably transfected cell line. The construction of the human diploid stably transfected cell line comprises the steps of construction of an hKREMEN1 lentivirus overexpression vector, virus packaging and stably transfected cell screening. The problem that proliferation of the CVA6 virus in human diploid cells is not ideal is solved, the human KREMEN1 gene is over-expressed in the human diploid cells (KBM17) by utilizing a lentivirus technology, and the proliferation capacity of the CVA6 virus on the KMB17 cells is improved by constructing the KMB17 cells capable of stably expressing hKREMEN1 receptors, so that a good foundation is laid for research and development of hand-foot-and-mouth disease vaccines; vaccine preparation and effectiveness evaluation are facilitated.

The present invention discloses a RAW 264.7 cell line stably overexpressing SRSF3, a preparation method thereof and an application thereof, belonging to the technical field of biomedicine. The present invention constructs a Lenti-Flag-hyg-SRSF3 plasmid overexpressing SRSF3, which forms a three-plasmid expression system with the psPAX2 and pMD2.G packaging vectors, co-transfects 293T cells, collects lentivirus, and then transfects RAW 264.7 macrophages with the lentivirus to obtain a stable cell line overexpressing the SRSF3 gene. The stable cell line overexpressing the SRSF3 gene is detected with a fluorescent antibody against Flag, and an LPS-induced inflammation model is used. Through Western Blot and qPCR detection, the stable cell line overexpressing SRSF3 can inhibit autophagy and reduce the expression level of inflammatory factors, and has the development and application prospects of inhibiting inflammation.

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 provides a method for producing engineered virus-like particles through a stable cell line and the engineered virus-like particles of the method, and particularly relates to a method for producing engineered virus-like particles through a stable cell line. The method comprises the following steps: constructing a BaEV retrovirus stable packaging cell line, transfecting a Gag-Cas9 plasmid to the BaEV retrovirus stable packaging cell line obtained in the step 1, constructing a self-inactivation delivery plasmid for a retrovirus packaging system, and selecting to obtain a stable cell line capable of producing engineering viroid particles, therefore, engineered virus-like particles can be produced through a stable cell line. According to the present invention, the eVLP stable cell line preparation method is innovatively developed, the existing production bottleneck is broken through, the stable, efficient and economic gene editing carrier solution is provided for scientific research and clinic, and the further development of the gene therapy field is promoted.

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 belongs to the technical field of air pollution health risk assessment, and particularly relates to an air pollutant health risk assessment method based on a cell stress effect. According to the invention, an immortalized normal human bronchial epithelial cell 16HBE is selected as a chassis cell, a luciferase reporter gene stably transfected cell strain ARE-luc2p-16HBE containing an ARE element and a luciferase reporter gene stably transfected cell strain p53RE-luc2p-16HBE containing a p53RE element are constructed, and the two cell lines are combined for use, so that the cell line can be used for preparing a stable cell line of the luciferase reporter gene. Therefore, the cell-irritable air pollutants have the capability of identifying and distinguishing extremely strong cell-irritable air pollutants from weak cell-irritable air pollutants. Moreover, the cell strain not only can be used for oxidative stress and genetic toxicity effect evaluation and health risk prediction of air pollutants, but also can be used for toxicity effect screening of general compounds, and has a relatively high application value.

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.

The invention relates to a modified mammalian glutamine synthetase comprising a mutation at amino acid position 10 and / or 298 in a mammalian glutamine synthetase, wherein the mutation is selected from the group consisting of R298K, N10S, N10T and N10Q and to an expression vector, a nucleic acid and a eukaryotic host cell encoding said modified mammalian glutamine synthetase and to its use as a selection marker. The invention further relates to methods for preparing stable cell lines, or for producing a protein of interest using said modified mammalian glutamine synthetase for selection.

The invention belongs to the technical field of gene engineering, and particularly relates to a bEnd.3 stably transfected cell strain for knocking down an Npc1 gene, an in-vitro cell model and a construction method of the in-vitro cell model. According to the invention, the bEnd.3 stably transfected cell strain knocking down the Npc1 gene is successfully and efficiently constructed, and comparative study shows that the constructed bEnd.3 cell strain knocking down the Npc1 gene can be used for studying the Npc1 gene function in vascular endothelial cells and the molecular mechanism of blood-brain barrier injury and leakage of the NPC1 type Niemann-Pick disease (NPC1). The bEnd.3 stably transfected cell strain for knocking down the Npc1 gene, established by the invention, provides an in-vitro cell model for deep research on blood-brain barrier dysfunction of the NPC1 disease, and provides theoretical and technical support for prevention and treatment of the NPC1 disease.

The disclosure pertains to tightly regulated inducible expression systems useful for the inducible production of one or more RNAs or proteins of interest, including the production of biologics such as recombinant proteins, vaccines, or viral vectors. Also provided are cell lines and kits useful for the production of said RNAs or proteins, as well as methods for making said cell lines and methods for inducing production of said RNAs or proteins.

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.

The present application relates to a kind of goldfish milt cell line and its application, belong to aquatic animal cell technical field.The preservation number of the present application goldfish milt cell line RcGFSB is: GDMCC NO:68000.The present application first successfully establishes the stable cell line from goldfish milt tissue, the cell line has good proliferation ability and is stable, can be long-term subculture and freeze storage;The goldfish milt cell line is sensitive to a variety of aquatic animal viruses, especially to the most serious virus pathogen of carp herpesvirus II type currently harmful to crucian carp and its variety, provides key experimental material for the isolation culture of the specificity virus and other aquatic pathogens of crucian carp and its variety, pathogenesis research and antiviral drug screening;The cell line has good exogenous gene expression capacity, can be used as fish bioreactor for recombinant protein production and gene engineering vaccine research and development, has important application prospect.

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.

The invention discloses an application of a CHO cell strain overexpressing Bag6 in improving the expression quantity of exogenous recombinant protein, and belongs to the technical field of bioengineering. Based on the fact that Bag6 protein is overexpressed in the cell, the innovation point is that the obtained modified cell has a remarkable enhancement effect on the expression of exogenous recombinant protein; the CHO cell strain for overexpressing the Bag6 in the application technology of the CHO cell strain for overexpressing the Bag6 in improving the expression quantity of the exogenous recombinant protein is characterized in that CHO cells are infected by lentivirus to enable the CHO cells to overexpress the Bag6 gene to construct a stable transfection cell line for overexpressing the Bag6, and the stable transfection cell line for overexpressing the Bag6 is used for expressing the exogenous recombinant protein (an antibody, hemagglutinin, enzyme, green fluorescent protein and the like). And the yield can be greatly increased by 1-10 times.

The invention relates to a method for constructing a stable cell strain capable of inducing gene silencing based on a Piggybac system. In particular, the present disclosure provides an engineered transposable element based on Piggybac comprising, from 5'to 3 ': a 5' ITR, a transposed region, a 3 'ITR. According to the present invention, the promoter, the Tet operon, the TetR, the shRNA design, the recombinant plasmid construction, the plasmid transfection and other aspects are optimized, such that the delivery, the integration and the validity verification of the Piggybac system-based induced gene silencing are achieved, the precise control and the reversible operation of the target nucleic acid expression level are achieved, and the quantitative data of the highly controllable silencing system is provided.

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

The invention discloses a reversible gene silencing stable cell line construction method based on inducible shRNA (short hairpin Ribonucleic Acid) expression, which belongs to the technical field of biology and comprises the following steps: (1) transferring a vector containing a drug inducible promoter system and a shRNA sequence of a targeted specific gene into a target cell through lentiviral transfection, screening through puromycin to obtain a monoclonal cell population of a stable integration vector; (2) adding doxycycline into the culture medium of the monoclonal cell population, and inducing shRNA expression to silence target protein; and (3) verifying the expression recovery of the target protein by removing doxycycline, and confirming the reversibility of the system. According to the invention, shRNA expression is controlled through the drug-induced promoter system, reversible silencing of specific genes is realized, and the drug-induced promoter system has the characteristics of high induction efficiency, low background expression and good stability.