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78 results about "Puromycine" patented technology
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Method for achieving HMGCR gene knockout based on CRISPR/Cas9 technology
The invention relates to a method for achieving HMGCR gene knockout based on the CRISPR / Cas9 technology. The method is characterized in that two CRISPR / Cas9 target sequence aiming at the HMGCR gene isdesigned, a gRNA single chain is synthesized in vitro, annealing is performed to obtain two gRNA double-chain DNA target insertion fragments, the insertion fragments are inserted into PX459 (pSpCas9(BB)-2A-Puro)V2.0 vectors to obtain the two different-locus plasmids of the target HMGCR gene; the two plasmids are transfected into PK15 cells, puromycin is used to process the cells, the processed cell genome DNA is extracted to perform PCR amplification, the PCR product is denatured, annealing is performed, and then T7E1 is used to perform HMGCR gene knockout identification. The method has the advantages that method can be used for analyzing the expression conditions of sequence and mRNA after the HMGCR gene knockout, whether an off-target phenomenon exists or not can be verified by using amethod combining PCR and T7E1 enzyme treatment, and accordingly the specificity based on target sequence HMGCR-gRNA can be determined; the method is applicable to cell and animal models to achieve fixed-point HMGCR gene knockout, has a reference value to the knockout of other genes, and is good in effect, simple, economical, short in time and the like.
Owner:HUNAN AGRICULTURAL UNIV
Method for inhibiting target gene expression by antisense lncRNA-mediated cis-regulation
The invention discloses a method for inhibiting target gene expression by antisense lncRNA-mediated cis-regulation. The method for inhibiting gene expression by antisense lncRNA-mediated cis-regulation is characterized in that by utilizing the characteristic that the lncRNA has the effect of competing endogenous target RNA, a strong promoter is inserted on a target gene by taking RNA as a genome positioning tool through a CRISPR Cas9 gene editing method, and a lot of antisense lncRNA complementary with the target gene is synthesized to realize in situ cis-competitive inhibition of expression of the endogenous target gene. The method comprises the following steps: firstly, constructing a targeting vector and a homologous template strand donor vector, simultaneously transfecting the constructed vectors into host cells, performing target cloning by puromycin and ganciclovir co-screening, and finally, performing detection and functional research on expression of the target gene.
Owner:JILIN UNIV
Conditional Cas9 expression induced swine trophoblastic cell line and establishment method and application thereof
InactiveCN105624194ATo achieve the purpose of conditional knockoutEasy to operateEmbryonic cellsFermentationPregnancyEmbryo
The invention discloses a conditional Cas9 expression induced swine trophoblastic cell line and an establishment method and application thereof. The method includes: subjecting a lentiviral vector, for controlling Cas9 gene expression, of a Tet-on system to virus packaging, concentrating and purifying; incubating lentivirus with a typical swine trophoblastic cell line, changing fluid, adding puromycin for drug screening, diluting cells to single ones, and culturing to obtain the single-cell-source swine trophoblastic cell line with Cas9 expression under Tet-on regulation. The swine trophoblastic cell line is simple in operation and convenient to use and has a potential utilization value in researching of key functional genes of some trophoblast cells and screening of swine gene target spots. The cell line is expected to be a significant cell material for researching swine placenta development and applicable to correlation researches on swine early-stage placenta development and uterine pregnancy mechanisms and also has a high reference value in research of human early embryonic development.
Owner:AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
Human pancreatic cancer nude mouse model construction method and use thereof
The invention relates to a novel human pancreatic cancer nude mouse model construction method for living imaging research and a use thereof. A human pancreatic cancer PANC-1 cell is stably transfected with a plasmid carrying a luc gene through a Lonza nuclear transfection system, a cell strain PANC-1-LUC for stable luciferase expression is screened through puromycin and PANC-1-LUC nude mouse transplant subcutaneous sarcoma is further constructed. An in vitro and vivo bioluminescence detection result proves that the PANC-1-LUC cell strain can stably express luciferase for a long time. Compared with a lentiviral vector-mediated bioluminescence pancreatic cancer nude mouse model, the built pancreatic cancer nude mouse model has a short tumor formation incubation period and a high tumor formation rate, satisfies PANC-1 transplantation tumor growth features and is suitable for living imaging research.
Owner:ZHEJIANG CHINESE MEDICAL UNIVERSITY
Preparation method and application of colon cancer cell strain expressed by stabilized silent PKM2 gene
InactiveCN103451222AEnhanced inhibitory effectMicrobiological testing/measurementTumor/cancer cellsLentivirusLarge Intestine Cancer
The invention provides a preparation method of a colon cancer cell strain expressed by stabilized silent PKM2 gene. The preparation method comprises the steps of constructing of shRNA lentiviral expression vector of human PKM2 gene, packaging and obtaining of lentivirus, DLD1 colon cancer lentivirus infecting, stabilized cell strain puromycin screening and Real-time PCR, Western blot identification. The experimental results show that the shRNA nucleotide sequence can be successfully inserted into the pLKO.1-puro expression vector, and the inhibitory effect on the expression of PKM2 gene is remarkable, endurable and stable. The prepared cell strain can be used as the experimental material for researching the regulating effect of PKM2 in malignant characteristics such as colon cancer cell energy metabolism, cell proliferation, cell migration and inflammation.
Owner:SHANXI UNIV
Oocyte activating solution and application thereof
ActiveCN111944744AEfficient activationNo biohazardClimate change adaptationCulture processIonomycinAnimal science
The invention belongs to the technical field of assisted reproduction, and relates to an oocyte activating solution and application thereof. The oocyte activating solution comprises 1.1 to 1.8 mg / L ofionomycin, 6 to 12 mg / L of calcium ion carrier A23187, 63 to 94 mug / L of puromycin, 19 to 43 mg / L of 6DMAP, 3.4 to 6.6 mg / L of actinone CHX, 28 to 53 mug / L of phorbol PMA, 7.8 to 12.7 mug / L of inositol triphosphate, 1.5 to 2.4 g / L of SrCl2, 16 to 25 mg / L of heparin, and basic culture medium. The oocyte activating solution can effectively activate oocytes, and is suitable for activating the oocytes after ICSI in an in-vitro fertilization assisted reproduction technology.
Owner:成都艾伟孚生物科技有限公司
Fibroblasts derived from multiple tissue sources of native dog through primary isolated culture and immortalization construction method of fibroblasts
PendingCN112574946AHigh activitySimple and fast operationCell dissociation methodsArtificial cell constructsDiseaseMuscle tissue
The invention provides fibroblasts derived from multiple tissue sources of a native dog through primary isolated culture and an immortalization construction method of the fibroblasts, and belongs to the technical field of cell culture. Heart tissue, lung tip muscle tissue or leg muscle tissue of the native dog are used as materials and subjected to enzymolysis and separation of pancreatin and / or collagenase II, and myocardial fibroblasts, lung fibroblasts and myofibroblasts with typical fibroblast morphological characteristics are obtained. The myocardial fibroblasts, the pulmonary fibroblastsand the myofibroblasts which are subjected to primary isolated culture are transfected by SV40T virus liquid, and then puromycin screening culture and passage are performed to obtain immortalized myocardial fibroblasts, pulmonary fibroblasts and myofibroblasts. Compared with primary culture cells, the immortalized constructed cells are higher in growth speed and remarkably improved in activity, and a material basis is provided for dogs in the aspects of scientific research, disease research and the like.
Owner:KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
Humanized intestinal cancer precancerous lesion immortalized epithelial cell line and construction method and application thereof
ActiveCN111172114AProliferative activity in vitroValue-added activity did not change significantlyGastrointestinal cellsGenetically modified cellsOncologyViral vector
The invention relates to a humanized intestinal cancer precancerous lesion immortalized epithelial cell line and a construction method and application thereof. The construction method comprises the following steps: firstly, transfecting primarily separated human colorectal adenoma polyp epithelial cells by using an SV40 overexpression lentiviral vector, then performing screening by using puromycin, and finally amplifying the screened cells to obtain the humanized intestinal cancer precancerous lesion immortalized epithelial cell line. The humanized intestinal cancer precancerous lesion immortalized epithelial cell line constructed by the invention overcomes the problems that conventional adenoma polyp cannot be subjected to passage in vitro, is low in cell proliferation and poor in cell activity, and cannot meet the cell passage requirement. According to the invention, the humanized intestinal cancer precancerous lesion immortalized epithelial cell line is established for the first time, an important cell experiment tool is provided for developing an in-vitro experiment of intestinal cancer precancerous lesions, and preclinical researches such as new drug screening and drug effectcomponents are also convenient to develop.
Owner:YUEYANG INTEGRATED TRADITIONAL CHINESE & WESTERN MEDICINE HOSPITAL SHANGHAI UNIV OF CHINESE TRADITIONAL MEDICINE
TALEN and pMD18 vector-based site-directed mutagenesis system and its application
InactiveCN104212778AStable passageAccurate passagingHydrolasesVector-based foreign material introductionDouble strandTarget gene
The invention relates to a TALEN and pMD18 vector-based site-directed mutagenesis system and its application. The system is composed of nuclease TALEN and a pMD18 vector, the nuclease TALEN is a transcription activator-like effect factor shearing a target gene target sequence, and the TALEN is composed of nuclease TALEN-L identifying the 5'-terminal of the target sequence and nuclease TALEN-R identifying the 3'-terminal of the target sequence; and the pMD18 vector is a pMD18 homologous recombinant template plasmid inserted into human AR site-directed mutagenesis gene, and the pMD18 vector is composed of corresponding upstream and downstream sequence of the target gene, and a puromycin screening marker. The system allows genome to be efficiently and accurately edited by using the homologous sequence provided by the Pmd18 vector during the cut-out of a double strand DNA molecule by the TALEN; and puromycin resistant gene is carried out when the introduced homologous sequence is adopted to edit the DNA sequence, so manual labor in the screening process is substantially reduced.
Owner:SHAOXING PEOPLES HOSPITAL
Method for quickly building CRISPR gene editing liver cancer cell strain and cell strain
PendingCN111254164AGood expression efficiencyStable knockoutHydrolasesGenetically modified cellsCas9Liver adenocarcinoma
The invention discloses a method for quickly building a CRISPR gene editing liver cancer cell strain. According to the method disclosed by the invention, a CRISPR / Cas 9 technique is improved, recombinant plasmids better in expression efficiency are constructed, quick monoclone culture is combined, and a stable gene knockout liver cancer cell strain is constructed. Required sgRNA is accurately obtained through primer synthesis, an inserting fragment is synthetized through two-step PCR, a carrier is loaded, and recombinant plasmids for knockout are constructed; after slow viruses are packaged, the packaged slow viruses and liver cancer cells are co-incubated, and sgRNA and Cas9 proteins of equal quantity are transmitted into the liver cancer cells at the same time through slow virus mediating; and liver cancer cells after gene editing are subjected to puromycin resistance screening and monoclone culture, and finally, a gene knock-out positive stable liver cancer cell strain is quickly obtained. According to the method disclosed by the invention, important experimental materials are provided for researching a molecular mechanism of the gene in generation and development of tumors, andreference is provided for in vitro cell modeling of liver cancer diseases.
Owner:DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
Method for constructing MLH1 gene knockout cell line
InactiveCN112501170AIncrease positive rateLow costGenetically modified cellsMicroorganism based processesTotal proteinGenetic engineering
The invention relates to a method for constructing an MLH1 gene knockout cell line, and relates to the technical field of gene engineering. According to the method, a CRISPR / Cas9 system is used to prepare an MLH1 gene knockout cell line, firstly designing two sgRNA sequences for the MLH1 gene, and constructing recombinant plasmids by utilizing molecular cloning technology; then transfecting the recombinant plasmids to HeLa cells, verifying the activity of sgRNA through PCR, carrying out puromycin drug screening and monoclonal treatment, extracting genome DNA and total protein of the monoclonalcell strain, and carrying out MSH1 gene level sequencing identification and expression detection of protein level to obtain an MSH1 gene knockout HeLa cell line. The MSH1 gene knockout HeLa cell lineconstructed by the invention is a cell line with stable inheritance of genes. The method provided by the invention can be used for directional knockout of the MSH1 gene to cause functional inactivation of the MSH1 gene, which has the characteristics of simplicity, convenience, high efficiency, quickness, low cost and the like, and has important significance for research on functions and related pathways of the MSH1 gene.
Owner:WUHAN AIBO TAIKE BIOTECH CO LTD
Construction method of stable cell line realizing FTH1 gene controlled expression
InactiveCN105349579AEfficient infectionReduce adverse effectsGenetic engineeringFermentationTiterNeuroblastoma cell
The invention provides a construction method of a stable cell line realizing FTH1 gene controlled expression. The construction method comprises the following steps: (1) acquiring FTH1 genes; (2) transferring the FTH1 genes into a Tet-On vector to obtain recombinant plasmids; (3) constructing the recombinant plasmids into a lentiviral vector; (4) inoculating human neuroblastoma cells, after the cell fusion reaches 40-50%, adding a safe medium containing lentivirus liquid 0.8 [mu]l / ml (the titer is 1X10<9>TU / ml) and polybrene 5 [mu]g / ml, and polyethylene glycol 0.5-1 [mu]g / ml, and placing the safe medium into an incubator for culturing for 20-24 h; and (5) after cell passage, and the cell fusion reaches 50-60%, adding a medium containing 8 [mu]g / ml puromycin and treating for 7d, then adding a medium containing 4 [mu]g / ml puromycin and screening for 3d, and thus obtaining the stable cell line capable of inducing expression of the FTH1 genes, and the stable cell line is named SK-N-SH / Tet-on / FTH1. With the adoption of the construction method, the expression level and the expression time of FTH1 are accurately regulated, so that FTH1 is expressed when MR imaging is needed, and is turned off when MR imaging is not needed.
Owner:CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
CCR5 deleted hematopoietic stem cell, preparation method and application thereof
InactiveCN102234628AAchieve the goal of treating AIDSMammal material medical ingredientsAntiviralsZinc finger nucleaseTherapy HIV
The invention discloses a method for preparing auto CCR5 deleted hematopoietic stem cells. The method comprises the steps that: CCR5 specific zinc finger nucleases (ZFNs) expression plasmids, and enhanced green fluorescent protein (EGFP) and puromycin recombinant plasmids are cotransfected into human induced pluripotent stem cells; human induced pluripotent stem cell CCR5 is specifically inactivated through gene insertion; and in vitro directional induction and differentiation are carried out, such that CCR5 deleted hematopoietic stem cells are obtained. The hematopoietic stem cells provided by the invention can be applied in the treatment of AIDS. With the hematopoietic stem cells provided by the invention, a novel method for treating AIDS stem cells can be provided.
Owner:GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI +1
Design and application for lentiviral expression vector
ActiveCN110982842AEfficient gene insertionMonitor transfection efficiency over timeBiological material analysisBiological testingTranscription startInsertion
The invention belongs to the fields of molecular biology and cytobiology, and specifically relates to a design and an application for a lentiviral expression vector. According to the vector designed by the invention, a CMV enhancer and a CMV promoter are used as transcription starting elements of an insertion gene; an EF1a-core promoter is adopted as a transcription starting element for screeningand marking puromycin and green fluorescent protein (GFP), and a puromycin resistance gene and a GFP sequence are connected and expressed in series through T2A; a lentiviral vector is adopted as a vector skeleton, and two above-mentioned elements are inserted into the skeleton vector; thus, the lentiviral expression vector is obtained by reasonable design and optimization. By adoption of the lentiviral vector designed by the invention, the inserted gene can be efficiently expressed, and the expression efficiency of the lentiviral expression vector is superior to the expression efficiency of conventional vectors with the same type of characteristics.
Owner:SHANXI UNIV
Suspension PK15 cell line for expressing pig CD163 and CD169 molecules
PendingCN111454910AIncreased susceptibilityReduce manufacturing costSsRNA viruses positive-senseGenetically modified cellsVaccine ProductionTGE VACCINE
The invention discloses a suspension PK15 cell line for expressing pig CD163 and CD169 molecules and a preparation method thereof, and belongs to the technical field of transgenosis. The method comprises the following steps: 1) constructing recombinant plasmids; 2) transfecting PK15 cells by using lipidosome; 3) screening puromycin; and 4) performing suspension culture and domestication by using aserum-free culture medium. The cell line prepared by the method is very sensitive to porcine reproductive and respiratory syndrome viruses, can be used for amplifying and reproducing a large number of porcine reproductive and respiratory syndrome viruses, is low in cost, and has a good application prospect in vaccine production practice.
Owner:成都史纪生物制药有限公司
Construction of cell strain capable of stably expressing porcine circovirus type 2 ORF3 protein and application thereof
PendingCN113265426AEasy to detectAntibody mimetics/scaffoldsVirus peptidesPorcine CircovirusesCloning Site
The invention discloses construction of a cell strain capable of stably expressing porcine circovirus type 2 ORF3 protein and an application thereof, and belongs to the field of biotechnology detection. The construction method comprises the following steps: (1) inserting a PCV2 ORF3 gene into multiple cloning sites of NheI and BamHI of a pLVZG-CMV-copGFP-Puro vector; (2) co-transfecting a 293T cell by using the constructed pLVZG-CMV-ORF3-copGFP-Puro recombinant plasmid and a virus packaging plasmid, culturing, discharging virus, collecting a virus supernatant, and filtering to obtain a recombinant lentivirus solution; and (3) infecting the packaged recombinant lentivirus with PK-15 cells, and screening and identifying through puromycin to obtain the cell strain for stably expressing the PCV2 ORF3 protein. According to the invention, a recombinant lentivirus system is utilized to prepare a cell strain which is fused with tag protein and can stably express PCV2 ORF3 protein, and the cell strain provides a platform for researching PCV2 pathogenesis and is beneficial to deep-level pathogenic mechanism research of PCV2.
Owner:LONGYAN UNIV
ICAM-1 (Intercellular Adhesion Molecule 1) gene knockout tumor cell strain and application thereof
InactiveCN108467864AImprove biological activityEfficient targetingGenetically modified cellsNucleic acid vectorHuman DNA sequencingCytokine
The invention relates to related study fields of cytobiology, molecular biology and the like and discloses an ICAM-1 (Intercellular Adhesion Molecule 1) gene knockout tumor cell strain and applicationthereof. The invention discloses sgRNA (Small Guide Ribonucleic Acid) which targets to a PAM (Protospacer Adjacent Motif) site of an ICAM-1 gene of a human genome; through transfection recombinant CRISPR plasmid transfection, the ICAM-1 gene of a HelA cell can be efficiently knocked out; through resistance screening with puromycin, a conventional limiting dilution method is not used in the screening process, but a relatively convenient and concise monoclonal picking method is used instead, and thus the experiment cycle is greatly shortened. With the ICAM-1 knockout cell strain, a novel research platform is established for studying adhesion and metastasis process of tumor cells, experiments show that the adhesion property of HeLa is remarkably degraded after ICAM-1 is knocked out, molecular mechanisms of mutual actions of ICAM-1 with different cell factors can be studied, and meanwhile treatment mechanisms and molecular targets related to anti-metastasis medicines can be well studied.
Owner:FUZHOU UNIV
Double-donor mediated drug/fluorescence collaborative screening-based diallelic editing system
PendingCN111850051AIncrease production capacityFacilitate the study of gene functionStable introduction of DNANucleic acid vectorFluorescent proteinBleomycinum
The invention discloses a double-donor mediated drug / fluorescence collaborative screening-based diallelic editing system. The system comprises two donor carriers, the donor vector sequence comprises aleft target gene homologous sequence, a right target gene homologous sequence, a point mutation site and an exogenous sequence located between the left target gene homologous sequence and the right target gene homologous sequence. The exogenous sequences are divided into a plurality of expression cassettes with thymokinase screening genes, green fluorescent proteins and puromycin resistance genesand a plurality of expression cassettes with thymokinase screening genes, red fluorescent proteins and bleomycin resistance genes. The gene editing system constructed by the invention can be used forcarrying out accurate and efficient diallelic editing on the genome.
Owner:NORTHWEST A & F UNIV
New application of drug screening cell model targeting HBV core promoter, Tricirine and structural analogue
ActiveCN113025651AInhibition of transcriptional activityCompound screeningApoptosis detectionHepatitis B immunizationHbv replication
The invention discloses a plasmid pcore-HU-SMAR, a framework vector of the plasmid pcore-HU-SMAR is plasmid PCMV-Gluc, a HiBiT-unaG fusion gene is inserted into the framework vector, the HiBiT-unaG fusion gene is connected with a puromycin resistance gene through a T2A sequence, an SMAR sequence is inserted between the puromycin resistance gene and a polyA sequence, and the HiBiT-unaG fusion gene is a fusion gene of a luciferase tag HiBiT and a fluorescent protein reporter gene UnaG. The invention further discloses a drug screening cell model of the targeted HBV core promoter, wherein the drug screening cell model is obtained by transfecting the AML12 with the plasmid pcore-HU-SMAR. The invention further discloses application of Tricirine and a structural analogue thereof to preparation of a medicine for inhibiting HBV core promoter transcription and / or HBV replication or application of the Tricirine and the structural analogue thereof to preparation of a medicine for treating hepatitis B.
Owner:CHONGQING MEDICAL UNIVERSITY
Cell strain and method of expressing reteplase (rPA) by human source cells
The invention belongs to the field of biopharmacy and particularly relates to a method and a cell strain of expressing reteplase by human source cells. The method specifically comprises the followingsteps of genetically synthesizing a cDNA sequence which contains a secretory signal and encodes the reteplase, inserting the cDNA sequence into a lentiviral expression vector, constructing a lentiviral vector capable of expressing the reteplase with secretion, packaging lentiviral particles, infecting the human source cells 293F with the lentiviral particles, performing resistance screeing on a high-copy monoclonal cell strain capable of stably expressing the reteplase with puromycin carried in the lentiviral vector, testing the expression content and the enzymatic activity of the reteplase, and verifying the established expression method. The method and the cell strain have the advantages that the reteplase expression cell strain established by the method is human source 293F cells, doesnot contain pyrogen endotoxin, is suspended, stably expresses the reteplase, is easy in large-scale amplification as well as is less in impurity and easy to purify as expressing the reteplase with secretion.
Owner:谢伟全
Construction method of immortalized forest musk gland epithelial cells
PendingCN113025661AReduce the impactGuaranteed growth rateCell dissociation methodsGenetically modified cellsBiotechnologyGlandular Epithelial Cells
The invention provides a construction method of immortalized forest musk gland epithelial cells. The construction method comprises the following steps: (1) collecting musk gland tissues of a male adult forest musk dead due to illness in a fragrance secretion period; (2) separating and purifying the forest musk gland epithelial cells and fibroblasts of the forest musk gland tissues to obtain purified lindel gland epithelial cells; (3) carrying out lentivirus transfection on the purified forest musk gland epithelial cells to obtain transfected forest musk gland epithelial cells; (4) screening out successfully transfected forest musk gland epithelial cells from the transfected forest musk gland epithelial cells through puromycin; and (5) carrying out multiplication culture on the screened successfully transfected forest musk gland epithelial cells to obtain the immortalized forest musk gland epithelial cells. According to the construction method of the immortalized forest musk gland epithelial cells, the immortalized forest musk gland epithelial cells are immortalized in a lentivirus transfection mode, the characteristics of the epithelial cells can be completely kept after 12 generations of culture, and the defect of epithelial cell apoptosis is overcome.
Owner:重庆市药物种植研究所 +1
Mesenchymal stem cell strain for overexpression of PD-L1 gene as well as a construction method and application of mesenchymal stem cell strain
PendingCN111961689ARelieve symptomsReduce joint damageAntipyreticAnalgesicsInflammatory factorsBiomedicine
The embodiment of the invention discloses a mesenchymal stem cell strain for overexpression of a PD-L1 gene as well as a construction method and an application of the mesenchymal stem cell strain, andbelongs to the technical field of biomedicine. The method comprises the following steps: 1) constructing a recombinant slow virus carrying a PD-L1 gene; and 2) transfecting mesenchymal stem cells with the recombinant slow virus carrying the PD-L1 gene, and carrying out puromycin screening, so as to obtain a mesenchymal stem cell strain for over-expressing the PD-L1 gene. After the mesenchymal stem cell strain for over-expressing the PD-L1 gene is injected into a rheumatoid arthritis mouse model intravenously, the symptoms of rheumatoid arthritis can be remarkably relieved, the joint injury degree is reduced, the expression of inflammatory factors is regulated, inflammatory response is inhibited, and the mesenchymal stem cell strain has a remarkable curative effect on treatment of rheumatoid arthritis diseases.
Owner:樊克兴
Directed evolution of multivalent glycopeptides that tightly bind to target proteins
ActiveUS20160304858A1Induce immune responseFusion with post-translational modification motifBiological material analysisProtein targetBioinformatics
The invention relates to a method for selecting a glycopolypeptide that binds to a target protein, the method including the steps of providing a pool of glycopolypeptides fused via puromycin linker to an encoding mRNA-cDNA duplex; combining the pool with a target protein to form a mixture; incubating the mixture for a period of time sufficient to allow any target protein to bind to one or more of the glycopolypeptides, thereby forming glycopolypeptide-target protein complexes; and isolating from the mixture the glycopolypeptide-target protein complexes, thereby identifying a plurality of selected glycopolypeptides.
Owner:THE GENERAL HOSPITAL CORP +1
Method for preparing porcine kidney epithelial cell line for stably expressing BE4 protein based on piggyBac transposon system
InactiveCN112280802AGood experimental basisAids in the study of biological functionsGenetically modified cellsMicroorganism based processesPiggyBac Transposon SystemPorcine kidney
The invention discloses a method for preparing a porcine kidney epithelial cell line for stably expressing BE4 protein, which comprises the following steps: by using porcine kidney epithelial cells (PK15) with good passage as host cells, co-transfecting a piggyBac transposon (PB-BE4max) vector containing a BE4 fusion protein sequence and a transposase expression vector plasmid, screening a positive cell population by puromycin, and selecting a monoclonal cell line; and verifying the editing activity amplifying the characteristic fragment of BE4 and transfecting active sgRNA, so as to obtain the transformant for stably expressing BE4 protein. The porcine kidney epithelial cell line prepared by the method can stably express BE4 protein, and has important application value in the porcine genetic engineering fields of porcine genome function research, transgenic pig preparation, drug-resistant target screening and the like.
Owner:HUAZHONG AGRI UNIV
Method for constructing cell line capable of highly expressing PRMT7 and cell line
InactiveCN111235182AGenetically modified cellsMicroorganism based processesPuromycinePulmonary metastasis
The invention discloses a method for constructing a breast cancer MCF7 cell line capable of stably over-expressing exogenous recombinant plasmid pCDH-puro-PRMT7. The full length of a PRMT7 gene is obtained; the pCDH-puro-PRMT7 plasmid is constructed; a virus is packaged, and cells are infected by the virus; puromycin is screened; and the MCF7 cell line capable of stably over-expressing the PRMT7 is obtained after amplification. The construction of the cell line is good for researching the cell biological functions of the PRMT7, such as the influences of the PRMT7 on the tumorigenic abilities of mice and the influences on the pulmonary metastasis abilities of the mice.
Owner:DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
Method for determining glucocorticoid mixture based on transgenic engineering cell strain
InactiveCN111500545AImprove throughputLow costAntibody mimetics/scaffoldsGenetically modified cellsEngineered geneticCytoplasm
The invention discloses a transgenic engineering cell strain and a method for determining a glucocorticoid mixture based on the cell strain. According to the cell strain, a green fluorescent protein GFP and a glucocorticoid receptor GR fusion protein gene are integrated into a Hela cell chromosome in a lentiviral infection mode, puromycin puro screening is conducted, and finally the Hela cell strain stably expressing GFP-GR is obtained. Based on a Hela cell strain for stable expression of GFP-GR, by measuring a migration ratio of a GFP-GR binding hormone ligand migrated from a cytoplasm regionto a cell nucleus region, the positive detection rate of the glucocorticoid mixtures in different samples can be rapidly detected in high flux and low cost, the sensitivity can reach 10<-9> mol / L interms of dexamethasone, the content of the glucocorticoid mixture in the environment and food can be rapidly screened, the harm degree is clear, and risk prevention and control are guided.
Owner:INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
Culture medium and method for evaluating embryo quality by using culture medium
ActiveCN110684721AIncrease abundanceEasy to detectCulture processCell culture active agentsProtein detectionEmbryo
The present invention belongs to the field of protein detection and particularly relates to a culture medium and a method for evaluating embryo quality by using the culture medium. The culture mediumcomprises fetal bovine serum proteins, an insulin-transferrin-selenium additive and puromycin. The method for evaluating the embryo quality by using the culture medium successively comprises the following steps of embryo culturing and embryo conditioned culture solution collecting, embryo conditioned culture solution thawing, endometrial stromal cell culturing, embryo conditioned culture solutionadding and protein detecting, differential protein of specific cell secreted protein is obtained and the embryo quality is evaluated. The evaluating method is very effective and accurate and makes upfor gaps in the market.
Owner:深圳中山泌尿外科医院
Cell line used for detecting hepatitis A virus titer as well as construction method and application thereof
ActiveCN107881152AShorten the production cycleQuality assuranceMicrobiological testing/measurementMicroorganism based processesTiterVirus detection
The invention relates to a cell line used for detecting hepatitis A virus titer as well as a construction method and an application thereof. According to the invention, by utilizing the characteristicthat 3ABC protease can interact with MAVS, MAVS C end and fluorescent protein are utilized to construct fusion protein which is packaged into lentivirus particles; after cells are infected, the expressed fusion protein is anchored to mitochondrion in cytoplasm to form dotted distribution; under the screening of puromycin, a stable cell line capable of stably expressing fusion protein can be obtained; after such a cell line is infected with HAV, fluorescent protein disperses in the whole cell or is positioned into a cell nucleus; and in the presence of methyl cellulose, fluorescent bacteriophage plaques can be observed, and the infectious titer of HAV can be detected by calculating the quantity of bacteriophage plaques. Compared with conventional methods, according to the method disclosedby the invention, only 1-4 days are needed for detecting HAV titer, and therefore, the virus detection cycle is remarkably shortened, the operation is simple, the sensitivity is high, infection courseof HAV can be intuitively observed, and popularization and application are easy.
Owner:INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
Compositions and methods for treating vascular malformation and related conditions
ActiveUS20180235994A1Reduce complexityImprove throughputOrganic active ingredientsMicrobiological testing/measurementDiseaseMedicine
In one aspect, the present invention features a method of inhibiting proliferation and / or reducing survival of a cell comprising a GNAQ polynucleotide or polypeptide having a R183Q or Q209L mutation, comprising contacting the cell with puromycin or a puromycin analog, thereby inhibiting proliferation and / or reducing survival of the cell. In another aspect, a method of treating a vascular malformation or related condition in a subject, comprising administering to the subject an effective amount of puromycin or a puromycin analog is featured. In another aspect, the present invention features a method of identifying a candidate agent that modulates a GNAQ R183Q or Q209L mutation-associated disease, comprising contacting a cell comprising a GNAQ polynucleotide or polypeptide having a R183Q or Q209L mutation with puromycin and a candidate agent and comparing viability of the contacted cell with a reference level of viability, wherein an alteration in viability indicates that the candidate agent modulates the GNAQ R183Q or Q209L mutation-associated disease.
Owner:DUKE UNIV OFFICE FOR TRANSLATION & COMMERCIALIZATION +1
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