75 results about "Genetic transfection" patented technology

The invention belongs to the technical field of immunology and in vitro diagnosis, and particularly relates to a rabbit-derived recombinant monoclonal antibody for specific recognition of type II grass carp reovirus VP4 protein, an eukaryotic expression method and application. According to the invention, a target gene S6 is transfected to an HEK293 cell for expression and purification, a New Zealand white rabbit is immunized in cooperation with a Freund's adjuvant, and the rabbit-derived recombinant monoclonal antibody capable of specifically recognizing the GCRV-II VP4 protein is obtained by using ELISA, single B cell screening and eukaryotic expression technologies. The antibody has strong specificity, provides support for further establishment of a specific II-type grass carp reovirus diagnosis technology, and has great application value in development of GCRV-II related scientific research.

The invention discloses a cartilage regeneration and repair method and system based on an irreversible electroporation technology. The method comprises the following steps: establishing an electroporation model of a cell membrane, simulating the distribution of an electric field and the potential change of the cell membrane, and predicting a membrane perforation effect; analyzing potential change according to an electroporation model, and combining energy consumption change of the cell membrane to obtain a critical condition of cell membrane pore formation; in combination with critical conditions, establishing an adaptive adjustment feedback mechanism, controlling the adjustment of electroporation parameters, and performing cartilage related gene transfection and cartilage repair; setting a comprehensive evaluation function, evaluating the repairing effect of the cartilage tissue, and adjusting electroporation parameters and a gene transfection strategy. The system comprises an electroporation model modeling module, a critical analysis module, an adaptive mechanism establishment module, a transfection repair module and an evaluation adjustment module. According to the method, the transfection efficiency of cartilage repair related genes can be improved and cartilage regeneration can be promoted by accurately regulating and controlling electroporation parameters and combining a self-adaptive feedback mechanism.

The invention relates to the technical field of biopolymer materials, in particular to a preparation method and application of a cationic polymer gene vector with a fluorinated end group. The invention provides a cationic polymer with a fluorinated end group. The structure of the cationic polymer is shown in the specification. According to the method, a fluorinated group is introduced through a fluorinated initiator by a one-step method to initiate synthesis of various terminal group fluorinated linear polyethyleneimine F-PEI with different molecular weights, the gene transfection efficiency is improved by introduction of the fluorine group, and high-efficiency DNA transfection is realized. The gene vector can achieve an efficient transfection effect in the cell transfection process, the toxicity generated to cells in the transfection process is small, gene molecules can be effectively and safely conveyed into the cells, and the gene transfection vector has the advantages of being efficient, low in toxicity, low in price, easy to synthesize and the like. The method has the advantages of simple reaction, easily available raw materials, low preparation cost, easily controlled molecular weight, high yield and simple post-treatment, and is suitable for large-scale production. # imgabs0 #

The application belongs to the technical field of immunology and in vitro diagnosis, and particularly relates to a rabbit-derived recombinant monoclonal antibody specifically recognizing type II grass carp reovirus VP4 protein, a eukaryotic expression method and application. The S6 gene in GCRV-II encodes VP4 protein, the application transfects the target gene S6 into HEK293 cells for expression and purification, cooperates with Freund's adjuvant to immunize New Zealand white rabbits, and uses ELISA, single B cell screening and eukaryotic expression technology to obtain a rabbit-derived recombinant monoclonal antibody specifically recognizing GCRV-II VP4 protein. The antibody has strong specificity, provides support for further establishment of specific type II grass carp reovirus diagnosis technology, and has great application value for development of GCRV-II related scientific research.

A preparation method for a micro-vehicle formed by enveloping siRNA-loaded polypeptide dendrimer nanogel with a cell membrane of engineered MSCs, and the use of same. The method comprises: using lentivirus-mediated gene transfection technology for constructing MSCs that highly express CXCR4 and PSGL-1; using cytokinin B for stimulating a cell to generate vesicles, and then performing centrifugation to collect the vesicles; by means of the electrostatic attraction between polypeptide dendrimer nanoparticles and siRNAs, attaching the siRNAs of IL-6 and HIF-1α into the polypeptide dendrimer nanoparticles; and by means of using a membrane extruder, enveloping the siRNA-loaded polypeptide dendrimer nanogel with the cell membrane. The prepared micro-vehicle exhibits a good property of aggregating at inflammatory blood vessel sites and a targeting function for joint cavities with rheumatoid arthritis. In addition, the loaded siRNAs can inhibit the expressions of IL-6 and HIF-1α in the joint cavities and inhibit the cell migration and angiogenesis. The micro-vehicle targeting inflammation sites has the advantages of excellent biocompatibility, ideal siRNA loading ratio, good inflammation targeting function, etc.

The invention discloses a cationic polyester, comprising a copolymer of monomer C, monomer S and monomer M, wherein the copolymer terminal group is modified with a group E; the monomer C comprises cholesterol; the monomer S comprises an organic acid having at least two carboxyl groups at the terminal group; the monomer M comprises a compound containing two hydroxyls and at least one secondary amine or tertiary amine; and the compound modified with the group E comprises at least one primary amine, secondary amine or tertiary amine group. The in vitro cell-level mRNA delivery efficiency of the cationic polyester of the invention is significantly better than that of the existing commonly used commercial gene transfection reagents, and the cytotoxicity is lower; the cationic polyester is soluble in ethanol, and is easy to be industrialized and clinically applied in preparations, and therefore has great practical application prospects.

The invention discloses an electroporation transfection protection liquid suitable for abalone embryonic cell gene editing and application thereof, and belongs to the technical field of gene editing. The electroporation transfection protection liquid comprises a basic liquid and a cell protection agent, the basic liquid is prepared from NaCl, MgSO4, CaCl2, KCl, NaHCO3, NaBr, K2HPO4, glucose and glutathione, and the pH value of the basic liquid is 7-8. The cell protective agent comprises polyethylene glycol 400. The electroporation transfection protection liquid can significantly reduce the malformation rate and death rate of abalone cells after an electroporation technology, improve the gene transfection and gene editing efficiency, reduce the gene editing experiment cost, and lay a foundation for accelerating marine organism research, promoting environmental protection, species conservation and aquaculture.

The invention discloses branched fluorine-containing poly (beta-amino ester) as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. The branched fluorine-containing poly (beta-amino ester) is prepared by polymerizing a diacrylate monomer, a triacrylate monomer, micromolecular organic amine and a fluorine-containing alkyl chain monomer through a Michael addition reaction and then blocking with an end-capping reagent. The preparation method is simple, and the process is controllable. The polymer has controllable molecular weight, branching degree and fluorine content, can be self-assembled with a gene drug to form a stable nano-composite, can mediate efficient gene transfection, has good in-vitro and in-vivo gene editing ability, and can be used for treating genetic metabolic diseases and the like.

The invention belongs to the technical field of gene transfection, and discloses a transfection enhancement method for improving transient transfection efficiency of plasmids, which comprises the following steps: step 1, plasmid extraction: extracting plasmids by using an endotoxin-removing plasmid extraction kit; 2, preparing cells before transfection; and the cells are recovered and cultured, so that the density of the cells reaches 70-90% during transfection. Step 3, transfection: 1) adding a proper amount of the serum-free culture medium into one centrifugal tube under a sterile condition, then adding a transfection reagent, and fully and uniformly mixing; 2) adding the serum-free culture medium and a reinforcing agent into the other centrifugal tube, fully and uniformly mixing, and then adding plasmids. According to the scheme, the transient transfection efficiency and effect of plasmids are comprehensively improved from plasmid extraction, cell preparation, transfection compound formation to subsequent treatment through a series of optimization steps. Meanwhile, cytotoxicity and stress reaction are reduced, and the cell survival rate and the target protein expression quantity are improved, so that the transfection method becomes an efficient and reliable transfection method.

Therefore, this disclosure provides a genetically engineered mesenchymal stem cell (MSC) population, including an expression vector containing the Akt or HGF gene and the PD-L1 gene. It also provides a method for synergistically improving the survival status and immunomodulatory capacity of mesenchymal stem cells or enhancing their proliferation, including transfecting mesenchymal stem cells with the Akt or HGF gene and the PD-L1 gene; and a method for preventing, improving, and / or treating ischemic conditions, enhancing neurogenesis, or reducing neuronal death, including administering an effective amount of the genetically engineered mesenchymal stem cell population of this disclosure to individuals in need.

The primary culture method of the porcine ovary granular cells comprises the following steps: step 1, taking a fresh porcine ovary, quickly putting the porcine ovary into a Hank's balanced salt solution preheated at 38 DEG C for transportation, repeatedly washing the ovary with the Hank's balanced salt solution to remove blood and impurities, and then extracting follicular fluid with a 5ml syringe under a sterile condition; according to the method, the granular cells can be effectively separated and cultured from the porcine ovary, and the porcine ovary granular cells with higher purity and better activity can be obtained, so that the subsequent cell biology experiment research, such as gene transfection, protein expression analysis and the like, is facilitated; a cell model is provided for researching the physiological functions (such as hormone secretion and follicle development regulation and control) of the porcine ovarian granular cells, the operation is relatively simple, the cost is relatively low, and the used reagents and instruments are common in a cell culture laboratory and are convenient to popularize and apply.

The invention discloses a method for improving transfection efficiency of chicken primordial germ cells, and belongs to the technical field of cell transfection. The invention provides application of an EGFR (epidermal growth factor receptor) activator NSC 228155 in improvement of plasmid uptake efficiency and cationic liposome transfection efficiency of chicken PGCs, and a method for efficiently transfecting the chicken PGCs is constructed. Experimental results show that compared with a control group, the gene transfection efficiency of the chicken PGCs treated by the EGFR activator NSC 228155 under a DMRIE-C transfection reagent and the gene transfection efficiency of the chicken PGCs treated by the EGFR activator NSC 228155 under a LipofectamineTM 3000 transfection reagent are both remarkably improved, which indicates that the EGFR activator remarkably improves the transfection efficiency of the chicken PGCs; and the EGFR activator does not influence the cell characteristics of the chicken primordial germ cells. The method lays a foundation for establishing an efficient gene editing chicken technical system, and has a wide application prospect.

An embodiment of the present invention discloses a gene transfection method and a gene transfection device, belonging to the field of cell biology. The present invention discloses a gene transfection method, including the following processes: providing an ultrasonic generating device and a heat generating device, where the ultrasonic generating device can emit ultrasonic waves, and the heat generating device can directly heat the biological system to be transfected; the ultrasonic waves and the heat generating device simultaneously stimulate the biological system to be transfected. By setting the ultrasonic generating device and the heat generating device, the present invention enables the combined action of ultrasonic waves and heat on the biological system to be transfected, generating a combined action including various effects such as mechanical effects, cavitation effects, and microfluidic effects on the biological system to be transfected, further shortening the time required for cell transfection, causing less damage to cells, and thus improving the survival rate after transfection.

The application relates to the technical field of biological macromolecular materials, in particular to a preparation method and application of a cationic polymer gene carrier with a fluorinated end group. The cationic polymer gene carrier with a fluorinated end group has the following structure. The fluorinated initiator is used to introduce fluorinated groups in one step to initiate the synthesis of linear polyethylene imine F-PEI with various molecular weights, the introduction of the fluorinated groups improves the gene transfection efficiency, and high-efficiency DNA transfection is realized. The gene carrier can achieve high-efficiency transfection effect in the cell transfection process, the transfection process has small toxicity to cells, gene molecules can be effectively and safely delivered into cells, and the gene carrier has the advantages of high efficiency, low toxicity, low price, simple synthesis and the like. The application has the advantages of simple reaction, easily-obtained raw materials, low preparation cost, easy control of molecular weight, high yield, simple post-treatment and suitability for large-scale production.

The invention relates to the technical field of nerve cell marking and regulation and control, in particular to a nerve cell living body selective protein modification probe capable of penetrating a blood brain barrier and application of the nerve cell living body selective protein modification probe. The protein body part specifically targets a living glutamic acid energetic neuron cell membrane, the protein body part and / or a modified part thereof can regulate and control the function of the glutamic acid energetic neuron, and a coding sequence of the protein body part comprises a sequence as shown in SEQ ID NO: 1 or a sequence having at least 85% identity with the SEQ ID NO: 1. The protein modified probe provided by the invention provides a new feasible thought for developing in-vivo nerve cell selective markers and regulation tools which are non-gene transfection of blood brain barrier penetration, are high in biological safety, are more stable and are easy to obtain and have nerve cell specific type selectivity.

The invention provides an electrode suitable for electroporation of early development chick embryos and a preparation method of the electrode. The electrode comprises an upper electrode and a lower electrode; the lower electrode comprises a root end, a middle end and a tail end which are connected with one another; the root end is a straight section which forms an angle of 35-45 degrees with the embryo; the middle end is arc-shaped, and the middle end is in a state of bending downwards and bending forwards at the same time relative to the root end; the tail end is a straight section horizontal to the embryo; the upper electrode is an electrode A suitable for small-range transfection or an electrode B suitable for large-range transfection; the electrode A comprises a straight section and a downward inclined section which are connected with each other and form an angle of 35-45 degrees with an embryo; and the electrode B comprises a straight section, a downward inclined section and a tail section which are connected with one another, wherein the straight section and the embryo form an angle of 35-45 degrees, and the tail section is horizontal to the embryo. By designing a reasonable electrode shape, accurate control of transfection area positioning is facilitated, and gene transfection in an ultra-small target range is realized.

The invention belongs to the field of biological medicine, and particularly relates to an engineered macrophage, an exosome, an engineered macrophage exosome delivery system and application thereof. The engineering macrophage is constructed by transfecting the PDCD1 gene and the Trp53 gene into the macrophage, and then the engineering macrophage is injected into the abdominal cavity of a mouse with colon cancer. The engineered macrophage continuously secretes exosomes containing expression products of the PDCD1 gene and the Trp53 gene in the abdominal cavity of a mouse and directly acts on colon cancer cells. Experiments show that the engineered macrophage has good killing ability and targeting ability, and can effectively inhibit tumor growth.

The invention discloses an in-vitro transcription system of theileria annulata transformed cells and a construction method of the in-vitro transcription system, the in-vitro transcription system comprises IVT-EGFP-mRNA, and the structure of the IVT-EGFP-mRNA is 5 'UTR-Cl-Kozak-EGFP-Pacl-3' UTR-polyA (120). According to the invention, mRNA for coding the EGFP gene is synthesized through in-vitro transcription, through capping, purification and agarose gel electrophoresis analysis, the expression condition of the target gene in TaNM1 cells is finally verified through an in-vitro transfection test and an indirect immunofluorescence technology, and the result shows that the in-vitro transcription system transfected and coded with the EGFP successfully expresses the EGFP, and the expression condition of the target gene in the TaNM1 cells is verified. The invention shows that a system for transfecting the EGFP gene into the theileria annulata transformed cell TaNM1 through an in-vitro transcription method is successfully established, and an important reference value is provided for design and research and development of theileria annulata RNA vaccines.

The present invention relates to methods for preparing carbon-based nanoparticles, also known as carbon quantum dots or carbon dots (CDs), in particular cationic carbon dots (CCDs), by incubating (1) CDs obtained from the hydrothermal treatment of an amine-free aqueous solution of citric acid with (2) polyethylenimine (PEI) (3) using stirring. The invention further relates to the use of said CCDs in the generation of gene delivery and gene transfection systems for the production of retroviral vectors.

The application relates to the technical field of new biological medical materials, in particular to a cinnamaldehyde modified high-molecular nucleic acid carrier and a preparation method and application thereof, wherein the cinnamaldehyde modified high-molecular nucleic acid carrier is a cinnamaldehyde modified polyethylene imine polymer. The high-molecular nucleic acid carrier provided by the application can significantly improve the DNA and RNA transfection efficiency in different cells, and can effectively improve the biocompatibility of the high-molecular carrier. The preparation steps of the high-molecular nucleic acid carrier are simple, the biocompatibility is good, the repeatability of batches is strong, and the high-molecular nucleic acid carrier has a wide application prospect in the development of nucleic acid carriers, gene transfection and nucleic acid treatment and the like.

The invention discloses adipose-derived mesenchymal stem cells (ADSCs) as well as a preparation method and application thereof. The ADSCs modified by the novel annular RNA-circAars gene have obviously enhanced osteogenic ability, and are suitable for the field of bone tissue engineering. The preparation method comprises the following steps: firstly, extracting adipose tissues from the groin of an SD (Sprague Dawley) rat, and digesting with collagenase to obtain ADSCs; then, circAars overexpression plasmids are synthesized, and circAars genes are transfected into the ADSCs by utilizing a lentivirus packaging system; and finally, screening and purifying the successfully transfected ADSCs through purinase. The ADSCs modified by the circAars gene show relatively strong osteogenic ability, are suitable for treatment of craniomaxillofacial bone defects, and also provide a new treatment strategy for repair of other bone defects. The method has great industrialization potential, and an efficient and safe treatment scheme is provided for bone defect repair.

The invention belongs to the technical field of biological materials, and particularly relates to an implant coating as well as a preparation method and application thereof. The invention aims to provide a material for an implant coating, which is characterized in that a cytoplasmic membrane and an oxidation response lipid peptide hybrid nanoparticle compress a gene through positive charge adsorption, and the oxidation response lipid peptide hybrid nanoparticle is a lipid peptide formed by connecting a branched basic amino acid head and a hydrophobic tail through an oxidation response bond, the hydrophobic tail part is a long-chain alkyl group or a saturated / unsaturated fatty acid chain, and the cytoplasmic membrane is an amphiphilic cytoplasmic membrane which has high functional protein expression capability after being subjected to hypoxia treatment. The invention provides an innovative combined strategy, namely fusion of gene engineering and bionic technology, and aims to improve the efficiency of coating gene transfection, actively regulate and control the peripheral microenvironment of the implant, accurately promote directional recruitment and enrichment of stem cells and effectively inhibit local inflammatory response, thereby establishing long-term stable bone-implant integration.

The application discloses an electroporation transfection protection fluid suitable for abalone embryo cell gene editing and application thereof, and belongs to the technical field of gene editing. The electroporation transfection protection fluid comprises a basic fluid and a cell protection agent; the basic fluid comprises NaCl, MgSO4, CaCl2, KCl, NaHCO3, NaBr, K2HPO4, glucose and glutathione; and the cell protection agent comprises polyethylene glycol 400. The electroporation transfection protection fluid can significantly reduce the deformity rate and mortality rate of abalone cells after electroporation technology, improve the gene transfection and gene editing efficiency, and reduce the cost of gene editing experiments, thereby laying a foundation for accelerating marine biological research and promoting environmental protection, species conservation and aquaculture.

The invention belongs to the technical field of biological gene modification, and particularly relates to transgenic moss, an ozone monitoring biosensor and a preparation method and application of the transgenic moss and the ozone monitoring biosensor. The transgenic moss is obtained by transfecting a target gene into moss cells, the target gene comprises a ZAT12 promoter, and the sequence of the gene ZAT12 is as shown in SEQ ID NO: 1. The transgenic moss is transferred into an oxygen response element ZAT12 promoter, and an ozone specific signal of the transgenic moss can be actively enhanced, so that high-sensitivity (5ppb) detection of ozone can be realized.

The invention provides a trachinotus ovatus spermatogonial stem cell line as well as establishment, cryopreservation and recovery methods and application thereof, and relates to the technical field of biologication.The method comprises the steps that tissue cells of trachinotus ovatus testis are placed in a complete culture medium to be cultured, and a fresh culture medium is replaced once every 2-4 days within 15 days before culture; culturing until the bottom of the bottle is full of cells, adding a trypsin solution to digest the cells, sucking out the digested cells every 30 seconds, respectively adding a complete culture medium for culturing for three times, screening out high-proportion spermatogonial stem cells for subsequent continuous culture, subculturing to a new culture bottle according to the proportion of 1: (1-3), and culturing to obtain the trachinotus ovatus spermatogonial stem cell line. The trachinotus ovatus spermatogonial stem cells with relatively high purity are obtained by a differential digestion method, and the trachinotus ovatus spermatogonial stem cell line is obtained by setting a passage proportion for culture and can be stably passed. And gene transfection research is carried out, so that technical support is provided for extensive research on spermatogonial stem cells of trachinotus ovatus.

The application provides a gene transfection device and a gene transfection method. The gene transfection device comprises an ultrasonic transfection chip for generating ultrasonic waves and a reaction channel in contact with the ultrasonic transfection chip to receive the ultrasonic waves, and the reaction channel is used for loading a cell solution. The device provides a novel, simple, high-throughput, fast, safe and efficient solution for cell transfection, has the characteristics of wide application range, simple operation, good repeatability, strong stability and the like, and can flexibly realize efficient transfection of adherent cells and suspension cells, and has good applicability to various difficult-to-transfect cells such as stem cells, primary cells and tumor cells.

The invention relates to the technical field of plant genetic engineering, in particular to a mutant OsPBL8 gene, a preparation method thereof and application of the mutant OsPBL8 gene in enhancing rice disease resistance. The mutant OsPBL8 gene comprises an OsPBL8 gene mutant promoter full length and an OsPBL8 gene coding sequence full length, and the nucleotide sequence of the mutant OsPBL8 gene is shown as SEQ ID No: 5. The mutant OsPBL8 gene sequence is loaded on a pZ469 (pZCB11-1300-3xMyc-GFP) vector subjected to BamHI single enzyme digestion, then agrobacterium tumefaciens is transferred, and the gene is transfected into an initial rice plant by an agrobacterium tumefaciens-mediated transformation method to form transgenic rice. By modifying the promoter of the OsPBL8 gene, the disease resistance of the rice can be remarkably improved, so that a mutant OsPBL8 gene overexpression plant still has excellent rice blast resistance in an environment with a relatively large day and night temperature difference.

The present invention provides an in situ gene transfection system based on a hydrogenated TiO2 nanotube composite coating, as well as its construction method and application. The in situ gene transfection system uses hydrogenated TiO2 nanotubes as a substrate and utilizes a layer-by-layer self-assembly technique to prepare a dopamine / graphene oxide / type IV collagen composite coating. This system can enhance the surface bioactivity of biomedical metal materials while also serving as an in situ gene transfection system for substrate-mediated local viral delivery, improving gene transfection efficiency and treatment safety, and enabling precise regulation of the treatment site. The in situ gene transfection system provides a new strategy for enhancing peri-implant epithelial sealing and preventing peri-implantitis.

The invention relates to the technical field of biological materials, in particular to lipid nanoparticles based on modified natural saponin, a preparation method and application of the lipid nanoparticles, and the modified natural saponin comprises natural saponin modified by metal complexes and / or natural saponin modified by targeting molecules. The natural saponin is selected from at least one of glycyrrhizic acid, aescin, soyasaponin, ginsenoside, ursolic acid and oleanolic acid. The modified natural saponin can effectively simulate the structural function of cholesterol compounds in lipid nanoparticles, can overcome the problems of unstable physicochemical properties of cholesterol, potential safety hazards of sources and the like, and can significantly improve the gene transfection and silencing efficiency of nucleic acid drugs as a drug carrier. And a new solution is provided for gene therapy and vaccine development. The preparation method of the lipid nanoparticles based on the modified natural saponin is mild in reaction condition, simple and convenient to operate, easy to control and beneficial to industrial production.

The invention discloses establishment and application of a human ovarian ectopic inner membrane cell strain (HOEEC) based on SV40T gene transfection, and relates to the technical field of biology, and the establishment comprises the following steps: S1, collecting a specimen; s2, isolated culture of human ovarian ectopic inner membrane primary cells; s3, amplification and extraction of plasmids; s4, performing lentivirus transfection to establish a human ovarian ectopic inner membrane cell strain; the method comprises the following steps: firstly, extracting and separating to obtain primary cells of the human ectopic ovarian endometrium, then establishing the human ectopic ovarian endometrium cell strain by transfecting SV40T, comparing the differences between the HOEEC and the primary cells in the aspects of cellular morphology, passage condition, multiplication capacity, invasion capacity, hormone receptor expression and estrogen reaction, and detecting the number and morphology of HOEEC chromosomes and the existence of in-vivo tumorigenicity. A new human ovarian ectopic endometrial cell strain is established by transfecting the SV40T gene for the first time, and an in-vitro model is provided for studying the pathogenesis of ovarian endometriosis.