46 results about "In vitro transfection" patented technology

The invention discloses a biodegradable cationic polymer micelle, preparation and application thereof. The biodegradable cationic polymer micelle consists of a triblock copolymer. The triblock copolymer at least comprises a cationic hydrophilic segment and a biodegradable hydrophobic segment, wherein the cationic hydrophilic segment forms a shell of the polymer micelle; and the hydrophobic segment forms a core of the polymer micelle. The polymer micelle can load siRNA through electrostatic compounding to obtain steady polymer / siRNA composite particles, and the particles can effectively inhibit the expression of specific genes during in vitro transfection experiments; and the polymer micelle loaded with a hydrophobic medicament can also load the siRNA through electrostatic compounding to realize a method for synchronously loading the medicament and the siRNA, and the method has the double effect on pharmacological treatment and gene therapy.

A composition for in vivo transfection of vertebrate male germ cells comprises a nucleic acid or transgene, and a gene delivery system, and optionally a protective internalizing agent, such as an endosomal lytic agent, a virus or a viral component, which is internalized by cells along with the transgene and which enhances gene transfer through the cytoplasm to the nucleus of the male germ cell. A pharmaceutical preparation and a transfer kit utilize the composition. A method for introducing a polynucleotide into vertebrate male germ cells comprises the administration of the composition to a vertebrate. A method for isolating or selecting transfected cells utilizes a reporter gene, and a method for administering transfected male germ cells utilizes male germ cells which have been transfected in vitro.

Novel compositions for the in vitro or in vivo production of specific proteins are provided. The compositions comprise components of vectors, such as a vector backbone, a promoter, and a gene of interest that encodes for the protein of interest, and the transposon-based vectors comprising these components. Also provided are methods of making these compositions and methods of using these compositions for the production of desired proteins in vivo or in transfected cells in vitro.

The invention relates to a method of efficiently separating embryonic stem cells of poultry. The method comprises the steps: 1) taking out an embryonic disc from a hatching egg; 2) digesting and cleaning the embryonic disc and removing yolk of the embryonic disc; 3) conducting primary culture to the cells of the digested embryonic disc; and 4) enriching and purifying the embryonic stem cells from the cells of the primarily cultured embryonic disc. The method is simple and easy, accurate and effective, high in repeatability and low in cost, equipment or reagent in the whole separation and culture system can be self-made or commercialized, a feeding layer is not prepared, enzymatic digestion and related processing are not needed, blood serum is not required, and about 20 thousand purified embryonic stem cells can be separated and cultured from one hatching egg, and can be used for in vitro transfection to conduct gene modification or apparent modification, thereby compacting the technological base for the transgenic poultry, installing a boost accelerator for development of a bioreactor and the realization of huge commercialization value, and having great scientific research values and popularization significance.

The invention discloses fusion protein for screening and evaluating an anti-enterovirus 71 (EV 71) medicine and application of the fusion protein. According to the fusion protein, firefly luciferase (Fluc) is used as a reporter gene, two micromolecule polypeptides which can be bonded tightly pepA and pepB are bonded with the N end and the C end of the firefly luciferase respectively, the middles of the firefly luciferase are connected by an EV713C protease effect substrate, and a fusion gene is inserted into an eukaryotic expression vector and is expressed to generate the fusion protein. Simultaneously, enhanced green fluorescent protein (EGFP) which is inserted reversely can indicate the transfection efficiency of the EGFP in cells which are transfected in vitro by observing the condition of green fluorescence. By utilizing an indication vector provided by the fusion protein, the reproduction condition of EV 71 can be indicated simply, quickly, flexibly and quantitatively, and the indication vector also can be applied to the screening and evaluation of the anti-EV 71 medicine, has high actual application value and has a broad application prospect in the field of medical science.

The invention relates to the technical field of T cell tumor treatment and in particular relates to a compound containing an anti-PD (Program Death)-1 gene and poly-spermidine and application of the compound to treatment of tumors. The compound contains a polyspermidine polymer and the anti-PD-1 gene at the mass ratio of (1 to 100) : 1. The compound is transfected to a T cell in vitro to prepare a self-secretion anti-PD-1 antibody-T cell and can generate a PD-1 antibody, so as to help to block a PD-1 signal and effectively help a depleted T cell, and promote the T cell capable of resisting tumor immune response to treat the tumors. Compared with the prior art, the secretion anti-PD-1 antibody-T cell provided by the invention can be used for effectively treating the tumors, and immune treatment of the tumors can be realized; an in-vivo toxicity problem does not exist.

The invention provides a preparation method of a lipidosome-protected nano-gold gene vector. The preparation method comprises the following steps: dissolving dimethyldioctadecylammonium bromide and dioleoyl phosphatidyl ethanolamine into an organic solvent, removing the organic solvent, dissolving into water, and performing ultrasonic treatment to obtain a phospholipid vesicle solution, wherein the molar ratio of the dimethyldioctadecylammonium bromide to the dioleoyl phosphatidyl ethanolamine is 1:10-10:1; and mixing the phospholipid vesicle solution with a compound containing gold ions, adding a reducing agent, and reacting to obtain the lipidosome-protected nano-gold gene vector. After being compounded with genes, the lipidosome-protected nano-gold gene vector is high in transfection efficiency and high in gene release speed in a specific environment; the lipidosome-protected nano-gold gene vector is high in stability in serum and is not aggregated, and a transfection process is simplified since serum in a culture solution does not need to be removed in an in-vitro transfection process; the carrying amount of DNAs (Desoxvribose Nucleic Acids) is high, the using quantity of the DNAs is reduced, and the immunogenicity of exogenous DNAs to cytotoxicity and organisms are lowered.

Oligomeric sulfonamides for use as endosomolytic reagents for transfection with polymeric or lipid-based vectors are described. A mixture of an oligomeric sulfonamide with a polymeric or lipid-based gene carrier and a nucleic acid results in a polyplex that exhibits 6-12-fold better gene expression than controls. A method of transfecting cells in vitro is carried out by contacting cultured mammalian cells with an effective amount of a polyplex.

The invention relates to a method for transfecting pig T lymphocytes in vitro by applying a nuclear transfection method, comprising the following steps of: introducing an expression vector containing foreign genes or mRNA (Messenger RNA) of the foreign genes into the pig T lymphocytes by applying an electroporation technology and a cell-specific nuclear transfection solution; culturing transfected cells, and separating the pig T lymphocytes expressing the foreign genes. The method disclosed by the invention has the advantages of high transfection efficiency, easiness and convenience for operation, less needed plasmid quantity and fast expression of target genes and is beneficial to researching the specific gene functions of T cells at activated and effective stages.

The invention provides a synthetic method for a self-luminous polymer microsphere. The synthetic method comprises the following steps: A, providing an aqueous polyethyleneimine solution and an emulsifier solution and mixing the two solutions so as to obtain an emulsified liquid; B, adding a cross-linking agent into the emulsified liquid obtained in the step A at a temperature of 15 to 25 DEG C and carrying out stirring so as to obtain an orange red emulsion; and C, removing an oil phase and excess cross-linking agent in the emulsion obtained in the step B so as to obtain the self-luminous polymer microsphere. The self-luminous polymer microsphere synthesized in the invention is in a regular spherical shape, has fluorescent properties and can meet requirements on biological tracing without fluorescein marks; moreover, the self-luminous polymer microsphere synthesized in the invention has the advantages of a low price, good in-vivo and in-vitro transfection effect, no immunogenicity, stable performance, no toxicity, etc.; and the synthetic method has the advantages of simple operation, low cost, mild reaction conditions and no toxicity.

The invention discloses a modified polyethyleneimine derivative as well as a synthesis method and application thereof. According to the invention, Triton X-100 is conjugated to polyethyleneimine, suchthat a PEI-Triton-N carrier is obtained. In-vitro cell evaluation shows that the carrier shows good safety and stability; cell transfection effect experiments show that the carrier shows efficient and low-toxicity gene delivery capacity, can significantly improve the gene transfection efficiency of various cells including skin immortalized cells HaCaT difficult to transfect, and is an excellent in-vitro transfection reagent; an in-vivo toxicity experiment shows that the carrier has no obvious systemic toxicity; and a gene delivery experiment result of mouse skin local administration shows that the carrier has good local administration gene delivery capability and is a good local administration gene delivery vector.

The invention relates to skin tissue engineering seed cells, a construction method, an adenovirus vector and a use thereof, and the construction method comprises the steps of culturing human skin fibroblasts and constructing the adenovirus vector containing double genes of CTLA4Ig-CD40Ig; and the in vitro transfection of the human skin fibroblasts is carried out by the adenovirus vector containing the double genes of CTLA4Ig-CD40Ig. The skin tissue engineering seed cells with low immunogenicity are produced by the above construction method. The adenovirus vector used for transfection of the human skin fibroblasts is the Ad5F35 chimeric adenovirus vector which contains CTLA4Ig gene and CD40Ig gene effectively connected with a promoter. The invention relates to the use of adopting the adenovirus vector in the preparation of the transfected human skin fibroblasts for repairing injured skin. The invention can effectively reduce the immunogenicity of the seed cells, and the transfected human skin fibroblasts do not affect the proliferation performance thereof in vitro.

The invention relates to a composite tumor gene vaccine taking a bacterial nano magnetosome as a carrier and a preparation method thereof. The range of a pH value of the optimal phosphate buffer system when the bacterial nano magnetosome is connected with the gene vaccine and magnetic field intensity and magnetic field acting time used by in-vitro transfection and in-vivo immunization are provided specifically, and treatment dosage range and immunization way are determined when the composite gene vaccine is used for treating a tumor model. A novel simple and convenient gene delivery mode is established, and a preparation condition and an immunization method of a new gene vaccine are provided for gene treatment.

The invention relates to the field of articular cartilage injury repair and particularly relates to an adipose-derived stem cell transfected by a magnetic nanoparticle mediated IGF-I gene and a preparation method thereof. The method comprises three steps of ADSCs separation and culture, Fe3O4 MNPs preparation and in vitro transfection. The mediated IGF-I gene with an iron oxide magnetic nanoparticle as a vector is used for transfecting ADSCs to achieve over expression of IGF-I, and the homing rate of stem cells is improved by a dynamically rotating magnetic field in vitro in a targeting manner, and the iron oxide magnetic nanoparticle used in the research is a non-virus gene vector, has the advantages of being safe, harmless, high targeting and efficient and stable expression of a target gene, and the like, can be used for greatly improving the gene transfection efficiency, and more importantly can be used for solving the problem of low homing rate of ADSCs in a scaffold in an implant via the dynamically rotating magnetic field in vitro, and positioning and tracking the stem cell in vivo through magnetic resonance technology.

The invention relates to a pseudo MERS-CoV virus packaging system, a method for preparing pseudo MERS-CoV viruses by in-vitro transfection of cells using the pseudo MERS-CoV virus packaging system, and a mouse model with knock-in of a hDPP4 (human dipeptidyl peptidase 4) gene infected by the pseudo MERS-CoV viruses in a biological laboratory lack of BSL-3 (biological safety level 3), so as to evaluate the in-vivo effects of an anti-virus agent and a vaccine.

The invention discloses a VSIG4 (V-set and Ig domain containing 4) and IGRP (glucose-6-phosphatase catalytic subunit-related protein) dual-gene coexpression recombinant adenovirus as well as a preparation method and an application thereof. The recombinant adenovirus genome contains a VSIG4 and IGRP dual-gene expression cassette which sequentially comprises a CMV (cytomegalovirus) promoter, a VSIG4 full length coding gene, a terminator, an internal ribosome entry site (IRES), an IGRP full length coding gene and a terminator from the upstream to the downstream. The preparation method of the recombinant adenovirus is simple. Dendritic cells of the recombinant adenovirus, which are transfected in vitro, are fed back in vivo, which can reduce the multiplication capacity of lymphocytes of NOD (Nonobese Diabetic) mice with diabete liability and the secretion capacity of cell factors, moreover, the onset time of the diabetes of the NOD mice is delayed, and the morbidity is reduced. The recombinant adenovirus is proved to be capable of effectively inducing the tolerance of specific T-cells and effectively inhibiting the breakage of pancreas islet beta cells and can be used for preparing dendritic cell vaccines for resisting type-1 diabetes.

The invention discloses a degradable polymer based on ring opening polymerization of a valerolactone derivative and a production method and application thereof. The compound is mainly produced throughMichael reaction, ROP reaction and thio-ene click reaction, and the structure of the compound is identified through nuclear magnetism and mass spectrometry. According to the degradable polymer, through testing means of gel electrophoresis, dynamic light scattering, SEM and the like, the fact that the degradable polymer can condense DNA is proven, and the diameters of formed nanoparticles are allsmaller than 200 nm. An in-vitro transfection experiment proves that lipidosome formed by the degradable polymer and dioleoyl phosphatidylethanolamine (DOPE) can be used as a non-viral gene vector.

The invention discloses a method for acquiring a solid tumor cell with successful in-vitro transfection, comprising the following operation steps: 1) taking single-cell suspension of an exponential phase, culturing for 20h at the temperature of 37 DEG C by serum culture solution; 2) mixing antisense oligonucleotide in a sterile EP tube; 3) mixing liposome in another sterile EP tube, placing at room temperature for 5min after mixing; 4) taking the above antisense oligonucleotide mixture and lipofectamine 2000 mixture; evenly mixing, placing at room temperature for 20min for later use; and 5) transfecting. The invention effectively solves the problem that an in-vitro transfection technology can not stabilize the cells in transfection three-dimensional auxocyte mass; the cells can be evenly transfected into the three-dimensional auxocyte to be transfected, thus achieving the highest transfection efficiency, and simulating the functions of the related molecules in vivo to the maximum.

The invention provides a mink enteritis parvovirus whole genome infectious clone and a construction method and application thereof. The method comprises the following steps of: carrying out enzyme digestion on a complete genome in an MEV replication form, and sequentially and directionally cloning to a vector pUC18 M in a segmented form to obtain a recombinant plasmid; and mixing the recombinantplasmid with a transfection reagent, and transfecting CRFK cells to obtain a rescue virus. The mink enteritis parvovirus infectious clone constructed by utilizing a reverse genetic technology transfects the CRFK cells in vitro, and can induce the cells to generate cytopathic effects and growth trends the same as those of a parent virus. The method is simple, rapid, time-saving and labor-saving, and has higher accuracy compared with a traditional PCR method, and base mutation can be rapidly and conveniently carried out at any position of the MEV genome by applying the cloning system, so that aneffective way and means are provided for subsequent research and development of molecular biology of MEV and research and development of vaccines.

The invention discloses cationic complex nanoparticles of poloxamer and / or poloxamine in combination with PEG lipids, and belongs to the field of gene therapy. The cationic complex nanoparticles comprise poloxamer and / or poloxamine and PEG lipids. Wherein the cationic complex nanoparticles comprise one or more of components disclosed in the invention; when the cationic complex nanoparticles are taken as a nucleic acid carrier, the cationic complex nanoparticles are lower in cost than a virus carrier; quality control is convenient; compared with a common nano-carrier, the cationic complex nanoparticles are high in transfection efficiency, low in toxicity, good in biocompatibility, free of liquid change after administration in an in-vitro experiment, particularly suitable for transfection ofisolated cells, effective in in-vivo and in-vitro transfection and simple to prepare, and creatively can solve the problem of safe and efficient in-vivo and in-vitro delivery of nucleic acid, especially mRNA.

The invention discloses a poloxamer and / or poloxamine and lipid compound, and relates to the field of gene therapy. The poloxamer and / or poloxamine and lipid compound comprises poloxamer and / or poloxamine and lipid. Wherein the compound is composed of any one of components disclosed in the invention with DOTAP and DOPE; the cost of the compound as a nucleic acid vector is lower than that of a virus vector; compared with a common nano-carrier, the compound is high in transfection efficiency, low in toxicity and good in biocompatibility, liquid does not need to be replaced after administration in an in-vitro experiment, the compound is particularly suitable for transfection of in-vitro cells, in-vivo and in-vitro transfection has high expression, preparation is easy, and the problem that nucleic acid, especially mRNA, is safely and efficiently delivered in vivo and in vitro is creatively solved.

The invention discloses cationic complex nanoparticles of poloxamer and / or a combination of poloxamer amine and PEG lipids, and relates to the field of gene therapy. The cationic complex nanoparticlescomprise poloxamer and / or poloxamer amine and PEG lipids, wherein the cationic complex nanoparticles comprise one or more of formulas as shown in the specifications. As a nucleic acid carrier, the cationic complex nanoparticle is lower in cost than a virus carrier, convenient for quality control, higher in transfection efficiency than a common nano carrier, low in toxicity, good in biocompatibility, free of liquid change after administration in an in-vitro experiment, particularly suitable for transfection of isolated cells, effective in in-vivo and in-vitro transfection, and simple to prepare, so that the problem in that nucleic acid, especially mRNA, is safely and efficiently delivered in vitro and vivo is creatively solved.

The invention provides highly acidic chitosan-nucleic acid polyplex compositions. The compositions may be used to transfect cells in vitro and in vivo, and are particularly useful for transfecting cells of mucosal epithelia.

The invention discloses a poly-valerolactone type amphiphilic polymer based on tetraphenyl ethylene as well as a preparation method and an application of the poly-valerolactone type amphiphilic polymer. The disclosed compound is mainly prepared through a Michael reaction, an ROP reaction and a thio-ene click reaction, and the structure of the compound is confirmed by nuclear magnetism and mass spectrum. The poly-valerolactone type amphiphilic polymer has an aggregation-induced emission (AIE) effect, gel electrophoresis, dynamic light scattering, SEM and other test means prove that the polymercan aggregate DNA, and the diameter of formed nanoparticles is smaller than 200 nm. In-vitro transfection experiments prove that liposome formed by the poly-valerolactone type amphiphilic polymer anddioleoyl phosphatidyl ethanolamine (DOPE) can be used as a non-virogene vector.

The invention provides a whole genome infectious clone of mink enteritis parvovirus and its construction method and application. The method is to digest the complete genome of the MEV replication form, and sequentially and directional clone it into the vector pUC18‑M in the form of segments to obtain the recombinant plasmid. After mixing the recombinant plasmid and the transfection reagent, the CRFK cells can be transfected to obtain the rescued virus. The mink enteritis parvovirus infectious clone constructed by using the reverse genetic technology in the present invention transfects CRFK cells in vitro, and can induce the cells to produce the same cytopathic and growth tendency as the parental virus. This method is simple, fast, time-saving and labor-saving, and has higher accuracy than the traditional PCR method. The application of this cloning system can quickly and conveniently carry out base mutations at any position in the MEV genome, thereby providing a basis for the subsequent development of MEV molecular biology. Research and the development of vaccines provide effective ways and means.

The invention discloses a nano cationic liposome carrying an FL gene recombinant plastid, and a preparation method and application of the nano cationic liposome, and belongs to the field of biological medicine. The liposome comprises Lipofectamine 2000 and a recombinant plasmid pEGFP-cl-FL carrying an FL gene according to an electrostatic adsorption principle, wherein the recombinant plasmid pEGFP-cl-FL carrying the FL gene is prepared by cloning the FL gene into a pEGFP-cl vector containing a green fluorescent protein reporter gene. The nano cationic liposome has the characteristics of simple preparation method, low toxicity, low immunogenicity, slow release and high-efficiency targeting, is particularly suitable for serving as a gene vector of in vivo and in vitro transfection and gene therapy, and has a good application prospect in the gene research aspect in the fields of tumor and others.

The invention relates to a method for differentiating and identifying dry cells under the action of exogenous neurotrophic factors. The method comprises the following steps: separating and amplifying spinal cord neural stem cells of an embryonic rat; culturing and differentiating neurosphere cells depending on EGF (epidermal growth factor) and FGF (fibroblast growth factor) under the action of different exogenous neurotrophic factors; constructing a recombinant adeno-associated virus containing HIF-1 (hypoxia-inducible factor-1) alpha and BDNF (brain-derived neurotrophic factor) sequences; performing in vitro transfection and analysis on the spinal cord neural stem cells of the rat, namely measuring an expression product and analyzing the bioactivity. The method is stable in performance.

The invention provides a preparation method of a liposome-protected nano-gold gene carrier: dissolving dioctadecyldimethylammonium bromide and dioleoylphosphatidylethanolamine in an organic solvent, and then removing the organic solvent After dissolving in water, ultrasonically obtain the phospholipid vesicle solution; the molar ratio of the dioctadecyldimethylammonium bromide and dioleoylphosphatidylethanolamine is 1:10~10:1; the phospholipid vesicle After the capsule solution is mixed with the compound containing gold ions, a reducing agent is added to react to obtain the nano-gold gene carrier protected by the liposome. After the liposome-protected nano-gold gene carrier of the present invention is combined with the gene, the transfection efficiency is high, and the gene release speed is fast in a specific environment; it has good stability in serum, no aggregation phenomenon, and does not need to be cultured during the in vitro transfection process. The serum in the liquid is removed, which simplifies the transfection process; the DNA load is high, which reduces the amount of DNA used, and reduces the cytotoxicity and immunogenicity of exogenous DNA to the body.

The invention discloses a degradable polymer based on ring opening polymerization of a valerolactone derivative and a production method and application thereof. The compound is mainly produced throughMichael reaction, ROP reaction and thio-ene click reaction, and the structure of the compound is identified through nuclear magnetism and mass spectrometry. According to the degradable polymer, through testing means of gel electrophoresis, dynamic light scattering, SEM and the like, the fact that the degradable polymer can condense DNA is proven, and the diameters of formed nanoparticles are allsmaller than 200 nm. An in-vitro transfection experiment proves that lipidosome formed by the degradable polymer and dioleoyl phosphatidylethanolamine (DOPE) can be used as a non-viral gene vector.