102 results about "Transient transfection" patented technology

A method of mRNA production for use in transfection is provided, that involves in vitro transcription of PCR generated templates with specially designed primers, followed by polyA addition, to produce a construct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ cap and/or Internal Ribosome Entry Site (IRES), the gene to be expressed, and a polyA tail, typically 50-2000 bases in length. This RNA can efficiently transfect different kinds of cells. This approach results in increased efficiency (fidelity and productivity) of mRNA synthesis and is less time consuming because it does not require cloning, and also consequently eliminates the unwanted errors and effects related to RNA made on DNA templates obtained with cloning techniques. The results of transfection of RNAs demonstrate that RNA transfection can be very effective in cells that are exceedingly difficult to transfect efficiently with DNA constructs. Further, the levels of gene expression following mRNA transfection are consistent from cell to cell in an experiment and these levels can be controlled over a wide range simply by changing the amount of mRNA that is transfected, and without obvious cytotoxic effects due to the levels of RNA per se. Due to high efficiency the cells can be simultaneously transfected with multiple genetic constructs. The method can be used to deliver genes into cells not- or only poorly transfectable for DNA, in vitro and in vivo.

The present invention relates in part to methods for suppressing the innate immune response of a cell to transfection with an exogenous nucleic acid, to methods for increasing expression of a protein encoded by an exogenous nucleic acid by repeated delivery of the exogenous nucleic acid to a cell, and to methods of changing the phenotype of a cell by differentiating, transdifferentiating or dedifferentiating cells by repeatedly delivering one or more nucleic acids that encode defined proteins. A method is provided for extended transient transfection by repeated delivery of an in vitro-transcribed RNA (“ivT-RNA”) to a cell to achieve a high and sustained level of expression of a protein encoded by an ivT-RNA transcripts.

The present invention relates to compositions and methods for transfecting eukaryotic cells with nucleic acid vectors. In particular, the invention relates to the uses of Matrix Attachment Region (MAR) elements to increase stable and transient transfection efficiency.

Compositions and methods of making cells using RNA, and cells made using the disclosed compositions and methods are also provided. In exemplary embodiments, RNA is transfected into cells to effect a molecular, biological, physiological, or histological change in the cells. In preferred embodiments, the RNA is prepared in vitro, more preferably using a DNA template according to the provided compositions and methods. Methods for treating or inhibiting a disorder or disease such cancer are also provided. The methods can include, for example, locally or systemically administering to the host an effective amount of one or more RNAs; or an effective amount of population of cells isolated from the subject or a syngeneic or histocompatible subject, contacted ex vivo with one or RNAs, and optionally expanded. The cells can be, for example, immune cells or stem cells.

The present invention relates to compositions and methods that allow the production of polypeptides and untranslated RNA molecules in host cells. More specifically, the invention provides nucleic acid molecules, expression systems, recombinant host cells, methods and kits, which enable the production and/or isolation and/or purification of polypeptides and untranslated RNA molecules. The compositions and methods of the invention can be applied in a regulatable expression system that is transiently transfected into mammalian host cells.

The invention provides a novel retroviral packaging system, in which retroviral packaging plasmids and packagable vector transcripts are produced from high expression plasmids after stable or transient transfection in mammalian cells. High titers of recombinant retrovirus are produced in these transfected mammalian cells and can then transduce a mammalian target cell by cocultivation or supernatant infection. The methods of the invention include the use of the novel retroviral packaging plasmids and vectors to transduce primary human cells, including T cells and human hematopoietic stem cells, with foreign genes by cocultivation or supernatant infection at high efficiencies. The invention is useful for the rapid production of high titer viral supernatants, and to transduce with high efficiency cells that are refractory to transduction by conventional means.

Recombinant proteins are of great commercial interest. Yet, most current production methods in mammalian cells involve the time- and labor-consuming step of creating stable cell lines. Production methods based on transient gene expression are advantageous in terms of speed and versatility, yet, thus far, those methods have not shown the specific productivity, batch yield and volumetric yield to be an economic alternative to stable cell lines. The inventors improved on the methodology of transient transfection and achieved commercially relevant yields in terms of specific productivity (exceeding 35 pg per cell per day), batch yield (exceeding 700 mg/l) and volumetric yield.

The invention belongs to the technical field of bioengineering and particularly relates to a novel double promoter structural unit. The novel double promoter structural unit disclosed by the invention comprises two CMV promoters shown in a nucleotide sequence SEQ ID NO. 2 and a sequence shown in a nucleotide sequence SEQ ID NO. 1. The double promoter structural unit is integrated by chromosomes, so that transcription or expression of a target gene in an expression system is increased. The promoter structural unit provided by the invention can be used for constructing expression vectors of cells of mammals and the constructed plasmid can be used for transient transfection for cells of mammals and construction of a stable expression cell strain. Compared with the conventional CMV promoter, the promoter unit provided by the invention is stronger in transcription efficiency.

The invention relates to a construction method and application of a DNA (Deoxyribonucleic Acid) vaccine for avian leukosis virus subgroup J. A recombinant eukaryotic expression plasmid pcDNA3.1-Fc-env of an Fc fragment gene for expressing chicken immunoglobulin G and an envelope protein (env protein) gene of the avian leukosis virus subgroup J is constructed; transient transfection and indirect immunofluorescence assay prove that the pcDNA3.1-Fc-env can be accurately expressed in a 293T cell; a great number of plasmids are extracted, purified and quantified to 1mg/ml, then, the recombinant plasmids are used for immunizing mice, each mouse is immunized three times, 100mu g of recombinant plasmids are used in each immunization, and one immunization is carried out every two weeks; the level of an ALV-J env protein-specific antibody in serum is detected to show that the pcDNA3.1-Fc-env has the effect of preventing the avian leukosis virus subgroup J.

The invention relates to a method for in-vitro screening inducers, particularly to a method for in-vitro screening of PXR (pregnane X receptor) activation characteristics, which comprises the following steps: constructing a reporter gene vector; culturing cells, and screening G418 working concentration; carrying out transient transfection of cells; screening stably transfected cell clones; inducing and identifying stably transfected cell strain with a tested drug (ligand drug); and screening the PXR activation characteristics. When the PXR is activated by the ligand drug, the PXR can regulate the expression CYP3A gene. PXR gene-deficient mice lose drug inductivity of CYP3A. Contrarily, hPXR (human pregnane X receptor) receptor in activation state expressed in liver of a transgenic mouse can lead to continuous high expression of CYP3A enzyme. The method of the invention establishes critical technology for high-throughput screening based on the passway and carries out screening of PXR activation characteristics from mass compounds in a compound library at the early stage of drug development, and can reduce the risk of adverse drug interactions after the new drug comes into the market, and greatly reduce development cost.

Provided is a method for improving transient expression of recombinant proteins through improvement of transfection efficiency, wherein the transfection efficiency is improved through changing of environment of cells before and after transfection. The method enables expression index of the transient transfection recombinant proteins to be improved by 37% to 56%.

The invention belongs to the field of bioengineering, and more specifically relates to a method for preparing serotype 8 recombinant adeno-associated virus, The method comprises the following steps: 1) using a serum-free medium for culturing 293T cell in a sheet carrier; 2) performing transient transfection on the cultured 293T cell by adeno-associated viral vector system plasmid to obtain the transfected cell; and the adeno-associated viral vector system plasmid comprises AAV expression vector plasmid, auxiliary plasmid pHelper and AAV capsid plasmid pRC8; and 3) continuously culturing the transfected cell, collecting and replacing the serum-free medium on time to obtain a medium containing the recombinant adeno-associated virus. The preparation method can guarantee production stability,preparation and purifying steps are saved, downstream virus purifying difficulty is obliviously reduced, and the preparation method is especially suitable for industrial scale-production of the serotype 8 recombinant adeno-associated virus.

The invention discloses a method for improving transient transfection of insect cell and stably expressing protein expression quantity. A traditional insect cell cultivation method adopts a phosphate buffer system mostly, CO2 is not required to add during the cultivation process, and the expression quantity of a target protein is relatively low. The method creatively finds that the protein expression quantity can be improved by adding CO2 in a cultivating box or changing an air permeable cover to be a sealing cover after transfection to increase CO2 concentration in a bottle upon the self-breathing effect of insect cells regardless of transient transfection or protein expression and production process of a stable cell pond when the insect cell is cultivated; moreover, the lifting effect is significant. The CO2 is added in the cultivation box during the insect cell cultivation process, or the cultivation bottle cover is changed to be a sealing cover, the method is simple and easy to practice, and the cost is low; the method is extensive in use and capable of significantly improving the protein expression quantity of the insect cells.

The invention provides a method for establishing a stable transgenic flounder embryo cell strain. The method for establishing stable transgenic flounder embryo cell strain comprises the following steps: transfecting flounder embryo cells by using plasmids which can widely express enhanced green fluorescent protein promoted by a beta-actin promoter in flounders and can express G418 resistant protein; after 24h, observing expression of the EGFP (express enhanced green fluorescent protein) under a fluorescent microscope, and through G418 screening, obtaining the embryo cells with stable genetic expression. By the method, an exogenous gene widely expressed in the flounders and promoted by the beta-actin can be effectively transfected into the flounder embryo cells, and can be genetically expressed in the flounder embryo cells stably, so that a novel method is provided for researching for flounder genes, the problem that difficult cellular-level gene function analysis and research caused by low transient transfection efficiency of the flounder cells is solved, and a novel technical means is provided for a molecular-level genetic breeding work of the flounders, and a transgenic method for stably inheriting a marine fish cell line is provided.

The invention provides a transient transfection reagent and a use thereof. The transfection efficiency of the transfection reagent is high and stable and is equivalent to the transfection efficiency of Lipofectamine 3000 (hereinafter referred to as lipo3000). The transient transfection reagent has high transfection efficiency both in plasmid single transfection and co-transfection and has very small cytotoxicity. The transient transfection reagent can be simply operated. Compared with lipo3000, the transient transfection reagent is free of plasmid dilution and reagent transfection, realizes transfection in 10min, has high solution stability, can be stored at a temperature of 4 DEG C for a long time, has a low cost, very high transfection efficiency and high stability, can be operated easily, is suitable for a wide range of host cells, is not influenced by antibiotics of the culture medium in transfection efficiency and is an excellent choice for a lab where a lot of transient transfection experiments are carried out.

The invention relates to a cell membrane particle expressing parafusin and preparation and application of the particle. VSVG is expressed on the cell membrane particle. Biomacromolecules and organelles are wrapped in the cell membrane particle. The cell membrane particle does not have a cell nucleus. The preparation method includes the steps that transient transfection is conducted on Ad293 cells through expression vesicular stomatits virus protein plasmids; the cells are digested through pancreatic enzymes 48 hours after transfection is carried out, and then centrifugation is carried out for 3 min; an obtained cell suspension is placed in a syringe, the syringe is installed in a syringe filter provided with a polycarbonate membrane, the syringe is pushed so that the cell suspension can be pushed from one side of the filter to the other side of the filter, and the cell membrane particle expressing VSVG is obtained. The cell membrane particle expressing parafusin is applied to biomacromolecule and organelle transfer. The membrane particle is larger, can wrap biomacromolecules and mitochondria, and transfers protein and the mitochondria in the aspect of function. Extra chemical reagents are not added, activity of VSVG is effectively maintained, mitochondrion release efficiency of the membrane particle is improved after the membrane particle enters a body, and accordingly mitochondrion transfer efficiency is improved.

The invention relates to an efficient genetic transformation method of agrobacterium tumefaciens mediated vacuum infected pigeon peas, and belongs to the technical field of molecular biology and planttransgenic engineering. Experimental steps of the method mainly comprise four parts: explant screening; preparing an agrobacterium infection solution; and performing vacuum infiltration and transgenosis identification on whole pigeon pea plants. According to the invention, a stable transgenic root system induced by stems can be obtained, and leaves of the pigeon peas can be used for rapid identification of gene expression after transient transfection. The inventor finds that transgenic roots can grow from stems of woody plant seedlings through agrobacterium tumefaciens vacuum infiltration forthe first time. The method not only can be directly applied to genetic transformation of the pigeon peas, but also can provide a new thought for a genetic transformation system of woody plants.