196 results about "Electroporation" patented technology

The invention discloses a targeted delivery method of a plant-derived biological regulator, and relates to the technical field of bioengineering, and the targeted delivery method comprises the following steps: in the application process of an electroporation technology, monitoring and acquiring cell membrane lipid change data information in real time through a biosensor technology; the method comprises the following steps: preprocessing original data acquired by a biosensor, and organizing and storing the preprocessed data according to rules to form a structured data set; the system can accurately control the state of the plant cell membrane by monitoring the lipid change of the cell membrane in real time and combining intelligent evaluation and dynamic adjustment of the electric field parameters. When membrane lipid is oversaturated, the electric field intensity and the pulse frequency are dynamically adjusted, membrane damage is reduced, cell integrity is protected, and meanwhile effective delivery of allogenic materials is ensured. According to the method, the stability and the accuracy of an electroporation technology are improved, the targeted delivery of the plant-derived biological regulator is optimized, and the application potential of the plant-derived biological regulator in the fields of agriculture and biomedicine is expanded.

The invention belongs to the field of biological medicines, and relates to a drug-loaded nano-vesicle as well as a preparation method and application thereof. The drug-loaded nano-vesicle comprises a vesicle core and a drug-loaded nano-vesicle, wherein the vesicle core comprises siRNA (small interfering Ribonucleic Acid) capable of specifically targeting and silencing an NR1D1 gene; the vesicle membrane is formed by fusing an erythrocyte membrane, a macrophage membrane, cardiolipin, cholesterol and lecithin. The drug-loaded nano-vesicle can specifically target macrophages in a sepsis immunosuppression stage, has an intracellular response release function, recovers BMAL1 and IGF2BP2-ATP6V1B2 / ATP6V0c axis functions by inhibiting NR1D1 expression, reconstructs a macrophage phagocytosis function and lysosome-dependent bacterium removal capability, and can be used for preparing a drug-loaded nano-vesicle with a specific targeting function. The survival rate of sepsis immunosuppression model animals is obviously improved; and the bacterial load is reduced. Compared with a traditional electroporation method, the preparation method disclosed by the invention has the advantage that the encapsulation efficiency of siRNA is remarkably improved.

The invention discloses a construction method of a nocardia seriolae mycolic acid deficient strain, which belongs to the technical field of aquaculture prevention and control, and comprises the following steps: strain and material preparation, culture and genome extraction, upstream and downstream fragment cloning to construct deletion plasmids, competent cell preparation, electrotransformation and screening, and biological characteristic and toxicity analysis. The nocardia seriola seriola mycolic acid defect type strain is constructed and compared with a wild strain and a back-filling strain for research, the effect of the mycolic acid in the aspects of growth, form, toxicity and the like of thalli can be defined, the relationship between the mycolic acid and the pathogenicity of the thalli can be deeply known, and an important clue is provided for revealing the pathogenic mechanism of the nocardia seriola seriola.

The invention discloses a CAR donor plasmid, a T cell as well as a preparation method and application of the CAR donor plasmid and the T cell, and belongs to the field of genetic engineering and immune cell therapy. An RNP compound and donor plasmid DNA are transferred into a T cell through electroporation, and a CAR expression unit is integrated into a T cell genome at a fixed point to prepare the CAR T cell. The donor plasmid DNA comprises a plasmid skeleton, a homologous arm sequence, an EF1 alpha core promoter, a CAR expression element and an SV40 poly A termination signal. The number of base pairs of the plasmid is 4544 bp, the toxicity of the plasmid to T cells after electroporation can be effectively reduced, and the T cell viability and the CAR integration efficiency are improved. Compared with dsDNA and ssDNA, the plasmid DNA is convenient to prepare and low in cost; therefore, the plasmid is taken as the donor template DNA, and the CRISPR / Cas9 technology is combined, so that the use of a virus vector can be avoided to improve the safety of the CAR T cell, and the production cost of the preparation of the CAR T cell can be effectively reduced.

Systems and methods for infection prevention and treatment in biological conduits using electroporation or pulsed field ablation (PFA). The system includes an elongate member, such as a dialysis catheter, vascular access device, or peritoneal dialysis conduit, with at least one electrode positioned along, within, or around the member. A control unit applies pulsed electric fields to inhibit microbial colonization, disrupt bacterial biofilms, and alter permeability of bacterial or endothelial cells to enhance sterility or fluid exchange. The system may use real-time impedance monitoring, microbial detection, or fluid composition analysis to adjust electroporation parameters. Electroporation may synchronize with dialysis cycles or antimicrobial lock solutions. The method includes automated, clinician-controlled, or sensor-triggered treatments to reduce infections and improve device longevity. The system provides an alternative or augment to antibiotics, reducing the risk of drug-resistant infections while improving the longevity and safety of medical implants.

The invention discloses an electrotransfection solution for pig in-vitro fertilization embryos and application thereof, and belongs to the technical field of pig embryo in-vitro editing. The objective of the invention is to solve the technical problems of low in-vitro electrotransformation efficiency and large damage of pig embryos. The invention provides an electrotransfection liquid for a pig in-vitro fertilized embryo. The electrotransfection liquid is prepared from the following components: a basic liquid which is Opti-MEM, N-acetylcysteine, mannitol and a small-molecule inhibitor Y-27632. According to the technical scheme, the embryo damage in the electrotransfection process can be remarkably reduced while efficient delivery of the gene editing tool is ensured, and a key technical support is provided for establishing a stable and efficient pig embryo gene editing platform.

Method of electroporating with cell culture aimed to improve cell viability wherein the method comprises electroporating cells of interest with an anti-apoptosis protein, treating the cells with DNase post-electroporating, and shifting the temperature of the cells from 37° C. to 32° C. while resting post electroporation.

Embodiments of the present invention include methods of targeting acidosis (low pH) within the tumor microenvironment (TME) through the use of cathodic electrochemical reactions (CER). Low pH is oncogenic by supporting immunosuppression. Electrochemical reactions create local pH effects when a current passes through an electrolytic substrate such as biological tissue. Electrolysis has been used with electroporation (destabilization of the lipid bilayer via an applied electric potential) to increase cell death areas. However, the regulated increase of pH through only the cathode electrode has been ignored as a possible method to alleviate TME acidosis, which could provide substantial immunotherapeutic benefits. Here, ex vivo modeling shows that CERs can intentionally elevate pH to an anti-tumor level and that increased alkalinity promotes activation of naïve macrophages. Embodiments of the invention include pairing CER treatment protocols with existing electric field-based cancer therapies or use as a stand-alone therapy.

A device, system and process involve conducting electroporation of microvesicles or exosomes or other target structures in a microfluidic arrangement at pressures that exceed atmospheric pressure. Single as well as multiple flow configurations can be employed. In some cases, the system and its operation are computer-controlled for partial or complete automation.

The present invention discloses a method for constructing acid-resistant engineered algae, belonging to the fields of biology and molecular biology. The specific method is as follows: Using Phatr3_J33543 with the sequence structure shown in SEQ ID No.1, Phatr3_J50516 with the sequence structure shown in SEQ ID No.2, or Phatr3_Jdraft1806 with the sequence structure shown in SEQ ID No.3 as the target gene, removing the stop codon of the target gene, and connecting the fluorescent protein gene without the start codon through the DNA sequence encoding glycine to obtain a recombinant gene; inserting the recombinant gene into the multiple cloning site of the pPhaNR plasmid to obtain a vector plasmid; introducing the vector plasmid into diatom cells by electroporation and then inoculating them onto the ESAW solid selection medium containing bleomycin, and growing brown colonies; selecting the colonies with fluorescent protein signals in the colonies, which are the acid-resistant engineered algae. Compared with the wild algal species, the acid-resistant ability of the engineered algae obtained by the present invention is significantly improved. The present invention has important reference significance for the cultivation of acid-resistant diatoms.

A recombinant strain of F. diplosiphon was made by transforming wild type F. diplosiphon with a pGEM-7Zf (+) plasmid containing the photolyase gene (phrA) via electroporation. The recombinant strain was designated B481-ViAnSa and overexpressed the phrA gene to result in enhanced UV tolerance compared to wild type F. diplosiphon.

The invention discloses application of amifostine to improvement of fixed-point insertion efficiency of a genome, and belongs to the field of biological medicine. Specifically, amifostine provided by the invention can significantly improve the CRISPR gene editing efficiency, and especially can be used for preparing non-viral site-specific integrated CAR T cells. Through cell experiments, it is found that amifostine has a remarkable promoting effect on the positive rate of CAR T cells after electrotransfection, and the tumor killing function of the T cells is greatly improved.

The invention belongs to the field of biological medicine, and particularly discloses a gene editing method for improving the HDR efficiency of THP-1 cells, which comprises the following steps: selecting THP-1 cells as target cells, designing CRISPR / Cas9 gRNA for specific gene loci, preparing an alpha-donor carrier containing target gene homologous arms, and introducing the gRNA, Cas9 protein and the alpha-donor carrier into the THP-1 cells by using an electroporation transfection technology. By optimizing electroporation transfection conditions and using a novel alpha-donor carrier, the survival rate and HDR efficiency of THP-1 cells are remarkably improved. In addition, the invention also provides a kit for improving the HDR efficiency of THP-1 cells, and the kit comprises required CRISPR / Cas9 gRNA, an alpha-donor carrier, Cas9 protein, and a buffer solution and a reagent which are required by electroporation transfection. The method provides powerful support for application of gene editing in animal cell model construction, drug target screening and gene function research.

A method for electroporation of biological cells, comprising the steps of: providing a shock tube for cell electroporation; filling up the cavity with the cell sample comprising cells and materials to be injected into the cells, wherein a bulged liquid surface is formed; securing the stopple to a second end of the tube by a positioning structure, while generating a compressive deformation to the elastic piece between the stopple and the second electrode; connecting the first electrode and the second electrode with a pulse power supply to perform electroporation.

The invention discloses a remediation method and system for soil heavy metal pollution based on a bamboo nano material, bamboo is cut in the axial direction of fibers, ultrasonic cleaning and vacuum drying are carried out, after energy gradient grinding is carried out in sequence, high-pressure homogenization and in-situ dispersion nanocrystallization treatment are carried out, and the porous bamboo nano material is obtained; the method comprises the following steps: inserting an anti-heavy metal target gene into a selected microbial expression vector, introducing the expression vector into microorganisms by using an electroporation method, detecting the expression level of the target gene, inoculating the microorganisms over-expressing the target gene into a culture medium, and determining the heavy metal removal rate based on microbial growth and the heavy metal removal rate. The microorganism with high heavy metal tolerance and biotransformation capability is obtained; the method comprises the following steps: uniformly distributing microorganisms under the assistance of a dispersing aid, carrying out low-temperature standing crosslinking by adopting a cross-linking agent, adsorbing the microorganisms on the surface of a bamboo nano material to obtain a microorganism and bamboo nano material composite repairing agent, and applying the microorganism and bamboo nano material composite repairing agent to polluted soil for heavy metal pollution repairing.

Methods of delivering pharmaceutical compositions containing one or more nucleic acids via electroporation are disclosed. The methods include (i) delivering a pharmaceutical composition containing one or more nucleic acids to a target site of the subject; and (ii) electroporating the target site of the subject where the pharmaceutical composition was delivered. Electroporating the target site facilitates the uptake of the nucleic acids by and expression of an immunogenic protein in cells of the subject. Typically the nucleic acid is RNA such as non-replicating mRNA or circular RNA. The methods can deliver two or more nucleic acids, wherein at least one of the nucleic acids is an mRNA or a circular RNA. The disclosed methods are suitable treating or preventing the development of one or more symptoms of a disease or disorder such as an infection or cancer in a subject.

The process provided in this invention transfects primary cells with gene editing reagents using a high-volume gas-permeable cell culture device and a flow-through electroporation device under conditions that improve gene editing performance, cell yield, and drug (DP) quality characteristics for cell therapy applications. As demonstrated in the examples, primary cells edited according to the process provided herein achieved improved double-strand break (DSB) formation rates, increased frequency of homology-directed repair (HR) and non-homologous end joining (NHEJ) combinations, increased frequency of bi-allelic and mono-allelic HR events, improved cell viability, proliferative capacity, and cell fitness after gene editing, and reduced manufacturing time.

The invention relates to the fields of molecular biology and virology, and provides a method for preparing a specific MHC haplotype-derived cell-binding virus. The method comprises the following steps: firstly, obtaining chick embryos of different haplotypes, separating CEF cells, transiently expressing exogenous fluorescent protein through electrotransfection, then carrying out virus inoculation, carrying out flow sorting to obtain a fluorescent positive cell population carrying MDV, and carrying out quantitative analysis on the cell population to obtain the specific MHC haplotype chicken Marek's Disease Virus (MDV), thereby obtaining the specific MHC haplotype chicken Marek's Disease Virus Marek's Disease Virus Marek's Disease Virus Marek's Disease Virus. And carrying out in-vitro propagation to obtain the purified MDV with a specific haplotype source. The purified virus can provide a test material for related research of haplotype on virus infection, and can be widely applied to in-vitro purification of different cell binding viruses and related research of specific MHC molecule presenting virus peptides. The method disclosed by the invention is beneficial to carrying out related research aiming at the host immune response of the virus and analyzing the hereditary basis of haplotype to MDV differential resistance, and is expected to be applied to preparation of vaccine virus.

The invention discloses a construction method of a Klf6 gene knockout mouse embryo model, which comprises the following steps: S1, preparing a gene editing solution containing Cas9 enzyme and sgRNA for targeting a third exon region and a fourth exon region of a mouse Klf6 gene by utilizing a CRISPR / Cas9 gene editing technology; s2, taking out the fertilized eggs of the mouse, transfecting the fertilized eggs of the mouse with a gene editing solution, and then performing in-vitro culture on the fertilized eggs; s3, carrying out embryo transplantation on the two-cell stage embryo obtained in the step S2 into a pregnant female mouse, and then carrying out conventional feeding; and S4, 7-8 days after embryo transplantation, taking out the uterus and stripping the embryo to obtain the Klf6 gene knockout mouse embryo model. According to the method, gene knockout is carried out by using CRISPR / Cas9 in the fertilized egg period in combination with an electrotransfection method, and compared with an existing method, the construction process time can be greatly shortened, the working efficiency is obviously improved, and the success rate is high.

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 discloses a method for converting a carbon source into ethylene glycol. The method comprises the following steps: providing a plastid, wherein the plastid comprises gene sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; implanting the plastid into blue-green bacteria by means of electroporation treatment to obtain modified blue-green bacteria; and providing the carbon source to the modified cyanobacteria, so that the modified cyanobacteria converts the carbon source into ethylene glycol, thereby producing chemicals with high economic value while treating the carbon-containing waste gas.

Provided are a universal cell system, a construction method therefor, and use thereof. Universal mesenchymal stem cells are constructed by means of the combination of electroporation and CRISPRoff gene editing, and the cells can survive stably in a pulmonary inflammatory pathological environment to provide sustained treatment, significantly improve multiple indicators of acute pneumonia, solve immune rejection, and provide new cell therapies and ideas for acute pneumonia. CRISPRoff is introduced into universal CTL cells by means of electroporation technology, targeting a specific region and inhibiting the expression of HLA-I in T cells; thus, T cells with low immunogenicity are constructed, and the cells can avoid allogeneic immune rejection. The universal cell system combines the advantages of both transient transfection and CRISPRoff gene editing, has great significance in the field of cell therapy, provides a more comprehensive and effective solution for the treatment of various diseases, and has a wide application prospect.

The present invention addresses the problem of providing a virus production method with which it is possible to increase the production of viruses. According to the present invention, provided is a virus production method comprising: a nucleic acid introduction step for introducing a nucleic acid into a cell by electroporation to obtain a cell into which the nucleic acid has been introduced; and a culturing step for culturing the cells into which the nucleic acid has been introduced, the CNET product defined in the specification being 1 * 104 to 1 * 107 (inclusive) in the electroporation.

The invention relates to the technical field of construction of conditional gene knockout models, and discloses a construction method of a mouse Oct4 conditional gene knockout model based on quadruple sgRNA collaborative targeting, which comprises the following steps: step 1, designing four sgRNAs, namely sgRNA1, sgRNA2, sgRNA3 and sgRNA4, in which the sgRNA1 and the sgRNA2 target 5'end and 3 'end non-coding regions of a second exon of an Oct4 gene, and the sgRNA3 and the sgRNA4 target structural domain conserved sequences; step 2, preparing an electrotransfection solution, wherein the electrotransfection solution is composed of Cas9mRNA, the four sgRNAs and an electrotransfection buffer solution; step 3, carrying out electroporation treatment on the fertilized eggs of the mice; step 4, culturing the fertilized eggs subjected to the electrotransformation treatment to a blastocyst stage; and 5, carrying out genotype identification on the cultured blastocyst. According to the scheme, the sgRNA combination and embryo electrotransfection system is optimized, the mouse Oct4 conditional gene knockout model is efficiently and accurately constructed, a reliable tool is provided for regulation mechanism research of Oct4 in mouse early embryo trigerm differentiation, and the medical transformation value of Oct4 is excavated.

Electroporation method for delivering a composition comprising at least one peptide with a molecular weight ranging from 500 Da to 20 kDa. The present invention relates to an electroporation method for delivering a composition through human keratinous materials, the composition comprising a peptide with a molecular weight ranging from 500 Da to 20 kDa. The electroporation method comprises exposing the composition in contact with said keratinous materials to a succession of pulses of an electric current. The electric current is supplied by an electroporation device having an electrode (2) and a counter electrode (3). Each pulse has a duration of between 1 ms and 5 s with a rest interval between them of between 0.1 and 5 s, and the electric current has a voltage amplitude of between 20 and 400 V and an intensity of between 0.1 and 10 mA. Figure for the abstract: Figure 1

The present disclosure provides systems, devices, and methods for electroporating at least one cell of a plurality of cells. A cartridge divided into a plurality of separation chambers is provided, wherein each chamber of the plurality of separation chambers is configured to contain a culture medium comprising the at least one cell. Each of the plurality of separation chambers is fluidly connected via a fluid exchange path and functionally separated by a constriction disposed within the fluid exchange path. Each of the plurality of separation chambers includes an electrode for applying a pulsed electric field to the at least one cell to effect electroporation of the cell and transfection of the cell with nucleic acid. The constrictions disposed between each chamber prevent electric field interference between adjacent chambers during an electroporation procedure.

The present disclosure relates to methods of producing engineered human cells. Methods may comprise providing a composition for inducing a modification of a target endogenous nucleic acid sequence in a nucleus of a human cell, wherein the composition comprises an amount of a Cas9 protein and an amount of a sgRNA in an in vitro environment free of the human cell. In some embodiments, the amount of sgRNA and the amount of Cas9 protein may be present in the composition at a weight ratio of 4:9 to 4:3. A Cas9 / sgRNA complex may form in the in vitro environment prior to being introduced into the human cell. Methods may further comprise introducing the Cas9 / sgRNA complex into the human cell via electroporation, wherein the Cas9 / sgRNA complex induces the modification of the target endogenous nucleic acid sequence in the nucleus of the human cell.

The application belongs to the field of biological medicine, and relates to a drug-loaded nanovesicle and a preparation method and application thereof. The drug-loaded nanovesicle comprises: a vesicle core comprising siRNA capable of specifically targeting a silenced NR1D1 gene; and a vesicle membrane fused by red blood cell membranes, macrophage membranes, cardiolipin, cholesterol and lecithin. The drug-loaded nanovesicle can specifically target macrophages in the immune suppression stage of sepsis, and has an intracellular response release function. By inhibiting the expression of NR1D1, the drug-loaded nanovesicle restores the functions of BMAL1 and IGF2BP2-ATP6V1B2 / ATP6V0c axes, reestablishes the phagocytosis function of macrophages and the lysosome-dependent bacterial clearance capacity, significantly improves the survival rate of animals in a sepsis immune suppression model and reduces the bacterial load. Compared with a traditional electroporation method, the preparation method of the application significantly improves the encapsulation rate of siRNA.

The invention relates to the field of electroporation, and provides an electroporation regulation and control method for exosome-loaded miRNA in order to solve the aggregation problem after electroporation and determine the optimal electroporation condition to improve the loading efficiency and the molecular stability, and the method comprises the following steps: S100, obtaining the relationship among the concentration of a to-be-detected substance, a pulse parameter and an aggregation parameter of the to-be-detected substance; the aggregation parameters comprise an aggregation speed and an aggregation rate; the to-be-detected object comprises an exosome and miRNA (micro Ribonucleic Acid); and S200, selecting the concentration and pulse parameters of the to-be-detected object corresponding to the aggregation rate less than the first preset value and the aggregation speed greater than the second preset value as the actually used electrical conversion parameters. According to the invention, the concentration of the to-be-detected object and the pulse parameter are continuously adjusted by detecting the aggregation parameter so as to obtain the optimal electrical conversion parameter, so that in the electroporation process, the optimal electrical conversion parameter is obtained through the migration condition and the aggregation condition, and the to-be-detected object has a certain migration rate and a relatively low aggregation rate; meanwhile, aggregation of the exosome is reduced, and the recovery rate of the exosome is increased.