84 results about "Electroporation" patented technology

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.

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.

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 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.

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.

This invention belongs to the field of fungal detection technology, specifically relating to a micro-platform for rapid electrolysis and highly sensitive electrochemical nucleic acid detection of Cryptococcus neoformans and its application. The micro-platform includes an integrated microfluidic chip, an external power supply, a sample introduction system, and / or an imaging system. The integrated microfluidic chip includes an electrolysis region, an electrochemical detection region, and an electrode array region. The electrolysis region includes multiple vertically arranged counter electrodes arranged in pairs, and multiple horizontally arranged hollow channels in a serpentine pattern, bonded to the electrode pairs above them. The connection point between every two electroporated channels is the electroporation lysis point. The electrochemical detection region consists of a working electrode, a reference electrode, and a counter electrode. The working electrode is modified with rGO / AuNPs nanomaterials and molecular probes.

The invention discloses a four-channel pulse source for pulsed electric field space-time coherent synthesis, which is used for performing pulse ablation on biological cells. The four-channel pulse source comprises four Marx main circuits, a low-voltage power supply module, a high-voltage charging module, an FPGA control panel and a control panel. According to the invention, far-zone focusing of electroporation is realized through time-space synthesis of a pulsed electric field. An effective electroporation effect region can be generated in a region far away from the electrode, so that a scheme is provided for arranging an electrode needle in vitro and ablating an in-vivo focus deeply.

The application provides a construction method of a cell model for screening hypoglycemic drugs, and belongs to the technical field of biotechnology.The method provided by the application comprises the following steps: cell verification, vector construction and plasmid extraction, cell electroporation, drug screening, stable strain amplification and Q-PCR verification.The application successfully constructs a stable fluorescence reporter model (Min6-ins2>EGFP) based on Min6 cells, and verifies the specificity and sensitivity of the model to insulin secretion promoting drugs through positive drug verification.The model is simple to operate, can be monitored in real time, is suitable for high-throughput screening, can be effectively used for hypoglycemic activity evaluation of complex components such as chemical drugs and traditional Chinese medicines, and provides a reliable tool for early screening of hypoglycemic drugs.

This invention belongs to the field of biomedical technology, specifically relating to a method and application for preparing CAR-T cells with enhanced persistence based on the piggyBac transposon system combined with electroporation. This invention, through specific piggyBac dual plasmid ratios and electroporation conditions, can improve transfection efficiency to over 80%, and the integration site is more likely to be located in a safe genomic region, supporting long-term stable CAR expression. Furthermore, cell expansion kinetics are significantly improved, with expansion exceeding 120-fold within 15 days. The results of the examples show that CAR-T cells prepared using the method described in this invention exhibit significantly enhanced tumor cell killing ability and cytokine secretion levels in in vitro models, and are rich in memory T cell subsets (Tcm / Tem) with greater persistence and self-renewal potential. This indicates that this invention not only changes the method of gene delivery but also shapes the intrinsic properties of cells, enabling them to acquire potentially superior in vivo persistence and long-term anti-tumor capabilities.

This application discloses a smart method for preparing gene-edited mammalian cells. The method includes: acquiring the target gene sequence information of the mammal to be observed and processing it according to preset screening rules to obtain the target sgRNA sequence; mixing an RNP complex suspension prepared based on the target sgRNA sequence with a suspension of embryonic stem cells to be edited, and performing electroporation transfection using multiple consecutive micropulses to obtain a transfected cell suspension; acquiring raw images of the transfected cell suspension and preprocessing them to obtain single-cell images, and using a trained lightweight convolutional neural network model to obtain the predicted gene-editing efficiency and prediction confidence after 48 hours; if the transfected cell suspension meets the quality control conditions, expanding and culturing the transfected cell suspension to obtain the prepared gene-edited cells. This method can shorten the cell culture cycle, reduce the waste of cell materials, improve the stability of transfection efficiency, and achieve precise preparation of gene-edited cells.

This invention discloses a Chlorella heptapeptide TGPLGSR with multi-target lipid-lowering activity, its preparation method, and its applications. The amino acid sequence of this heptapeptide is Thr-Gly-Pro-Leu-Gly-Ser-Arg. The preparation method includes: performing ultraviolet and electroporation combined mutagenesis on wild-type Chlorella proteoglycans to obtain a high-protein-content mutant algal strain; obtaining algal protein through fermentation and synergistic extraction and purification with alcohols; directionally digesting the protein with alkaline protease, followed by enriching the active peptide components through ultrafiltration and gel chromatography; and finally, screening the heptapeptide using liquid chromatography-mass spectrometry (LC-MS), computer-aided prediction, and molecular docking techniques. This heptapeptide exhibits potent inhibitory activity against pancreatic lipase and can dose-dependently reduce the accumulation of triglycerides and cholesterol in 3T3-L1 cells. Its mechanism involves multiple target pathways, including inhibition of MMP-9, activation of AMPK phosphorylation, and downregulation of PNLIP, and it can be used to develop lipid-lowering functional foods or health products.

This invention provides a method for detecting AAV antibody titers, belonging to the field of biomedical technology, comprising: S1 preparing serum-free AAV vectors with different MOI values; preparing a mixture containing different volumes of test serum samples and AAV vectors with known MOI values ​​for co-incubation; S2: adding plasmids containing the CRISPR-Cas9 gene editing system to electroporation buffer, performing electroporation on a known number of cells, adding the serum-free AAV vectors and mixture from step S1 to the electroporated cells respectively, and detecting the expression efficiency of the reporter gene after electroporation; S3: calculating the AAV antibody titer in the test serum sample based on the detection results. The main advantage of this invention is that it can display AAV antibody titers digitally and quantitatively, facilitating understanding and communication, and obtaining stable and reliable results using different cell lines and different AAV transduction doses. This method is easy to standardize and validate, saving time, effort, and detection costs compared to traditional methods, and directly reflects antibody neutralizing activity.

This invention belongs to the field of genetic engineering technology, specifically disclosing a method for preparing and applying gene-modified homozygous cells of chicken progenitor cells (PGCs). Using a dual-fluorescent marker gene, two donor plasmids carrying green fluorescent protein (EGFP) and red fluorescent protein (mCherry), respectively, are transfected into PGCs. Cells simultaneously expressing red and green fluorescence are obtained using flow cytometry, thus forming homozygous cell lines. Then, the dual-fluorescent marker gene is deleted using the Cre-LoxP system combined with electroporation, and residual LoxP fragments are deleted using ssODN as a donor template, achieving traceless gene editing in recipient chicken embryos. This invention does not introduce any exogenous genes, increasing the safety of gene-modified chickens and is of great significance for eliminating the biosafety risks associated with gene-edited chickens.

The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to an extraction method of a high-activity ginseng extract, which is used for improving the conversion and extraction efficiency of rare ginsenoside Rg1, Rg3, Rg5, Rh2 and Compound K. The extraction method combines microbial fermentation, electrostatic field wall breaking and supercritical CO2 extraction, the high-voltage pulse electric field induces ginseng cells to generate an electroporation effect, permeability of cell membranes and cell walls is improved, supercritical CO2 extraction has high cell permeability, the extraction rate and purity of ginsenoside are improved, and in addition, a catalytic promoter added in the extraction process is beneficial to conversion catalysis of rare ginsenoside components.

The invention discloses a group of nocardia seriolae mycolic acid synthase genes and a construction and application method of a deletion strain of the nocardia seriolae mycolic acid synthase genes, and belongs to the technical field of disease prevention and control. Comprising the following steps: strain culture and whole genome extraction, specific primer design and gene fragment cloning, deletion plasmid construction, competent cell preparation, electrotransformation and deletion strain screening, and deletion strain characteristic verification. By analyzing the synergistic function of nocardia seriola seriola mycolic acid synthesis key genes, the core effect of the nocardia seriola seriola mycolic acid synthesis key genes in a synthesis pathway is clear to fill the blank of multi-gene association research, and a core theoretical basis is provided for revealing the pathogenic mechanism of nocardia seriola seriola mycolic acid.

This invention discloses the application of an electrode material for reducing intracellular resistance genes in drug-resistant bacteria. A platinum-doped dendritic TiO2 nanowire structure based on a three-dimensional material is formed by growing a micron-sized TiO2 backbone array and platinum-doped nano-sized TiO2 branches on the surface of a porous electrode. The prepared dendritic nanowire three-dimensional electrode material has a larger specific surface area, providing more reactive sites and strong electric field sites. Platinum doping enhances the conductivity and electrocatalytic performance of the nanowires. Furthermore, by combining it with chloride salts / oxidants, the electroporation and electrocatalysis of the nanowire electrode surface are coupled. The strong electric field confined at the nanowire tip induces perforation of the drug-resistant bacterial cell structure, strengthening the contact and reaction between the oxidant and the resistance gene. The high electrocatalytic activity confined at the nanowire tip induces the generation of strong oxidizing free radicals, achieving the goal of destroying the drug-resistant bacterial cell structure and efficiently reducing intracellular resistance genes.

The invention provides a method for rescuing a novel measles recombinant coronavirus. The pGEM3Zf (+)-T7RNAP plasmid constructed by the invention can be subjected to in-vitro reverse transcription processing to obtain T7RNA polymerase mRNA (messenger Ribonucleic Acid). The method comprises the following steps: by taking an MRC-5 cell as a cell matrix, co-transfecting the MRC-5 cell by using a pT7-MVEZ-COV2S.B.1.617.2 plasmid constructed by the invention and a helper plasmid in a physical electric shock manner, and simultaneously transfecting T7RNA polymerase mRNA (messenger ribonucleic acid). T7RNA polymerase mRNA translates and expresses T7RNA polymerase in an MRC-5 cell, the T7RNA polymerase further drives a T7 promoter, transcription of a full-length plasmid containing all genetic information of the virus and expression of N, P and L genes are started, virus packaging and proliferation are completed in a host cell, and the paramyxovirus with infection activity is obtained.

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 present invention provides a flow path device and an electroporation method that have high productivity. Provided is a flow path device that is used for electroporation and that, by applying an electric field via an electrode pair to a suspension which contains biological matter and a bioactive substance, introduces the bioactive substance into the biological matter. The flow path device comprises: a main flow path through which a suspension flows; a sheath liquid flow path through which flows only a sheath liquid that is a liquid other than the suspension and which causes the sheath liquid to flow into the main flow path; and an electrode pair which apply an electric field to the suspension flowing through the main flow path. The sheath liquid flow path has a sheath liquid expansion part that expands the flow path of the sheath liquid in the width direction, and a first flow path that is downstream of the sheath liquid expansion part and that is continuous with the sheath liquid expansion part. The total of the length of the first flow path and the length of the sheath liquid expansion part is not less than 0.8 times the width of the main flow path between the electrode pair.

An object of the present invention is to provide a virus production method capable of improving an amount of virus production. According to the present invention, there is provided a virus production method including a nucleic acid introduction step of introducing a nucleic acid into cells via electroporation to obtain cells into which the nucleic acid has been introduced; and a culture step of culturing the cells into which the nucleic acid has been introduced, in which a CNET product defined in the present specification in the electroporation is 1×104 or more and 1×107 or less.

The invention discloses an electrotransfection module for organ conformal, a bioelectronic patch and a preparation method and application of the bioelectronic patch, an electrotransfection module body is of a paper-cut structure and comprises a substrate layer, an electrode layer, a medicine storage layer and a nanopore layer, and the substrate layer, the electrode layer, the medicine storage layer and the nanopore layer are sequentially stacked together from bottom to top; cutting according to a paper-cut pattern conformal with the target organ to form inlaid paper-cut units connected through hinges; and integrating a wireless communication module to obtain the bioelectronic patch. A paper-cut conformal theory is adopted, a paper-cut structure electrotransfection module and a bioelectronic patch for organ conformal are designed and prepared on the basis of the characteristic slope of a target organ, seamless covering of the target organ is achieved, and meanwhile safe, controllable and efficient intracellular delivery at a target part is achieved; it is ensured that the electrotransfection module and the bioelectronic patch have the structural conformity and the high coverage area of a target organ, and therefore efficient and uniform whole-organ delivery is achieved, and the electrotransfection depth is increased.

The application discloses a method for constructing a genetically engineered Kluyveromyces lactis strain for producing miR162a, which comprises co-transferring two plasmids expressing DCL1, HYL1 and SE genes from Arabidopsis thaliana into a Kluyveromyces lactis GG799 strain by electroporation, so that the yeast obtains the ability to cut plant-derived precursor miRNA, one of the plasmids synchronously expresses a precursor of miR162a from Lycium ruthenicum Murr, and the genetically engineered Kluyveromyces lactis strain for stably producing miR162a is obtained after acetamide screening. The application further discloses the genetically engineered Kluyveromyces lactis strain constructed according to the above method and application thereof. The genetically engineered strain constructed by the method has a high yield of miR162a at a laboratory flask level, and can stably produce miR162a after continuous subculture, the genetically engineered Kluyveromyces lactis strain exhibits the ability to promote osteogenic differentiation in a cell experiment, and can effectively improve the osteoporosis symptoms of mice in an animal experiment.

The utility model provides a kind of food processing device and cooking equipment.The food processing device includes electrode, electric field excitation unit, purification chamber and stirring unit, electrode is installed in purification chamber, and electrode is electrically connected in electric field excitation unit to generate disinfecting electric field, purification chamber has purification cavity and purification cavity is located in the action range of disinfecting electric field, and stirring unit is used to disturb food in purification cavity.The food processing device carries out sterilization treatment to food that is cleaned and impurities in purification chamber by disinfecting electric field, and disinfecting electric field directly acts on the cell membrane of bacteria and other harmful organisms, and bacteria and other harmful organisms mixed in food produce electroporation and death.

The present disclosure relates to a high throughput, scalable system for electroporation of biological cells. The two part system comprises an electroporation reaction array that interfaces with a control unit, providing electrical pulse, temperature control, and mixing of the sample contents. The control unit automates the entire process; electroporation, cell recovery and outgrowth are performed in a electroporation array assembly. The bottom of each reaction well of the electroporation array assembly contains a pair of coplanar electrodes. The bottom surface containing the electrodes is treated in a way to render it hydrophilic. This results in increased wetting of the electrodes thereby increasing transformation efficiency. The electrode configuration allows for processing of smaller sample volumes, reducing the consumption of expensive biological reagents by several orders of magnitude compared to conventional cuvette-based electroporation devices.