228 results about "Electroporation" patented technology

The invention provides an intracellular mycobacterium tuberculosis in-situ nucleic acid detection method based on CRISPR / Cas and application, and the method comprises the following steps: S1, designing crRNA and a primer pair of a mycobacterium tuberculosis target gene sequence, and synthesizing a single-stranded DNA fluorescent probe; s2, preparing a standard solution containing a target gene sequence, and incubating with a CRISPR / Cas12a system; s3, extracting nucleic acids of different types of bacteria, uniformly mixing the nucleic acids of different types of bacteria with the CRISPR / Cas12a system, and incubating; s4, incubating an attenuated strain of mycobacterium tuberculosis and the DNA fluorescent probe, infecting macrophages, and incubating to obtain infected cells; s5, electrically transferring a CRISPR / Cas12a system into the infected cells in S4 by utilizing an electroporation technology, incubating, and detecting the intensity of a fluorescence signal; and S6, performing quantitative and positioning analysis on the detected fluorescence signal to complete detection, and directly introducing a CRISPR / Cas12a system into the macrophage by adopting an electroporation technology to realize intracellular in-situ detection, thereby avoiding the operation complexity of a nucleic acid extraction link and the loss of detection sensitivity in the prior art.

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 present technology relates to an electroporation based antimicrobial apparatus The electroporation based antimicrobial apparatus includes a negative electrode and a positive electrode powered by an energy source, operatively coupled to an air inlet structure, such that upon applying 10 an external electric field across the negative electrode and the positive electrode, a lightning-rod effect is generated that causes permanent damage to outer structure of microbes passing along with air through the air inlet structure, thus achieving instant disinfection. The energy source includes a 15 respiration driven triboelectric Nanogenerator (R-TENG) for harvesting wasted biomechanical energy from the periodic motion of respiration. The R-TENG includes a pair of nanofiber films comprising a first nanofiber film composed of a tribopositive material and a second nanofiber film 20 composed of a tribonegative material. The tribopositive and tribonegative materials are with opposite polarity and the R-TENG serves as a filtration system concurrently. Most illustrative diagram:

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

The invention discloses 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.

The invention discloses a construction method and application of an iPSC-derived mesenchymal stem cell strain for overexpressing a VEGFA gene based on a CRISPR / Cas9 technology, and relates to the technical field of biological cells, the construction method of the iPSC-derived mesenchymal stem cell strain for overexpressing the VEGFA gene based on the CRISPR / Cas9 technology comprises the following steps: S1, designing sgRNA and ssDNA for an insertion site AAVS1, and detecting the cutting efficiency of the sgRNA; s2, introducing a CRISPR (clustered regularly interspaced short palindromic repeats) system into the cells in an electroporation manner to obtain an iPSC cell strain into which an exogenous gene VEGFA is inserted at a fixed point; s3, carrying out directional induced differentiation on the iPSC cell strain to the iMSC to obtain an iMSC cell strain with overexpression of VEGFA (Vascular Endothelial Growth Factor A) protein; the iMSC of the overexpressed VEGFA protein can well stimulate vascular endothelial growth and promote angiogenesis, and more possibilities are provided for repair treatment of ischemia-related diseases.

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.

The invention discloses a preparation method and application of a porous GelMA microneedle for delivering AIE molecules based on HSF derived exosome. The preparation method comprises the following steps: S1, preparing TTMN molecules; s2, dispersing DPPC and Chol in chloroform, performing water bath evaporation at 50 DEG C to form a thin film, hydrating the thin film in PBS, incubating to enable DPPC and Chol to form a liposome structure again, and uniformly dispersing TTMN molecules in the liposome structure to obtain TL; s3, extracting an exosome from the HSF cells; s4, loading the TL into the exosome by utilizing an electroporation technology, so as to obtain TE; s5, preparing the GelMA; and S6, mixing the GelMA with the TE, so that the TE is uniformly dispersed in the GelMA to obtain a mixed solution, injecting the mixed solution into a microneedle mold, and irradiating with 405nm laser to obtain the porous GelMA microneedle. According to the invention, high-efficiency treatment of hypertrophic scars is realized, and the system has a remarkable drug delivery effect, an innovative photodynamic therapy, a high-efficiency scar tissue remodeling effect and good biological safety and stability, and has important clinical application value and market prospect.

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.

According to the cell electroporation method and device, an electroporation loading part formed by buckling a first electrode plate and a second electrode plate is arranged, and the number of electrode plates welded to each electrode plate is the same as the number of cell culture pore cavities of a standard cell porous plate; the adjacent first electrode plates and second electrode plates are combined to form a complete positive and negative electrode group, and the positions of the positive and negative electrode groups are in one-to-one correspondence with the cell culture pore cavities, so that a standard cell culture porous plate for bearing a cell suspension can be embedded into the electroporation loading part for direct electroporation without transferring the cell suspension; the non-contact butt joint between the standard cell culture porous plate and the electroporation loading part is realized, the damage of electroporation to cells is smaller, the cell survival rate is improved, a cell porous plate of a special specification does not need to be customized, plug-and-play is realized, the problem that an existing electroporation instrument is not matched with the standard cell culture porous plate is solved, the cost is reduced, the operation process is simplified, and the operation efficiency is improved. And the high-flux electrotransfection is realized.

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.