10 results about "Stable transfection" patented technology

The invention discloses a monoclonal cell strain screening method based on fluorescence intensity, which comprises the following steps: determining a cell line, and stably transfecting fluorescent protein to cells of the cell line; a microwell plate is selected, a datum hole is set, the cells stably transfected with the fluorescent protein are separated and put into the microwell plate, a plurality of cells are put into the datum hole, and only one cell is put into the other holes; screening out single-cell pores with only one cell from the rest of pores except the reference pore; screening out clone group holes capable of growing clone groups; respectively digesting cells in the cloning group pores and then transferring the cells into new pores for culturing; screening out fluorescent screening holes; splitting proliferation holes are screened out; and determining the cells in the division proliferation hole as the screened monoclonal cell strain. According to the monoclonal cell strain screening method provided by the invention, the monoclonal cell strain which is high in fluorescence intensity and can proliferate at a normal speed can be screened, compared with fluorescence microscope observation and imaging, the method is convenient and rapid, and errors possibly generated by comparing the fluorescence intensity with human eyes are reduced.

The invention provides an organoid for simulating a dormancy and recurrence process of breast cancer cells as well as a construction method and application of the organoid. The construction method comprises the following steps: culturing breast cancer cells, and carrying out flow sorting on surface markers CD44 and ITGB4 to obtain an ITGB4 low-expression (ITGB4low) cell population; packaging a lentiviral vector, infecting a cell population with low expression of ITGB4, and carrying out flow sorting to obtain a stably transfected cell population; and carrying out induction treatment on the stably transfected cell population and culturing to obtain the organoid. The organoid provided by the invention provides powerful support for prevention and / or treatment of metastatic breast cancer, and shows extraordinary application prospects in accurate and efficient screening of drugs for targeting dormancy-recurrence process.

This invention relates to a detection kit and preparation method for enhancing the detection efficacy of autoimmune encephalitis (CBA) antibodies. The kit comprises genetically engineered 293T cells stably integrating and co-expressing the eIF2α S52A mutant and HSP70, and transiently transfected with CBA-associated antigens. The preparation method includes constructing a co-expression vector, establishing a stable transfection cell line, stress preconditioning treatment, antigen transfection, and kit assembly. A method for assessing antibody affinity maturation using this kit is also provided. By synergistically modifying detection cells at three levels—translational regulation, protein folding, and anti-apoptosis—the invention significantly increases antigen expression, reduces apoptosis, and improves detection sensitivity and specificity. Furthermore, it integrates antibody affinity assessment into CBA detection for the first time, providing auxiliary information for clinical staging.

The application relates to a cell line stably expressing an orthogonal aminoacyl tRNA synthetase / tRNA pair, a HEK293T cell line capable of stably expressing an EcTyr orthogonal aminoacyl tRNA synthetase / BsTyr tRNA pair is constructed, and a construction method of the cell line is disclosed. The construction method is to stably integrate the orthogonal EcTyr aminoacyl tRNA synthetase and BsTyr tRNA genes into the HEK293T cell genome by means of a PiggyBac transposon system and plasmid stable transfection. The constructed cell line genome is integrated with the EcTyr orthogonal aminoacyl tRNA synthetase and BsTyr tRNA genes, and provides a stable platform for efficiently introducing unnatural amino acids at any site of a protein. The application has the effects of further laying an experimental foundation for the exploration of novel protein structures and functions and the research and development of protein drugs containing certain specific functions.

The invention discloses an application of a construction method of a novel stably transfected cell pool in degradation research of a virulent pathogen. Belongs to the technical field of stable transfection cell pool application, and the application can be used for carrying out virulent pathogen research in a low-grade laboratory or an unprotected environment, so that the research and development cost is reduced, and the application value and strategic value are further improved.

The invention discloses a cell line for stably expressing porcine delta coronavirus S protein, a porcine delta coronavirus IgA antibody detection antigen and a kit. The method comprises the following steps: based on an S protein sequence of a CH / XJYN / 2016 strain, selecting an extracellular domain part D20-N1077, adding a human source septoglobin family 1D meber 1 signal peptide sequence MRLSVCLLLTLALCCYRANA to an amino terminal of the S protein, adding a 6 * His tag sequence to a carboxyl terminal, carrying out codon optimization according to a CHO expression system, and constructing into a pCDNA3.1 (+) eukaryotic expression vector to obtain a recombinant eukaryotic expression plasmid pcDNA3.1 (+)-PDCoV-S; then transfecting CHO cells with the plasmid, and carrying out two rounds of heredity mycin G418 pressurized screening, limited dilution cloning and continuous subculture to obtain an expiCHO-PDCoV-S stably transfected cell line through screening; and finally, constructing the PDCoV IgA antibody detection kit based on the cell line. The kit has no cross reaction with other common porcine disease virus positive serum, has intra-batch and inter-batch variation coefficients of less than 10%, has good specificity, repeatability and sensitivity, and can be used for early prevention and control of PDCoV.

The application discloses a monoclonal cell strain screening method based on fluorescence intensity, and the method comprises the following steps: determining a cell line, and stably transfecting cells of the cell line with fluorescent proteins; selecting a microwell plate and setting a reference hole, and separating the cells stably transfected with the fluorescent proteins into the microwell plate, wherein the reference hole is filled with multiple cells, and the remaining holes are filled with only one cell; screening single-cell holes with only one cell in the remaining holes except the reference hole; screening clone group holes capable of growing clone groups; transferring the cells in the clone group holes to new holes for culture after being respectively digested; screening fluorescence screening holes; screening division and proliferation holes; and determining the cells in the division and proliferation holes as screened monoclonal cell strains. The monoclonal cell strain screening method provided by the application can screen monoclonal cell strains with strong fluorescence intensity and normal proliferation speed, is convenient and fast compared with fluorescence microscope observation and imaging, and also reduces errors possibly caused by human eye comparison of fluorescence intensity.

This invention discloses a method for gene transfection of fibroblasts. The method includes: obtaining a fibroblast suspension with a cell concentration of 1-2 cells / µL; preparing cell suspension droplets in a cell culture dish, each droplet consisting of 10±0.5µL of the fibroblast suspension; incubating the cell culture dish containing the cell suspension droplets in a microenvironment, and adding cell culture medium after cell adhesion; obtaining a plasmid solution containing a linearized target gene with a concentration of 150-200 ng / µL; aspirating the plasmid solution into an injection needle, adjusting the angle between the injection needle and the horizontal plane to 30-60°, and injecting it sequentially into each cell; performing single-cell culture after injection to obtain stably transfected transgenic cell clones. This method aims to obtain a cell line without selection markers while achieving high-efficiency and stable transfection.

The invention belongs to the field of stem cell genetic engineering, and provides application of NLRP12 in anti-inflammatory osteogenesis promotion of periodontal ligament stem cells. According to the NLRP12 overexpression PDLSCs, the anti-inflammatory gene is applied to periodontal regeneration treatment, and the anti-inflammatory capacity and the tissue defect repairing capacity after the PDLSCs are transplanted into a body are effectively improved. An exogenous gene is transferred into PDLSCs through a virus vector, stable transfection and efficient and continuous expression of an anti-inflammatory gene are achieved, and the repair effect of periodontitis tissue defects is promoted. In-vitro experiments show that NLRP12 overexpression in an inflammatory microenvironment alleviates the inflammatory response and osteogenic differentiation inhibition of PDLSCs, which shows that the expression of proinflammatory factors IL-6 and IL-8 is down-regulated, the expression levels of anti-inflammatory factors IL-10 and osteogenic related proteins RUNX2, COL1 and BMP2 are up-regulated, ALP dyeing and alizarin red dyeing are deepened, and mineralized nodule formation is increased. The NLRP12 overexpression PDLSCs is applied to a rat experimental periodontitis model in vivo, inflammatory reaction is remarkably relieved, and periodontal tissue regeneration of periodontitis rats is promoted. Mechanism research shows that the NLRP12 alleviates the inflammatory response of PDLSCs and osteogenic differentiation inhibition by inhibiting an NF-kappa B pathway. The NLRP12 overexpression PDLSCs are suitable for repairing periodontal tissue defects, and have a good clinical application prospect.

The invention provides RNA (Ribonucleic Acid), shRNA (Short Hairpin Ribonucleic Acid) and a DNA (Deoxyribose Nucleic Acid) coding sequence of the shRNA for reducing the expression of a motor neuron survival gene SMN, and a delivery vector or a cell containing the RNA, the shRNA and the DNA coding sequence of the shRNA. Furthermore, the invention successfully constructs a stable transfection cell line with SMN-targeted shRNA transcriptional expression regulated by a tet-on regulation system, expression of the target gene SMN is successfully knocked down through DOX induction, a cell viability decline phenotype appears, an in-vitro cell model of the SMA disease is successfully constructed, and a better platform is provided for high-throughput screening of SMA treatment drugs. Furthermore, the invention provides a method for detecting the biological activity of the SMN protein, the method is short in period, low in cost and simple to operate, and an important means for rapidly evaluating the biological activity is provided for drugs for expressing or promoting expression of the SMN protein, including gene therapy drugs.