46results about How to "Phenotype stable" patented technology

The invention provides a method for in vitro induction and amplification of a human antigen nonspecific regulatory T cell. A human peripheral blood CD4<+> T cell with an abundant source is separated, and a CD4<+>CD25<-> T cell is induced and amplified into a CD4<+>CD25<+>CD127<dim> antigen nonspecific regulatory T cell with high purity and high regulation efficiency within a short period through a simple induction method meeting the Clinical Good Manufacturing Practice (GMP). An enough therapeutic dose of Treg can be obtained through induced cultivation of a week (6-7d), the phenotype and regulation function of the induced Treg are stable, furthermore, the incidence rate of events, such as cell activity reduction and microbial contamination due to long-term culture in vitro is greatly reduced, and the quality control method is specific and fast and is very suitable for clinical application.

The invention relates to a method for obtaining tissue engineering cartilage through directionally inducing bone-marrow mesenchymal stem cells (BMSCs). At present, a large amount of exogenous growth factors and culture in vitro are needed before culturing the BMSCs and the cartilage cells in vivo to promote the differentiation of the BMSCs towards the cartilage cells. The method comprises the following steps of: mixing the sub-cultured P3-generation BMSCs, the cartilage cells and a large extracellular matrix aggregate (cell brick), resuspending the mixture into a platelet rich plasma to prepare a cell compound of the sub-cultured P3-generation BMSCs, the cell brick and the platelet rich plasma, injecting the compound into the skin on the back of a nude mice when the compound condenses gradually, and culturing to obtain the new tissue engineering cartilage. The completely biological and injectable tissue engineering cartilage is obtained on the basis of coculturing the BMSCs and the cartilage cells. The differentiation of the BMSCs towards the cartilage cells can be kept stably, the hypertrophy of the tissue engineering cartilage in vivo can be inhibited effectively, and the defects in the background art can be overcome.

Engineered human tissue constructs are provided that are suitable for use in making humanized animals for use in pharmaceutical development. Humanized animals having the constructs implanted in vivo are provided. Methods of making and using the tissue-engineered constructs and humanized animals are also provided.

The invention discloses a method for expanding and culturing peripheral blood T cells in vitro. In the method, a culture medium consists of a basic culture medium, fetal bovine serum, interleukin-2, interleukin-15, a human CD3 monoclonal antibody and a human CD28 monoclonal antibody. According to the method, T cells isolated from peripheral blood are activated and cultured in one cycle, a sufficient amount of cells for in vitro functional experiments can be obtained; after 7 days of expansion, number of the T cells can reach 40-50 times the initial amount; after 14-22 days of expansion, the number of the cells can reach 80-200 times the initial amount; furthermore, the CAR-T cells difficult to be expanded can be expanded to 60-80 times the initial amount on the 22nd day after in vitro infection, and the number is much greater than the initial isolation amount. According to the method, activation / expansion of the T cells and CAR-T cells is realized through stimulation of monoclonal antibodies and cytokines in the culture medium, no antigen-presenting cells or specific antigens or magnetic beads are needed, operation is simple, and culture cost is greatly reduced.

The invention relates to a genetic modification method for regulating and controlling animal endogenous gene expression. Tetracycline response elements are inserted into proper positions of endogenous genes of animals to be regulated and controlled according to fixed points through the gene recombination technology; and regulation and control genes are transferred into animal genomes by a transgene method, transgene animals are obtained, and the reversible expression regulation and control on the endogenous genes is realized under the effect of inductors of tetracycline or other analogues. A mouse endogenous gene induction expression regulation and control model built according to the method can be widely applied to the fields of gene function study, disease animal model building and the like.

The invention relates to a preparation method of a skin cell damage repairing reagent; P4-generation umbilical cord mesenchymal stem cells have a large number of cells, phenotypes are stable, a lot of mixed cytokines can be secreted, and the requirements of mass production of the cell damage repairing reagent can be met, so that the purpose that the cell damage repairing reagent is applied in clinical treatment of skin damage repairing is realized.

The invention provides an organoid technology-based individualized lung cancer primary cell culture method. According to the method, high-throughput screening is carried out tumor cell models in vitrothrough organoid models established in tumor tissues of patients, so as to instruct individualized treatment of the patients. Specifically, excised lung cancer fresh tissues are utilized to carry outorganoid culture to establish individualized lung cancer primary cell models and carry out authentication, so that basis is provided for accurate treatment evaluation of lung cancer.

The invention discloses a method for preparing beef cattle with double muscled similar to naturally mutated Belgian blue cattle. The invention provides a method for preparing MSTN biallelic mutant cells, which comprises the following steps of: performing genome editing on a target region of a first exon of an MSTN gene biallelic of a bovine in vitro fibroblast genome, so that an exon 1 forms a termination codon in advance to terminate expression; obtaining an MSTN biallelic mutant cell; the invention establishes a method for preparing (MSTN-4 / -4) knockout cattle without any exogenous DNA integration and with double MSTN allele small fragment deletion. Importantly, the cattle have a very similar double muscled phenotype with naturally existing Belgian blue cattle.

The invention relates to an expression vector containing a calpain-1 gene. The expression vector comprises a Tet operon, an alpha-MHC promoter nucleotide sequence, a mitochondrial signal peptide sequence and a gene segment of calpain-1 which are connected in sequence. The invention further provides a construction method of an inducible transgenic mouse cardiomyopathy animal model. The constructionmethod comprises the following steps: importing the expression vector containing the calpain-1 gene into oosperms of a mouse, and transferring the oosperms into the uterus of the receptor mouse for gestation, thus obtaining the positive offspring transgenic mouse; and mating the positive offspring transgenic mouse with a transgenic mouse with specific expression of tTA or a transgenic mouse withspecific expression of rtTA, and picking out the inducible transgenic mouse cardiomyopathy animal model from the hybrid offsprings. The invention further discloses applications of the transgenic mousecardiomyopathy animal model obtained through the construction method in screening medicines for treating cardiomyopathy. According to the technical scheme provided by the invention, the modeling success rate is high, the phenotype is stable, the cost is ow, and whether inducting for the generation of cardiomyopathy is needed or not can be selected according to the actual demands.

Engineered human tissue constructs are provided that are suitable for use in making humanized animals for use in pharmaceutical development. Humanized animals having the constructs implanted in vivo are provided. Methods of making and using the tissue-engineered constructs and humanized animals are also provided.

A male sterile mouse model with high genetic stability and phynotype stability is disclosed. Its preparing process features that the gene Kif18a is removed from genome or the exogenous gene is inserted in the gene Kif18a to make it be deactivated. Said model can be used to research the generation and development of sperm, the gene associated with the development of sperm, the transplantation of stem spermatogonium, reconfiguration of sperm, the migration and differentiation of reproduction cells, and the medicine for contraception.

The invention discloses a serum-free medium of human adipose stem cells. The serum-free medium comprises main components such as amino acids, inorganic salt components, a growth factor and albumin. The invention provides the serum-free medium of the human adipose stem cells, which does not contain animal-derived components and can completely realize serum-free culture of the stem cells. By adopting the serum-free medium provided by the invention to culture the human adipose stem cells, a large number of high-purity, safe and high-quality mesenchymal stem cells can be obtained in vitro; meanwhile, the cell survival rate can be increased, and the stem cell characteristics can be maintained. The serum-free medium can solve the problem of insufficient cells, and the cell quantity, survival rate and immunophenotype of each batch of the adipose stem cells are more stable.

The invention relates a method for inducing in-vitro expansion of CD8<+> regulatory T cells by immunosuppressants. According to the method, expansion of CD8<+> regulatory T cells is jointly induced by TGF-beta and RAPA (rapamycin). The invention further provides a CD8<+> Treg (regulatory T cell) with an immunosuppression function and application of the CD8<+> Treg with the immunosuppression function. The method has the advantages that the TGF-beta and the RAPA are added into a polycloning in-vitro expansion system of the CD8<+> regulatory T cells for the first time, and a great number of CD8<+> Tregs with the remarkable immunosuppression function are obtained successfully; as multi-round expansion is conducted, the CD8<+> Tregs can increase exponentially so as to meet the demands of clinical cell treatment. The CD8<+> Tregs can be kept stable in the aspects of activity, purity, phenotype, nullipotency, suppression function and the like and can be used for treating various autoimmune diseases through adoptive infusion, thereby being huge in clinical application potential.

The invention discloses a double-gene co-expression plasmid pIRES2-Nrf2-DKK1, a preparation method and an application thereof. The plasmid includes CDS sequences of an original expression vector pIRES2-zs Green, Nrf2 and DKK-1 genes. The plasmid is 7151 bp in overall length and is represented in the SEQ ID No.1; wherein the Nrf2 gene sequence is located from the 619th to the 2436th loci, and the DKK-1 gene sequence is located from the 3016th to the 3816th loci. The double-gene co-expression plasmid can be high-effectively expressed in human adipose mesenchymal stem cells and human umbilical cord mesenchymal stem cells, and can significantly increase anti-oxidizing, anti-inflammation and anti-apoptosis capabilities of the mesenchymal stem cells. In-vivo test proves that by transplanting mesenchymal stem cells transfected by the plasmid can significantly improve liver damage of mice. The plasmid can improve the transplanting treatment effect of the mesenchymal stem cells and has great application prospect.

The invention discloses an in-vitro amplification kit for regulatory T cells in human peripheral blood. The kit comprises an activating agent I, an activating agent II, a screening culture medium andan amplification culture medium. The invention further discloses an in-vitro amplification method for the regulatory T cells in human peripheral blood. According to the method, mononuclear cells of peripheral blood are directly obtained and are paved in culture containers coated with different antibodies for culture, and activators such as interleukin and AICAR are added to promote cell proliferation. The method has the advantages of low cost, simple operation, and capability of obtaining of a large number of regulatory T cells with high cell viability in a short time; and the obtained cells are stable in phenotypes and functions and high in biological safety.

Phototropin is a blue light receptor, which mediates a variety of blue-light elicited physiological processes in plants and algae. In higher plants these processes include phototropism, chloroplast movement and stomatal opening. In the green alga Chlamydomonas reinhardtii, phototropin plays a vital role in progression of the sexual life cycle and in the control of the eye spot size and light sensitivity Phototropin is also involved in blue-light mediated changes in the synthesis of chlorophylls, carotenoids, chlorophyll binding proteins. We compared the transcriptome of phototropin knock out (PHOT KO) mutant and wild-type parent to analyze differences in gene expression in high light grown cultures (500 μmol photons m−2 s−1). Our results indicate the up-regulation of genes involved in photosynthetic electron transport chain, carbon fixation pathway, starch, lipid, and cell cycle control genes. With respect to photosynthetic electron transport genes, genes encoding proteins of the cytochrome b6f and ATP synthase complex were up regulated potentially facilitating proton-coupled electron transfer. In addition genes involved in limiting steps in the Calvin cycle Ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO), Sidoheptulose 1,7 bisphosphatase (SBPase), Glyceraldehyde-3-phosphate dehydrogenase (3PGDH) and that mediate cell-cycle control (CDK) were also up regulated along with starch synthase and fatty acid biosynthesis genes involved in starch and lipid synthesis. In addition, transmission electron micrographs show increased accumulation of starch granules in PHOT mutant compared to wild type, which is consistent with the higher expression of starch synthase genes. Collectively, the altered patterns of gene expression in the PHOT mutants were associated with a two-fold increase in growth and biomass accumulation compared to wild type when grown in environmental photobioreactors (Phenometrics) that simulate a pond environment. In conclusion, our studies suggest that phototropin may be a master gene regulator that suppresses rapid cell growth and promotes gametogenesis and sexual recombination in wild type strains.

The invention discloses a construction method of a zebra fish animal model for cardiovascular disease drug screening, and specifically relates to the field of animal model construction methods. The method comprises the following specific steps: selecting a gRNA target site of a zebra fish heg1 gene and synthesizing gRNA in vitro; in order to obtain the target site, firstly, downloading genome dataof the target gene heg1 from a zebra fish genome database; and selecting a 529bp long target sequence near a first exon of the heg1 gene and inputting the sequence into a target point website to obtain a target point primer. According to the invention, a cardiovascular malformation zebra fish mutant with genetic stability can be obtained; by using the zebra fish embryo transparency and the autonomous oxygen dissolving property, the mutant can be used for screening and verifying the influence of different cardiovascular drugs on cardiovascular development; and the zebra fish animal model construction method provides important physiological significance and specificity for screening of cardiovascular disease treatment drugs.

The invention belongs to the technical field of cell biology and particularly relates to culture and three-line differentiation induction methods of adipose tissue-derived mesenchymal stem cells. Theculture method specifically includes: placing adipose tissue into a centrifugal tube, adding normal saline, pipetting with a pipet, standing, sucking out liquid on the lower layer of the tissue, adding collagenase type I / PBS digestive juice with the concentration being 0.1% to perform digestion, and placing the culture bottle into a culture tank of 37 DEG C and with 5% CO2; taking out cells when the cells grow to 80% and merge, adding TrypLE digestive juice, terminating the digestion when the cells start retracting and rounding, and performing subculture and multiplication culture. The differentiation induction method has the advantages that differentiation time is shortened, the method needs 10 days to differentiate the cells into adipose and bones and needs 14 days to differentiate the cells into cartilage, and differentiation efficiency is increased greatly as compared a common induction method which needs 14 days to differentiate cells into adipose and needs 21 days to differentiate the cells into bones and cartilage.

The invention belongs to the field of biological technology. The wild-type cinerea cinerea strain is used as the background strain to knock out the gene of a key enzyme cylindroxone dehydratase in the melanin synthesis pathway, and construct a gene deletion mutation of the cinerea cinerea capable of producing a large amount of cylindroxone The main steps of the strain Δbcscd1 are as follows: use fusion PCR technology to fuse the 5'-UTR and 3'-UTR of the target gene bcscd1 with the upstream and downstream coding regions of the hygromycin fragment Hph to obtain the upstream and downstream fusion fragments, and use PEG-mediated The protoplast transformation method was used to transform the fusion fragment into wild-type protoplasts for double-crossover homologous recombination, and the gene deletion mutant strain Δbcscd1 was obtained through screening and identification. The gene deletion mutant strain constructed by the method of the invention has high genetic stability and low toxicity. The present invention also proposes a method for specifically purifying cylindroporin through steps such as extraction, rotary steaming and column chromatography.

The invention discloses application of miR-21 and a target gene thereof in preparation of reagents or drugs diagnosing and / or controlling osteoarthritis, relating to the technical field of biology. The reagent or drug diagnosing and / or controlling osteoarthritis is prepared from an inhibitor and / or inhibitor composition downregulating activity of miR-21 or blocking activity of miR-21, and the miR-21 inhibitor regulates a key transcription factor PIK3C2A gene related to differentiation and phenotype stability of cartilage cells in cartilage cells so as to stabilize the phenotype of the cartilage cells and prevent matrix outside the cartilage cells from degrading. Therefore, the invention provides application of miR-21 and / or antisense oligonucleotide thereof and / or target gene of miR-21 inpreparation of reagents or drugs diagnosing and / or controlling osteoarthritis.

The invention provides an antisense RNA sequence combination and application thereof in production of abortive tilapia, and belongs to molecular biology and breeding biology technologies. The antisense RNA sequence combination comprises antisense RNA of steroid-producing factors SF1-1 and SF1-2; and nucleotide sequences of Anti-SF1-1-II, Anti-SF1-2-I and Anti-SF1-2-II are respectively shown as SEQID No. 1 to SEQ ID No. 4. According to the method, an antisense RNA fragment is introduced into an ovum through a fertilization hole, so that effective and accurate targeted intervention is realizedto regulate expression of genes; and the method is simple to operate, extremely small in ovum damage, high in success rate and stable in representative type after breeding, and has extremely strong application prospect.

The invention provides a method for rapidly establishing an animal model of Parkinson's disease through substantia nigra gene knockout. The invention discloses sgRNA capable of efficiently specifically knocking out PINK1 genes, and further provides a method for establishing the animal model of Parkinson's disease by virtue of targeted knockout of PINK1 genes in substantia nigra of animals. The method specifically comprises the following steps: injecting sgRNA and CRISPR nuclease into a substantia nigra part of the animal brain, and causing target fragment deletion to the PINK1 genes. Obvious typical dyskinesia symptoms of Parkinson's disease can occur within 3-4 weeks, and the dyskinesia cannot restore without drug therapy. The method directly causes neuronal death of the substantia nigrapart, does not have any side effect, has the mold establishment success rate of 90% or higher, has phenotypic stability and excellent applicability and repeatability, provides important models for screening drugs for the Parkinson's disease, performing stem cell treatment and gene defect repair treatment, researching pathogenesis of deletion of the PINK1 genes and the like, and has huge economic value and preclinical study significances.

The invention provides a sgRNA combination of a targeted AHRR gene and application of the sgRNA combination, the sgRNA combination of the targeted AHRR gene comprises sgRNA1 and / or sgRNA2, the sgRNA1 comprises a nucleic acid sequence as shown in SEQ ID No.1, and the sgRNA2 comprises a nucleic acid sequence as shown in SEQ ID No.2. The invention also provides an AHRR gene editing system, a recombinant cell and a construction method thereof, and a construction method of an obese animal model, through gene editing and screening, the obtained homozygous mouse without the AHRR gene has typical obesity signs, larger body type, heavier body weight, higher fat content in vivo, and can be used for screening blood sugar and lipid reducing related drugs, and has extremely wide application value.

The present invention is directed to methods and compositions for expanding and stabilizing the phenotype of natural regulatory T cells. In particular, the present invention provides methods and compositions for treating natural regulatory T cells that renders the cells resistant to factors present in the inflammatory milieu and stabilizes the suppressive properties of the cells.

The invention provides a phenotype maintaining and in-vitro high-activity three-dimensional amplification method of hepatic stem cells. The method comprises the following steps: 1, preparing a medium, wherein the medium comprises 1L of DMEM / F12, 15-20 mg / L of a fibroblast growth factor, 10-15 mg / L of insulin, 0.1-0.2 mg / L of glutamic acid, 0.5-0.8 mg / L of nicotinamide, 0.1-0.2 mg / L of seleninic acid, 0.05-0.1 mg / L of beta-mercaptoethanol, 0.1-0.6 mg / L of alpha-actin, 0.2-0.3 mg / L of sodium acetate, 0.001-0.003 mg / L of sodium pyruvate and 2-3 wt% of fetal calf serum; and 2, immersing sterilized porous gel in the medium, inoculating the porous gel with hepatic stem cells, and culturing the hepatic stem cells. Stem cells with stable phenotype and high activity are obtained in the invention, and the good characteristics of the stem cells are kept.

The invention discloses an in-vitro amplification culture method of NK cell bodies. The method comprises the following steps: (1) adding a stimulating factor and a proportion regulator with a final concentration of 0.1-10nM into treated blood containing NK cells, and culturing for 3-4 days; and (2) adjusting the cell density to 0.5-1.5*10 < 6 > cells / mL, continuously adding a final proportion regulator and cell factors, and continuously culturing for 20-30 days. In the process of culturing the NK cells, allogeneic cells do not exist, the related factors are factors harmless to a human body inthe clinical level, the safety performance is high, the phenotype and number of the cultured cells are stable, and the culture method is simple, convenient and feasible.

The invention relates to an expression vector containing a calpain-1 gene. The expression vector comprises a Tet operon, an alpha-MHC promoter nucleotide sequence, a mitochondrial signal peptide sequence and a gene segment of calpain-1 which are connected in sequence. The invention further provides a construction method of an inducible transgenic mouse cardiomyopathy animal model. The constructionmethod comprises the following steps: importing the expression vector containing the calpain-1 gene into oosperms of a mouse, and transferring the oosperms into the uterus of the receptor mouse for gestation, thus obtaining the positive offspring transgenic mouse; and mating the positive offspring transgenic mouse with a transgenic mouse with specific expression of tTA or a transgenic mouse withspecific expression of rtTA, and picking out the inducible transgenic mouse cardiomyopathy animal model from the hybrid offsprings. The invention further discloses applications of the transgenic mousecardiomyopathy animal model obtained through the construction method in screening medicines for treating cardiomyopathy. According to the technical scheme provided by the invention, the modeling success rate is high, the phenotype is stable, the cost is ow, and whether inducting for the generation of cardiomyopathy is needed or not can be selected according to the actual demands.