153results about How to "Stable passage" patented technology

The invention provides a method for performing GINS2 gene editing on mesenchymal stem cells by adopting a CRISPR-cas 9 system, and particularly relates to a method for constructing a GINS2 gene-knocked mesenchymal stem cell line. A new enhancin CREnhancer 1.0 is used, so that the editing efficiency of the CRISPR-cas 9 gene in cells can be remarkably improved. The GINS2 knockout plasmid for the mesenchymal stem cells provided by the invention has high genetic stability.

The invention provides a method using a CRISPR-Cas system to perform GING2 gene editing on an epidermal stem cell and particularly relates to a method for building an epidermal stem cell line with theGING2 gene being knocked out. Two specific gRNAs are built and acquired, and the GINS2 gene editing efficiency of CRISPR / Cas9 in the epidermal stem cell can be increased evidently. An epidermal stemcell GINS2 knockout plasmid is good in hereditary stability and high in targeting efficiency.

The invention provides CASP3 gene editing performed on a mesenchymal stem cell through a zsystem, and particularly relates to the establishment of a mesenchymal stem cell line for constructing CASP3 gene knockout. Novel enhancing CREnhancer1.0 is adopted, and CRISPR-cas9 gene editing efficiency in a cell can be obviously improved. Bone mesenchymal stem cell CASP3 gene knocked out plasmid has goodhereditary stability.

The invention discloses a method for primary culture of tumor cells and relates to the field of cell biology. According to the method disclosed by the invention, a tumor sample is prepared into a single-cell suspension liquid by use of an enzyme method or a mechanical method, and then tumor cell bladders are prepared by use of a serum-free suspension culture method, wherein alkaline fibroblast growth factors, epidermal growth factors, insulin and bovine serum albumin need to be added during a culturing process, then the growth factors are removed, and the culture is implemented in a general culture medium to obtain adherent cells capable of realizing continuous passage. The method disclosed by the invention is short in culture period and has the effect of increasing the success rate of the primary culture of tumors.

The invention discloses a 3D culture, passage, cryopreservation, recovery and identification method for in-vitro organoids based on small intestines of different segments of mice. The method comprisesthe following steps: (1) separating recesses of duodenum, jejunum and ileum segments of mice; (2) performing 3D culture on the recesses of the duodenum, jejunum and ileum segments of mice, and forming organoids; (3) performing passage on organoids of small intestines of mice; (4) performing cryopreservation on the organoids of small intestines of mice; (5) performing recovery on the organoids ofsmall intestines of mice; (6) preparing frozen sections of the organoids of small intestines of mice; and (7) performing immunofluorescence labeling and identification on the frozen sections of the organoids of small intestines of mice. Compared with the prior art, the method disclosed by the invention optimizes a recess extraction manner, an in-vitro culture system and a culture medium based on the recesses of the small intestines containing stem cells, and comprises the steps of complete culture, passage, cryopreservation, recovery and identification. The small intestine organoids obtained by the method are capable of performing repeated passage, and the passage organoids have character stability.

The invention discloses a small-molecular compound combination for differentiated cell reprogramming, and a reagent kit and an application of the small-molecular compound combination. The small-molecular compound combination includes the following components: a TGF-beta inhibitor, a WNT / beta-catenin agonist, a cAMP agonist and a PKC inhibitor; furthermore, the small-molecular compound combinationalso includes at least one of an RAR agonist, a DNMT inhibitor, an HMT inhibitor, a histone demethylase inhibitor, ascorbic acid, a JNK inhibitor, an ROCK inhibitor and a lysine deacetylase inhibitor.Differentiated cells can be reprogrammed into mesenchymal stem cells by phased induction with the small-molecular compound combination, all steps can be subjected to precise quality control, and standardization operation and large-scale production are convenient. The method provided by the invention has wide donor sources, a patient himself can be used as a donor, and the mesenchymal stem cells needed for basic research, clinical treatment or tissue engineering production can be obtained in relatively short time.

The invention provides a hair follicle stem cell separation culture method. The method utilizes an organ culture method, adopts the William's E complete medium to culture the hair follicle, separate and purify to get hair follicle stem cells. The method only uses very small skin materials, the rat whisker follicle is separated with a stereoscope through a micro separation method, then the whole whisker follicle is cultured, the hair follicle stem cells grow out from the projection part and form clone, so the cell clone forming efficiency is high, the purification is easy, long-term passage can be realized, the characteristics of the hair follicle stem cells are kept unchanged, and only one whisker follicle can obtain enough hair follicle stem cells for long-term use.

The present invention belongs to the technical field of cell biology and particularly relates to a technology for regulating and controlling a Jak-Stat pathway to differentiate, dedifferentiate and rejuvenate cells, and an application of the technology. Rejuvenated cell products and / or cell products of different lineage types are obtained by small molecule compound combination, cytokine combination or recombinant protein combination, a gene editing technology and a transgenic technology to quantitatively and / or regularly regulate and control gene or protein targets of the Jak-Stat signaling pathways in cells. The provided technology for regulating and controlling the Jak-Stat signal pathway, and the product / derivatives of the cell product can be used for re-programming cells, tissues, organs and organisms, construct tissue engineering materials, repair damages of tissues and organs of mammals and the aged and degenerated tissues and organs, delay or reverse aging processes of the cells, tissues, organs and organisms, and can be used for immunoregulation of the cells, tissues, organs and organisms.

The invention discloses an egg yolk antibody for preventing novel goose astrovirus and a preparation method thereof. The egg yolk antibody contains a goose astrovirus strain resistance antibody; the preservation number of the goose astrovirus strain is CCTCC NO:V201808. The prepared egg yolk antibody is good in safety, and no any partial or whole adverse reaction is caused by the egg yolk antibody; infections caused by novel goose astrovirus can be effectively prevented and / or treated, and the good commercialization development prospect is achieved.

The invention discloses a method for producing trehalose synthase by taking integrated recombinant bacillus subtilis as a strain and producing trehalose from the trehalose synthase. The method specifically comprises the following steps of: integrating a trehalose synthase expression element in a bacillus subtilis chromosome to construct integrated recombinant bacillus subtilis, and fermenting in a nutrient culture medium to produce the trehalose synthase by taking the recombinant bacillus subtilis as a strain, wherein via simple separation, the trehalose synthase in the fermentation liquor can be directly used for manufacturing trehalose. The method disclosed by the invention has the advantages that the trehalose synthase is produced by virtue of a food safety expression system, the exogenous gene contained in the strain with integrated expression can be used for stable passage and expression, and the expressed trehalose synthase is secreted to the outside of cells, has no antimicrobial activity, and achieves the requirements of food applications for enzymic preparation, thus being beneficial to further manufacturing for trehalose.

The invention relates to a multidrug resistant cell strain for oophoroma, which is characterized in that a human oophoroma SKOV3 cell strain is selected as a parental cell; the action concentration of chemotherapeutics of etoposide (VP16) is gradually increased from 0.1 mu g / ml to 3 mu g / ml; and drug resistant SKOV3 / VP16 cell strain is established. The SKOV3 / VP16 cell can stably grow, transfer and reanimate in 3 mu g / ml VP16, makes the VP16 generate drug resistant index (RI) of 21.548, and performs intersect drug on MTX, DOX, DDP, VCR, 5-Fu and MMC except the VP16. The invention aims to provide a drug resistant tumor cell model for relative study on the tumor.

The invention provides establishment and application of a hepatocellular carcinoma cell line HCC-LY10. Specifically, the hepatocellular carcinoma cell line HCC-LY10 provided in the invention has stable traits, can be subcultured stably and repeatedly, and is applicable to established animal models of hepatocellular carcinoma, thus being an ideal cell line for the basic and preclinical phase application study of hepatocellular carcinoma. In addition, the hepatocellular carcinoma cell line HCC-LY10 partially expresses hepatocellular carcinoma stem cell marker EpCAM. The invention also provides application of the cell line in establishing model animals, screening drugs, and other aspects.

The invention discloses a preparation method and applications of a young repair-type fibroblast, and belongs to the technical field of cell biology. The JAK-STAT signal path in the repair-type fibroblast provided by the invention is inhibited, and the cell has the characteristic of being young. The preparation method adopts a small molecular compound, a cytokine combination or a recombinant protein combination to inhibit a corresponding signal path in a common fibroblast, and can be used for the reprogramming of cells, tissues, organs and organisms or the rejuvenation of cells, tissues, organsand organisms. The repair-type fibroblast and a cell product thereof can be used for constructing tissue engineering materials, repairing the damage of mammalian tissues and organs and repairing aging and degenerative tissues and organs, delaying or reversing the aging process of cells, tissues, organs and organisms, and can be used for the immunoregulation of cells, tissues, organs and organisms. The repair-type fibroblast prepared by the technology has the characteristics of skin fibroblasts, and has the characteristics of mesenchymal stem cells, so the fibroblast is named as the repair type skin fibroblast.

The invention discloses genetic engineering pseudomonas putida and a construction method and application thereof. The genetic engineering pseudomonas putida is characterized in that gene modification is performed for pseudomonas putida XPSN to construct genetic engineering pseudomonas putida P-HSP with inactivated 6-hydroxy-3-succinyl pyridine 3-hydroxylase gene. The construction method comprises the following steps: designing a primer; performing PCR amplification; constructing recombinant plasmid pK18mob-hspB for knockout; transferring the recombinant plasmid into escherichia coli S17-1; then performing parent hybridization. The genetic engineering pseudomonas putida P-HSP is also applied to nicotine conversion reaction as a biocatalyst to produce 6-hydroxy-3-succinyl pyridine. The genetic engineering strain constructed by the method is high in conversion efficiency, simple and convenient to operate, and stable in subculture, and can be recycled three times, so that the reaction cost is reduced, and high industrial application value is brought.

The invention discloses a cell line resistant to an A subgroup avian leukosis virus as well as a construction method and application thereof, and belongs to the technical field of agricultural biology. The cell line resistant to the A subgroup avian leukosis virus is named after an anti ALV-A chicken embryo fibroblast cell line DF-1 / A, and is conserved in Wuhan University, China of China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2014180 from October 15th, 2014; the cell line resistant to the A subgroup avian leukosis virus is applied to the diagnosis of the A subgroup avian leukosis virus. The cell line resistant to the A subgroup avian leukosis virus disclosed by the invention can achieve stable passage and be stored for a long term, is helpful for accurately identifying subgroup classification of ALV by using the characteristic of ALV superinfection, and is applied to the diagnosis of the A subgroup avian leukosis virus.

The invention relates to a recombination infectious haematopoietic necrosis virus (rIHNV HLJ-09) strain and a construction method and application thereof, and belongs to the field of biotechnologies. The microorganism preservation number of the rIHNV HLJ-09 strain is CGMCC No.8606, and the passage experiment of cells infected with recombinant virus shows that the virus can achieve passage stability. The rIHNV HLJ-09 is used in the intraperitoneal inoculation of juvenile rainbow trout, the result leads to the disease and death of the juvenile rainbow trout, and the lethallty rate can reach 90%, so that it shows that the virus has the similar pathogenic character as parental generation HLJ-09 strain virus. Furthermore, a rIHNV HLJ-09 reverse genetic system is used for replacing reporter gene green fluorescent protein (EGFP) encoding genes with NV ORF in the rIHNV HLJ-09, the virus is successfully rescued, and the rescued virus is named as rIHNV-EGFP. When a laser scanning confocal microscope is used for observing the cells infected with the rIHNV-EGFP virus, obvious green fluorescent can be observed, and it shows that the rIHNV HLJ-09 virus strain can be used for expressing foreign proteins.

The invention discloses a method for quickly constructing an IBV (Avian Infectious Bronchitis Virus) reverse genetic strain, and belongs to the technical field of coronavirus reverse genetics. The constructing method comprises the following steps: quickly completing construction containing IBV genomic full-strength cDNA (Complementary Deoxyribose Nucleic Acid) clone by taking a BAC (Bacterial Artificial Chromosome) vector as a framework and applying an in-vitro homologous recombination technology, directly transfecting cells by a constructed recombinant plasmid, and transcribing in the cells, thus obtaining a transcript having infectivity; completing virus packaging; inoculating SPF (Specific Pathogen Free) chick embryo to a mixed solution of the cells and a culture medium and passing from generation to generation, thus obtaining the IBV reverse genetic strain. The constructing method disclosed by the invention has the advantages of simple operation and high positive cloning efficiency, the obtained IBV reverse genetic strain has passage stability, and an effective tool is provided for researching pathogenesis of the virus in vitro, developing a novel vaccine and the like; according to the method disclosed by the invention, transcription is carried out in the cells by utilizing a CMV (Cytomegalovirus) promoter added on a 5' terminal, and the rescue efficiency of the virus is greatly increased by utilizing an HDVR (Hepatitis Delta Virus Ribozyme) sequence added on a 3' terminal.

The application belongs to the technical field of construction of animal tumor models, and particularly relates to a stably passaged immunodeficient mouse model constructed by utilizing an NK / T lymphoma cell strain and a construction method of the model. The method comprises the following steps: pretreating an innoculation object and a lymphoma cell strain respectively, preparing tumor suspensionfor injecting into a mouse, enabling tumor cells to passage and the like. The immunodeficient mouse model can be used for drug screening or therapeutic effect evaluation in medical science. The construction method for constructing the lymphoma animal model by utilizing the NK / T lymphoma cell strain, which is provided by the application, utilizes the fixing effect of matrigel, can be used for fixing lymphoma cells beneath skin, so that the tumor formation rate of the mouse is ensured, and the technical advantages of fast tumor formation and high tumor formation rate are manifested; on the otherhand, in the process of passage, the method cooperates with the cryopreserved operation of a relevant tumor tissue culture solution, and the lymphoma animal model for passage can be recovered at anymoment and directly constructed.

The invention provides a cell culture composition used for primary culture of tumor cells and application thereof. According to application of the cell culture composition in primary culture of tumor cells from epitheliums, the cell culture composition comprises a serum-free horn cell medium and a serum-free additive for culture of nerve cells, and does not contain serum. The cell culture composition provided by the invention can realize successful in vitro primary culture of the tumor cells from epitheliums, substantially increases the success rate of primary culture of the tumor cells from epitheliums and can inhibit growth of stromal cells like fibroblasts and endothelial cells in cancer tissue; for example, primary culture of the tumor cells from epitheliums succeeds within 10 d by using the cell culture composition provided by the invention, and the success rate of culture is mostly more than 80%; the cultured tumor cells can be stably passed to more than 8 generations, and a cell line can be successfully established.

The invention discloses a human-derived rotavirus attenuated strain with wide-spectrum immunogenicity and a vaccine prepared from the human-derived rotavirus attenuated strain. The human-derived rotavirus attenuated strain with wide-spectrum immunogenicity is named as HRV-LZ-2013, has a gene type of G10P8, is preserved in the China general microbiological culture collection center and has a strain preservation number of CGMCC N0. 9452. An experiment proves that the rotavirus attenuated strain keeps low pathogenicity to animals. An immunization protection experiment on animals and especially on rotavirus-caused diarrhoea model rabbit and pig proves that the attenuated live vaccine or inactivated vaccine prepared from the human-derived rotavirus attenuated strain has good immunizing protection properties. An immunizing protection test on cattle and sheep proves that the vaccine provided by the invention has a wide immunizing protection range and can be used for preventing cattle, pig, horse, sheep and dog diarrhoea caused by rotaviruses.

The invention relates to a drug-resistant cell strain of human bile duct cancer. The drug-resistant cell strain has a collection number of CCTCC NO:C 201441. A human bile duct cancer RBE cell strain is selected as a parent cell and is added into 5-FU for culture, the action concentration is gradually increased according to a concentration sequence of 25, 50, 75 and 100mu g / ml, and the drug-resistant cell strain of human bile duct cancer is established. The drug-resistant cell strain RBE / 5-FU of the human bile duct cancer can provide a drug-resistant tumor cell model for researching a drug-resistant tumor mechanism, analyzing the sensitivity of chemotherapeutics, screening and evaluating the chemotherapeutics, developing drug-resistant tumor reversing drugs and researching more effective tumor treatment methods.

The present invention relates to the field of microbial animal cells, and specifically a hepatoma cell line STL-C1 derived from human hepatoma para-carcinoma tissue. The cell line has a preservation number CCTCC No:C201630. The present invention also provides an establishment method and application of the cell line. The established hepatoma cell line STL-C1 is derived from human hepatocellular hepatoma para-carcinoma tissue, and can be cultured in vitro for a long term and has stable passage; and in vivo experiments show that the cell line still has good tumorigenic property, and is completely different from the currently existing established common hepatoma cell lines and even from the hepatoma cancer embolus cell line. The hepatoma cell line STL-C1 has important usage and reference value to the study of the formation of satellite lesions, and biological behaviors suahc as recurrence and transfer of primary hepatic carcinoma, is a novel cell model for basic and clinical research of hepatoma, and has broad application prospects.

The invention discloses a mouse prostate cancer circulating tumor cell line and a prostate cancer circulating tumor cell isolating and culturing method. The preservation number of the circulating tumor cell line is CCTCC C201601. The isolating and culturing method comprises the following steps: establishing an in-situ prostate cancer animal model, taking blood, lysing red blood cells, and conducting isolating and culturing and passage. Obtained circulating tumor cells are uniform in quality; based upon immunofluorescence as well as scratch and membrane-penetrating experiments, the obtained circulating tumor cells conform to the properties of the circulating tumor cells and the circulating tumor cells are relatively strong in transfer and invasion capacities; and the circulating tumor cells can serve as a good experimental tool for researching a prostate cancer transfer mechanism in a laboratory and can also provide a cell research model for preventing prostate cancer transfer in the clinical field. The circulating tumor cell isolating and culturing method provided by the invention is simple and easy to operate, and the isolated cells can undergo stable passage, so as to facilitate such experiments as researching the properties of the circulating tumor cells and the like in the laboratory. The circulating tumor cell line and the circulating tumor cell isolating and culturing method provided by the invention can have a broad application scope in the field of researching tumor transfer.

The invention provides a serum-free feed layer-free mouse induced pluripotent stem cell (iPSC) induction medium and a culture method using the same. The medium is a serum-free feed layer-free medium,can be used as an induction medium for mouse somatic cell-induced formation of iPSCs, improves iPS cell induction efficiency, shortens the induction time and can be used for culturing mouse iPSCs. Inthe culture system, after 20 passages of mouse iPSCs, the mouse iPSCs retain stem cell pluripotency and the normal cell karyotype. An internal teratoma experiment result shows that after culture for 20 passages, the mouse iPSCs in the culture system retain a trilaminar differentiation potential of PSCs.

The invention relates to a bacterial wilt control technology, in particular to a bacterial wilt resistance biocontrol strain and an application thereof. Biocontrol strain SY-SF-Streptomyces venezuelaebelonging to the genus Streptomyces (Streptomyces) is isolated from Venezuela on 14 May 2018, Deposited in China Typical Culture Collection Center, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. Deposited No.: CCTCC M2018265. A Streptomyces venezuelanensis strain resistant to bacterial wilt is obtain through screening in northeast woodland and identify as Streptomyces venezuelanensis, which can antagonize bacterial wilt pathogen Ralstonia solanacearum, and that fermentation metabolite thereof can inhibit the growth and multiplication of pathogen and preventthe occurrence of bacterial wilt.

The invention provides establishment and application of a hepatocellular carcinoma cell line HCC-LY5. Specifically, the hepatocellular carcinoma cell line HCC-LY5 provided in the invention has stable traits, can be subcultured stably and repeatedly, and is applicable to established animal models of hepatocellular carcinoma, thus being an ideal cell line for the basic and preclinical phase application study of hepatocellular carcinoma. In addition, a very small part of the hepatocellular carcinoma cell line HCC-LY5 expresses hepatocellular carcinoma stem cell marker CD133. The invention also provides application of the cell line in establishing model animals, screening drugs, and other aspects.

The invention provides a method for separately culturing hippocampus neural stem cells and neurons. The method comprises the following steps: adopting an improved newborn rate nerve cell culturing process and an improved culture medium for performing primary culture on newborn rate hippocampus tissue; directly preparing hepatocytes growing on glass coverslips; extracting cell culture supernatant after 24 hours and transferring to another culture system for directly culturing neural stem cells; and replacing the improved neuron culture medium for the adherent cells in original system, thereby culturing the neurons. According to the invention, the stable culturing for neural stem cells and neurons is completed on the basis of killing an animal at one time, so that the experimental animals can be saved; the neurons with excellent culture density and form can be cultured and an in vitro experimental system related to the neurons can be constructed; the cultured neural stem cells have excellent proliferation, generation stability and excellent differentiation potential.

The invention discloses [delta]Tlrgt2 trichoderma engineering bacteria with high yield of peptaibols and a construction method and application thereof. A Tlrgt2 knockout vector is constructed by upstream and downstream homologous arm sequences of a Tlrgt2 gene and a hygromycin resistance gene hph sequence, the [delta]Tlrgt2 trichoderma engineering bacteria is prepared by a gene knockout method, aTlrgt2 gene is knocked out, and the peptaibols production capacity of the bacteria can be effectively improved. The stable passage of the constructed [delta]Tlrgt2 trichoderma engineering bacteria canbe achieved, the yield of the trichoderma peptaibols can be significantly increased, and the production of the peptaibols by the [delta]Tlrgt2 trichoderma engineering bacteria is about 12 times higher than that of an original strain. The application of the trichoderma peptaibols in medicine and agriculture is facilitated.

The invention discloses a method for constructing a monascus strain capable of achieving high yield of acid protease. The method is characterized by firstly amplifying an Asp fragment of an acid protease gene in monascus from a monascus genome, then connecting the Asp fragment obtained through amplification to an empty vector to construct a recombinant expression vector of acid protease in monascus, transforming the recombinant expression vector of acid protease in monascus into agrobacterium tumefaciens to obtain recombinant agrobacterium tumefaciens and then guiding the recombinant agrobacterium tumefaciens into monascus by utilizing an agrobacterium tumefaciens-mediated method, thus obtaining the monascus strain capable of achieving high yield of acid protease. The monascus transformant strain capable of achieving high yield of acid protease, which is obtained by a gene recombination method, has the characteristics of high-efficiency expression, accuracy in processing and genetic stability of the transformant and can achieve passage stability under non-selective pressure. The expression quantity of the acid protease gene in the monascus strain capable of achieving high yield of acid protease is 3.30 times the expression quantities of wild type genes, so that high yield of acid protease can be achieved.