73 results about "Puromycine" patented technology

The invention discloses a method for inhibiting target gene expression by antisense lncRNA-mediated cis-regulation. The method for inhibiting gene expression by antisense lncRNA-mediated cis-regulation is characterized in that by utilizing the characteristic that the lncRNA has the effect of competing endogenous target RNA, a strong promoter is inserted on a target gene by taking RNA as a genome positioning tool through a CRISPR Cas9 gene editing method, and a lot of antisense lncRNA complementary with the target gene is synthesized to realize in situ cis-competitive inhibition of expression of the endogenous target gene. The method comprises the following steps: firstly, constructing a targeting vector and a homologous template strand donor vector, simultaneously transfecting the constructed vectors into host cells, performing target cloning by puromycin and ganciclovir co-screening, and finally, performing detection and functional research on expression of the target gene.

The invention discloses a conditional Cas9 expression induced swine trophoblastic cell line and an establishment method and application thereof. The method includes: subjecting a lentiviral vector, for controlling Cas9 gene expression, of a Tet-on system to virus packaging, concentrating and purifying; incubating lentivirus with a typical swine trophoblastic cell line, changing fluid, adding puromycin for drug screening, diluting cells to single ones, and culturing to obtain the single-cell-source swine trophoblastic cell line with Cas9 expression under Tet-on regulation. The swine trophoblastic cell line is simple in operation and convenient to use and has a potential utilization value in researching of key functional genes of some trophoblast cells and screening of swine gene target spots. The cell line is expected to be a significant cell material for researching swine placenta development and applicable to correlation researches on swine early-stage placenta development and uterine pregnancy mechanisms and also has a high reference value in research of human early embryonic development.

The invention provides a preparation method of a colon cancer cell strain expressed by stabilized silent PKM2 gene. The preparation method comprises the steps of constructing of shRNA lentiviral expression vector of human PKM2 gene, packaging and obtaining of lentivirus, DLD1 colon cancer lentivirus infecting, stabilized cell strain puromycin screening and Real-time PCR, Western blot identification. The experimental results show that the shRNA nucleotide sequence can be successfully inserted into the pLKO.1-puro expression vector, and the inhibitory effect on the expression of PKM2 gene is remarkable, endurable and stable. The prepared cell strain can be used as the experimental material for researching the regulating effect of PKM2 in malignant characteristics such as colon cancer cell energy metabolism, cell proliferation, cell migration and inflammation.

The invention belongs to the technical field of assisted reproduction, and relates to an oocyte activating solution and application thereof. The oocyte activating solution comprises 1.1 to 1.8 mg/L ofionomycin, 6 to 12 mg/L of calcium ion carrier A23187, 63 to 94 mug/L of puromycin, 19 to 43 mg/L of 6DMAP, 3.4 to 6.6 mg/L of actinone CHX, 28 to 53 mug/L of phorbol PMA, 7.8 to 12.7 mug/L of inositol triphosphate, 1.5 to 2.4 g/L of SrCl2, 16 to 25 mg/L of heparin, and basic culture medium. The oocyte activating solution can effectively activate oocytes, and is suitable for activating the oocytes after ICSI in an in-vitro fertilization assisted reproduction technology.

The invention provides fibroblasts derived from multiple tissue sources of a native dog through primary isolated culture and an immortalization construction method of the fibroblasts, and belongs to the technical field of cell culture. Heart tissue, lung tip muscle tissue or leg muscle tissue of the native dog are used as materials and subjected to enzymolysis and separation of pancreatin and/or collagenase II, and myocardial fibroblasts, lung fibroblasts and myofibroblasts with typical fibroblast morphological characteristics are obtained. The myocardial fibroblasts, the pulmonary fibroblastsand the myofibroblasts which are subjected to primary isolated culture are transfected by SV40T virus liquid, and then puromycin screening culture and passage are performed to obtain immortalized myocardial fibroblasts, pulmonary fibroblasts and myofibroblasts. Compared with primary culture cells, the immortalized constructed cells are higher in growth speed and remarkably improved in activity, and a material basis is provided for dogs in the aspects of scientific research, disease research and the like.

The invention relates to a humanized intestinal cancer precancerous lesion immortalized epithelial cell line and a construction method and application thereof. The construction method comprises the following steps: firstly, transfecting primarily separated human colorectal adenoma polyp epithelial cells by using an SV40 overexpression lentiviral vector, then performing screening by using puromycin, and finally amplifying the screened cells to obtain the humanized intestinal cancer precancerous lesion immortalized epithelial cell line. The humanized intestinal cancer precancerous lesion immortalized epithelial cell line constructed by the invention overcomes the problems that conventional adenoma polyp cannot be subjected to passage in vitro, is low in cell proliferation and poor in cell activity, and cannot meet the cell passage requirement. According to the invention, the humanized intestinal cancer precancerous lesion immortalized epithelial cell line is established for the first time, an important cell experiment tool is provided for developing an in-vitro experiment of intestinal cancer precancerous lesions, and preclinical researches such as new drug screening and drug effectcomponents are also convenient to develop.

The invention relates to a TALEN and pMD18 vector-based site-directed mutagenesis system and its application. The system is composed of nuclease TALEN and a pMD18 vector, the nuclease TALEN is a transcription activator-like effect factor shearing a target gene target sequence, and the TALEN is composed of nuclease TALEN-L identifying the 5'-terminal of the target sequence and nuclease TALEN-R identifying the 3'-terminal of the target sequence; and the pMD18 vector is a pMD18 homologous recombinant template plasmid inserted into human AR site-directed mutagenesis gene, and the pMD18 vector is composed of corresponding upstream and downstream sequence of the target gene, and a puromycin screening marker. The system allows genome to be efficiently and accurately edited by using the homologous sequence provided by the Pmd18 vector during the cut-out of a double strand DNA molecule by the TALEN; and puromycin resistant gene is carried out when the introduced homologous sequence is adopted to edit the DNA sequence, so manual labor in the screening process is substantially reduced.

The invention discloses a method for quickly building a CRISPR gene editing liver cancer cell strain. According to the method disclosed by the invention, a CRISPR/Cas 9 technique is improved, recombinant plasmids better in expression efficiency are constructed, quick monoclone culture is combined, and a stable gene knockout liver cancer cell strain is constructed. Required sgRNA is accurately obtained through primer synthesis, an inserting fragment is synthetized through two-step PCR, a carrier is loaded, and recombinant plasmids for knockout are constructed; after slow viruses are packaged, the packaged slow viruses and liver cancer cells are co-incubated, and sgRNA and Cas9 proteins of equal quantity are transmitted into the liver cancer cells at the same time through slow virus mediating; and liver cancer cells after gene editing are subjected to puromycin resistance screening and monoclone culture, and finally, a gene knock-out positive stable liver cancer cell strain is quickly obtained. According to the method disclosed by the invention, important experimental materials are provided for researching a molecular mechanism of the gene in generation and development of tumors, andreference is provided for in vitro cell modeling of liver cancer diseases.

The invention relates to a method for constructing an MLH1 gene knockout cell line, and relates to the technical field of gene engineering. According to the method, a CRISPR/Cas9 system is used to prepare an MLH1 gene knockout cell line, firstly designing two sgRNA sequences for the MLH1 gene, and constructing recombinant plasmids by utilizing molecular cloning technology; then transfecting the recombinant plasmids to HeLa cells, verifying the activity of sgRNA through PCR, carrying out puromycin drug screening and monoclonal treatment, extracting genome DNA and total protein of the monoclonalcell strain, and carrying out MSH1 gene level sequencing identification and expression detection of protein level to obtain an MSH1 gene knockout HeLa cell line. The MSH1 gene knockout HeLa cell lineconstructed by the invention is a cell line with stable inheritance of genes. The method provided by the invention can be used for directional knockout of the MSH1 gene to cause functional inactivation of the MSH1 gene, which has the characteristics of simplicity, convenience, high efficiency, quickness, low cost and the like, and has important significance for research on functions and related pathways of the MSH1 gene.

The invention provides a construction method of a stable cell line realizing FTH1 gene controlled expression. The construction method comprises the following steps: (1) acquiring FTH1 genes; (2) transferring the FTH1 genes into a Tet-On vector to obtain recombinant plasmids; (3) constructing the recombinant plasmids into a lentiviral vector; (4) inoculating human neuroblastoma cells, after the cell fusion reaches 40-50%, adding a safe medium containing lentivirus liquid 0.8 [mu]l/ml (the titer is 1X10<9>TU/ml) and polybrene 5 [mu]g/ml, and polyethylene glycol 0.5-1 [mu]g/ml, and placing the safe medium into an incubator for culturing for 20-24 h; and (5) after cell passage, and the cell fusion reaches 50-60%, adding a medium containing 8 [mu]g/ml puromycin and treating for 7d, then adding a medium containing 4 [mu]g/ml puromycin and screening for 3d, and thus obtaining the stable cell line capable of inducing expression of the FTH1 genes, and the stable cell line is named SK-N-SH/Tet-on/FTH1. With the adoption of the construction method, the expression level and the expression time of FTH1 are accurately regulated, so that FTH1 is expressed when MR imaging is needed, and is turned off when MR imaging is not needed.

The invention discloses a method for preparing auto CCR5 deleted hematopoietic stem cells. The method comprises the steps that: CCR5 specific zinc finger nucleases (ZFNs) expression plasmids, and enhanced green fluorescent protein (EGFP) and puromycin recombinant plasmids are cotransfected into human induced pluripotent stem cells; human induced pluripotent stem cell CCR5 is specifically inactivated through gene insertion; and in vitro directional induction and differentiation are carried out, such that CCR5 deleted hematopoietic stem cells are obtained. The hematopoietic stem cells provided by the invention can be applied in the treatment of AIDS. With the hematopoietic stem cells provided by the invention, a novel method for treating AIDS stem cells can be provided.

The invention belongs to the fields of molecular biology and cytobiology, and specifically relates to a design and an application for a lentiviral expression vector. According to the vector designed by the invention, a CMV enhancer and a CMV promoter are used as transcription starting elements of an insertion gene; an EF1a-core promoter is adopted as a transcription starting element for screeningand marking puromycin and green fluorescent protein (GFP), and a puromycin resistance gene and a GFP sequence are connected and expressed in series through T2A; a lentiviral vector is adopted as a vector skeleton, and two above-mentioned elements are inserted into the skeleton vector; thus, the lentiviral expression vector is obtained by reasonable design and optimization. By adoption of the lentiviral vector designed by the invention, the inserted gene can be efficiently expressed, and the expression efficiency of the lentiviral expression vector is superior to the expression efficiency of conventional vectors with the same type of characteristics.

The invention discloses construction of a cell strain capable of stably expressing porcine circovirus type 2 ORF3 protein and an application thereof, and belongs to the field of biotechnology detection. The construction method comprises the following steps: (1) inserting a PCV2 ORF3 gene into multiple cloning sites of NheI and BamHI of a pLVZG-CMV-copGFP-Puro vector; (2) co-transfecting a 293T cell by using the constructed pLVZG-CMV-ORF3-copGFP-Puro recombinant plasmid and a virus packaging plasmid, culturing, discharging virus, collecting a virus supernatant, and filtering to obtain a recombinant lentivirus solution; and (3) infecting the packaged recombinant lentivirus with PK-15 cells, and screening and identifying through puromycin to obtain the cell strain for stably expressing the PCV2 ORF3 protein. According to the invention, a recombinant lentivirus system is utilized to prepare a cell strain which is fused with tag protein and can stably express PCV2 ORF3 protein, and the cell strain provides a platform for researching PCV2 pathogenesis and is beneficial to deep-level pathogenic mechanism research of PCV2.

The invention discloses a plasmid pcore-HU-SMAR, a framework vector of the plasmid pcore-HU-SMAR is plasmid PCMV-Gluc, a HiBiT-unaG fusion gene is inserted into the framework vector, the HiBiT-unaG fusion gene is connected with a puromycin resistance gene through a T2A sequence, an SMAR sequence is inserted between the puromycin resistance gene and a polyA sequence, and the HiBiT-unaG fusion gene is a fusion gene of a luciferase tag HiBiT and a fluorescent protein reporter gene UnaG. The invention further discloses a drug screening cell model of the targeted HBV core promoter, wherein the drug screening cell model is obtained by transfecting the AML12 with the plasmid pcore-HU-SMAR. The invention further discloses application of Tricirine and a structural analogue thereof to preparation of a medicine for inhibiting HBV core promoter transcription and/or HBV replication or application of the Tricirine and the structural analogue thereof to preparation of a medicine for treating hepatitis B.

The invention belongs to the field of biopharmacy and particularly relates to a method and a cell strain of expressing reteplase by human source cells. The method specifically comprises the followingsteps of genetically synthesizing a cDNA sequence which contains a secretory signal and encodes the reteplase, inserting the cDNA sequence into a lentiviral expression vector, constructing a lentiviral vector capable of expressing the reteplase with secretion, packaging lentiviral particles, infecting the human source cells 293F with the lentiviral particles, performing resistance screeing on a high-copy monoclonal cell strain capable of stably expressing the reteplase with puromycin carried in the lentiviral vector, testing the expression content and the enzymatic activity of the reteplase, and verifying the established expression method. The method and the cell strain have the advantages that the reteplase expression cell strain established by the method is human source 293F cells, doesnot contain pyrogen endotoxin, is suspended, stably expresses the reteplase, is easy in large-scale amplification as well as is less in impurity and easy to purify as expressing the reteplase with secretion.

The invention provides a construction method of immortalized forest musk gland epithelial cells. The construction method comprises the following steps: (1) collecting musk gland tissues of a male adult forest musk dead due to illness in a fragrance secretion period; (2) separating and purifying the forest musk gland epithelial cells and fibroblasts of the forest musk gland tissues to obtain purified lindel gland epithelial cells; (3) carrying out lentivirus transfection on the purified forest musk gland epithelial cells to obtain transfected forest musk gland epithelial cells; (4) screening out successfully transfected forest musk gland epithelial cells from the transfected forest musk gland epithelial cells through puromycin; and (5) carrying out multiplication culture on the screened successfully transfected forest musk gland epithelial cells to obtain the immortalized forest musk gland epithelial cells. According to the construction method of the immortalized forest musk gland epithelial cells, the immortalized forest musk gland epithelial cells are immortalized in a lentivirus transfection mode, the characteristics of the epithelial cells can be completely kept after 12 generations of culture, and the defect of epithelial cell apoptosis is overcome.

The invention discloses a method for preparing a porcine kidney epithelial cell line for stably expressing BE4 protein, which comprises the following steps: by using porcine kidney epithelial cells (PK15) with good passage as host cells, co-transfecting a piggyBac transposon (PB-BE4max) vector containing a BE4 fusion protein sequence and a transposase expression vector plasmid, screening a positive cell population by puromycin, and selecting a monoclonal cell line; and verifying the editing activity amplifying the characteristic fragment of BE4 and transfecting active sgRNA, so as to obtain the transformant for stably expressing BE4 protein. The porcine kidney epithelial cell line prepared by the method can stably express BE4 protein, and has important application value in the porcine genetic engineering fields of porcine genome function research, transgenic pig preparation, drug-resistant target screening and the like.

The invention discloses a method for constructing a breast cancer MCF7 cell line capable of stably over-expressing exogenous recombinant plasmid pCDH-puro-PRMT7. The full length of a PRMT7 gene is obtained; the pCDH-puro-PRMT7 plasmid is constructed; a virus is packaged, and cells are infected by the virus; puromycin is screened; and the MCF7 cell line capable of stably over-expressing the PRMT7 is obtained after amplification. The construction of the cell line is good for researching the cell biological functions of the PRMT7, such as the influences of the PRMT7 on the tumorigenic abilities of mice and the influences on the pulmonary metastasis abilities of the mice.

The invention discloses a transgenic engineering cell strain and a method for determining a glucocorticoid mixture based on the cell strain. According to the cell strain, a green fluorescent protein GFP and a glucocorticoid receptor GR fusion protein gene are integrated into a Hela cell chromosome in a lentiviral infection mode, puromycin puro screening is conducted, and finally the Hela cell strain stably expressing GFP-GR is obtained. Based on a Hela cell strain for stable expression of GFP-GR, by measuring a migration ratio of a GFP-GR binding hormone ligand migrated from a cytoplasm regionto a cell nucleus region, the positive detection rate of the glucocorticoid mixtures in different samples can be rapidly detected in high flux and low cost, the sensitivity can reach 10<-9> mol/L interms of dexamethasone, the content of the glucocorticoid mixture in the environment and food can be rapidly screened, the harm degree is clear, and risk prevention and control are guided.

The present invention belongs to the field of protein detection and particularly relates to a culture medium and a method for evaluating embryo quality by using the culture medium. The culture mediumcomprises fetal bovine serum proteins, an insulin-transferrin-selenium additive and puromycin. The method for evaluating the embryo quality by using the culture medium successively comprises the following steps of embryo culturing and embryo conditioned culture solution collecting, embryo conditioned culture solution thawing, endometrial stromal cell culturing, embryo conditioned culture solutionadding and protein detecting, differential protein of specific cell secreted protein is obtained and the embryo quality is evaluated. The evaluating method is very effective and accurate and makes upfor gaps in the market.

The invention provides a lentivirus-based porcine SPP1 gene vector as well as construction and application thereof and belongs to application technologies in the technical field of biology and modern agriculture. The porcine SPP1 gene vector of a recombinant lentivirus comprises an ampicillin resistance gene AmpR sequence; a prokaryotic replicon pUC Ori sequence; a virus replicon SV40 ori sequence; a WPRE-enhanced-type marmot hepatitis B virus post-transcriptional control element which is used for enhancing gene expression efficiency; a puromycin resistance gene Puro sequence; a T2A "self-cleavage" peptide sequence; a fluorescent protein expression tag CopGFP sequence; a lentivirus packaging cis element cis; a human cytomegalovirus (CMV) promoter; and a porcine SPP1 gene sequence, wherein the lentivirus packaging cis element cis adopts a third-generation lentivirus vector. The vector can directly infect eukaryotic cells, so that the purpose of co-expression of a porcine SPP1 gene in the eukaryotic cells is achieved, and a foundation is laid for further research on functions of the porcine SPP1 gene.

The invention discloses a suspension Marc145 cell line for expressing porcine CD163 and CD169 molecules and a preparation method thereof, and belongs to the technical field of transgene. The method briefly comprises the steps of 1) recombinant plasmid construction; 2) Marc145 cell transfection with liposome; 3) puromycin screening; 4) serum-free medium suspension culture and domestication. The cell line prepared by the method is very sensitive to porcine reproductive and respiratory syndrome virus, can be used for amplifying and breeding a large amount of porcine reproductive and respiratory syndrome viruses, is relatively low in cost, and has bright application prospects in vaccine production practice.