109 results about "Puromycin" patented technology

The invention mainly belongs to the technical field of gene editing and particularly relates to an SSA (single-strand annealing) repair-based gene seamless editing method utilizing a CRISPR / Cas9 technology. According to the gene seamless editing method, CRISPR / Cas9 expression vectors CCR5.CRISPR / Cas9 and eGFP.CRISPR / Cas9 targeting CCR5 and eGFP are constructed firstly, a donor CCR5-Donor vector is constructed, a CCR5-Donor plasmid vector adopts the pXL-BACII-L-arm-CAG-TK-PGK-Puro<R>-T2A-eGFP-bGH pA-R-arm structure, L-arm and R-arm are left and right homologous arms, and CAG-TK-PGK-Puro<R>-T2A-eGFP-bGH pA between L-arm and R-arm is a screening marking component; through two times of transfection, seamless editing is completed by using HR and SSA repair mechanisms in the CRISPR-Cas9 system and cells through positive and negative screening of puromycin and ganciclovir of the cells.

The invention provides a humanized PD-L1 tumor cell line MC-38-hPD-L1, a builtanimal tumor model with the same and a method for constructing the humanized PD-L1 tumor cell line. The method particularly includes knocking out animal-origin PD-L1 by the aid of CRISPR-CAS9; carrying out amplification and cultivation to obtain knocked-out cell banks; extracting DNA (deoxyribonucleic acid) and carrying out PCR (polymerase chain reaction) amplification; recycling and cloning amplification products; carrying out over-expression on human-origin PD-L1 in MC-38 cell lines of mPD-L1 KO by the aid of lentivirus systems; packaging lentivirus and screening Puromycin to obtain the humanized MC-38 cell line of PD-L1. The humanized PD-L1 tumor cell line, the animal tumor model and the method have the advantages that as shown by results, high killing efficiency and multiplication capacity are obviously presented by tumor infiltration CD8 T lymphocytes after antibody treatment is carried out, tumor infiltration Treg cells can be obviously inhibited after antibody treatment is carried out, and accordingly the method is proved to be effective and feasible from the aspect of molecular mechanisms.

This invention relates to pork liver cell immortalities and its producing method. Procedures are showed: high live ratio fresh original pork liver cell is got by dispase-collagenase perfusion method; it is cultured for 24 hours. Then cell upper heat removing of recombination retronituse that contains SV40 big T antigen is used to infect the pork cell under condition of polybrene which concentration is 8ug / ml. Then it is medicine pressure filtrated by 500ug / ml G418 after one week, single clone cell is picked for large culture when the cell clone appears and grows to 1.0-2.0cm to get pork cell that can passage. Reinfection is done under condition of 8ug / ml polybrene. Then it is medicine pressure filtrated by 2ug / ml puromycin after one week. Single clone cell is picked for large culture when the cell clone appears and grows to 1.0-2.0cm to get immortality pork cell.

The invention discloses a method for obtaining transgenic bovine fetal fibroblast by using Cas9 cutting nuclease-mediated Ipr1 fixed point insertion. The bovine fetal fibroblast is cotransfected by adonor vector and a CRISPR / Cas0 expression vector for a target locus through an electroporation method, an MSR1 promoter in the donor vector can realize specific expression in full-time phagocytic cells, and after puromycin drug screening, the targeted positive clone cell is obtained through PCR authentication. The transgenic positive clone cell is used as a donor cell so as to obtain a transgenicclone embryo, the transgenic clone embryo is further transplanted into the uterus of a rutting acceptor calf, and finally a living Ipr1 fixed point insertion transgenic cloned calf is obtained.

The invention discloses a stable knockout single plasmid vector with coordination of transposon and a CRISPR / Cas9 system and application of stable knockout single plasmid vector and belongs to the field of gene engineering. The single plasmid vector is a double-stranded circular plasmid containing an IRDR-L-IRDR-R box, and the IRDR-L-IRDR-R box comprises an IRDR-L sequence, a promoter, a gRNA scaffold sequence, a Cas9 protein sequence, a resistance screening gene sequence and an IRDR-R sequence. The single plasmid vector provided by the invention can realize expression of sgRNA and a Cas9 protein only needing once construction and once transfection. A method is simple in process, is efficient and rapid, greatly simplifies a plasmid construction flow, shortens an experiment period and improves the working efficiency. The vector provided by the invention carries a transposase recognition sequence, does not use a virus, can conveniently, rapidly and safely establish a gene knockout stablecell line and is beneficial to screening stable cell lines by comprising the puromycin screening resistance.

The invention relates to a method for achieving HMGCR gene knockout based on the CRISPR / Cas9 technology. The method is characterized in that two CRISPR / Cas9 target sequence aiming at the HMGCR gene isdesigned, a gRNA single chain is synthesized in vitro, annealing is performed to obtain two gRNA double-chain DNA target insertion fragments, the insertion fragments are inserted into PX459 (pSpCas9(BB)-2A-Puro)V2.0 vectors to obtain the two different-locus plasmids of the target HMGCR gene; the two plasmids are transfected into PK15 cells, puromycin is used to process the cells, the processed cell genome DNA is extracted to perform PCR amplification, the PCR product is denatured, annealing is performed, and then T7E1 is used to perform HMGCR gene knockout identification. The method has the advantages that method can be used for analyzing the expression conditions of sequence and mRNA after the HMGCR gene knockout, whether an off-target phenomenon exists or not can be verified by using amethod combining PCR and T7E1 enzyme treatment, and accordingly the specificity based on target sequence HMGCR-gRNA can be determined; the method is applicable to cell and animal models to achieve fixed-point HMGCR gene knockout, has a reference value to the knockout of other genes, and is good in effect, simple, economical, short in time and the like.

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.

Synthesis of many proteins is tightly controlled at the level of translation and plays an essential role in fundamental processes such as cell growth and proliferation, signaling, differentiation or death. Methods that allow imaging and identification of nascent proteins allow for dissecting regulation of translation, both spatially and temporally, including in whole organisms. Described herein are robust chemical methods for imaging and affinity-purifying nascent polypeptides in cells and in animals, based on puromycin analogs. Puromycin analogs of the present invention form covalent conjugates with nascent polypeptide chains, which are rapidly turned over by the proteasome and can be visualized and specifically captured by a bioorthogonal reaction (e.g., [3+2]cycloaddition). The methods of the present invention have broad applicability for imaging protein synthesis and for identifying proteins synthesized under various physiological and pathological conditions in vivo.

The invention relates to a building method of a cell strain for stably expressing NS1 (non-structural 1) protein. An NS1 gene is inserted in an EcoR I polyclone site of a pLenti-CMV-EGFP-3Flag-PGK-Puro carrier; a built pLenti-CMV-NS1-EGFP-3Flag-PGK-Puro recombinant plasmid and a virus packaging plasmid are used for 293T cell cotransfection; culture, toxin elimination, virus liquid supernatant collection and filtering are performed to obtain recombinant lentivirus liquid. Packaged recombinant lentivirus infection A549 cells are subjected to puromycin screening, immunofluorescence and Western blot identification to obtain the cell strain for stably expressing NS1 protein. A recombinant lentivirus system is used for preparing the cell strain fusing tag protein capable of stably expressing the NS1 protein. The cell strain provides a good tool for studying the biological function of the NS1 protein.

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 belongs to the field of biotechnology and particularly relates to a method for constructing a stable cell strain secreting an expression protein. The method comprises the following steps: with a recombinant lentiviral vector pCDH-S-His as a skeleton, constructing a target protein gene to the recombinant lentiviral vector pCDH-S-His to obtain a recombinant plasmid pCDH-S-P-His; co-transfecting the recombinant plasmid and helper plasmids pSPAX2 and pMD2.G with 293T cells by using a PEI transfection method to obtain a recombinant lentivirus; transducing the obtained lentivirus to the 293T cells, and performing puromycin screening to obtain a plurality of monoclonal cell strains; detecting the content of target protein in each cell supernatant by an Elisa method; determining a high-expression cell strain. Compared with a traditional plasmid transfection method, the method provided by the invention has the advantages of short experimental period, high positive rate and good stability of the cell strain.

This invention is for an improved process to co-encapsulate hydrophobic drugs and hydrophilic drugs in phospholipid liposomes. Non-toxic supercritical or near-critical fluids with / without polar cosolvents are utilized to solubilize phospholipid materials and hydrophobic drugs, and form uniform liposomes to encapsulate hydrophobic drugs and hydrophilic drugs.DNA topoisomerase I (Top1) is the target of camptothecin, and novel Top1 inhibitors are in development as anticancer agents. Top1 inhibitors damage DNA by trapping covalent complexes between the Top1 catalytic tyrosine and the 3′-end of the broken DNA. Tyrosyl-DNA phosphodiesterase (Tdp1) can repair Top1-DNA covalent complexes by hydrolyzing the tyrosyl-DNA bond. Inhibiting Tdp1 has the potential to enhance the anticancer activity of Top1 inhibitors and to act as antiproliferative agents. It has been recently reported that neomycin inhibits Tdp1 more effectively than the related aminoglycosides paromomycin and lividomycin A. Inhibition of Tdp1 by neomycin is observed both with single- and double-stranded substrates but is slightly stronger with duplex DNA, which is different from aclarubicin, which only inhibits Tdp1 with the double-stranded substrate. Inhibition by neomycin can be overcome with excess Tdp1 and is greatest at low pH. Aminoglycoside antibiotics and the ribosome inhibitors thiostrepton, clindamycin-2-phosphate, and puromycin are the first reported pharmacological Tdp1 inhibitors. The development of Tdp1 inhibitors as anticancer agents can be envisioned as combinations of Tdp1 and Top1 inhibitors. Moreover, Tdp1 inhibitors might also be effective by themselves as anticancer agents. In addition, Tdp1 inhibitors might be valuable as anti-infectious agents.This invention can produce a co-encapsulated combination drug product consisting of a topoisomerase 1 inhibitor such as camptothecins including neat camptothecin and its derivatives irinotecan, topotecan and other derivatives, and a tyrosyl-DNA phosphodiesterase (Tdp1) such as aminoglycoside antibiotics including neomycin and tetracycline, and the ribosome inhibitors thiostrepton, clindamycin-2-phosphate, and puromycin.

The invention relates to bioengineering technologies, and particularly relates a method of inducing differentiation of neural stem cells into dopaminergic neurons by using recombinant slow virus. The method comprises the following steps: constructing a recombinant slow virus vector; separating, culturing and identifying the neural stem cells; transfecting the recombinant slow virus into NSCs; identifying the NSCs transfected by the recombinant slow virus, wherein the process of transfecting the recombinant slow virus into the NSCs comprises the following steps: digesting NSCs after being subjected to three times of passages to prepare a single-cell suspension, and inoculating into a culture plate; transfecting the TH+Brn4 gene recombinant slow virus; adding the slow virus and ploybrene, uniformly shaking, putting the culture plate into an incubator to culture for 1-2 hours, and replacing fresh differential culture medium; meanwhile, screening by using puromycin to obtain stable transfected cells. The method has the advantages as follows: by constructing the slow virus vectors of target genes TH and Brn4, exogenous genes after transfecting the NSCs can be stably expressed in cells; high proportion of TH positive dopaminergic neuron is generated after differentiation; the Brn4 can promote high expression of neurotrophic factors and has the function of inhibiting apoptosis.

The invention belongs to the technical field of cell research, and particularly relates to a method for constructing a laryngeal cancer cell line for stably expressing the green fluorescent protein. The method comprises the following steps: (1) designing and synthesizing a primer; (2) digesting the gene sequence of pLenti-puro carrier plasmids with BamHI and XhoI according to the gene sequence andpLenti-puro sequence of the green fluorescent protein, and connecting digested products to obtain a target carrier pLenti-puro-GFP; (3) verifying the carrier pLenti-puro-GFP with endotoxin extraction, digesting the pLenti-puro-GFP plasmids with BamHI and XhoI, and detecting the gene sequence by agarose gel electrophoresis, and sequencing the carrier pLenti-puro-GFP plasmids to detect the sequencesituation of the GFP-LC3; and (4) co-transfecting a 293T cell with the carrier pLenti-puro-GFP plasmids and a lentivirus package carrier, collecting virus supernatant, and transfecting a laryngeal cancer cell Hep-2 with the virus supernatant, infecting for 24 hours, then adding a culture medium containing puromycin, screening the cell, and observing under high content until a stable strain is obtained.

The invention discloses a cell line for stable expression of a peste des petits ruminants virus receptor Nectin-4 and a construction method of the cell line. According to the invention, by virtue of asynthetic Nectin-4 gene and on the basis of a lentiviral vector system, a recombinant plasmid pLOV-eGFP-Nectin-4 is constructed. The recombinant plasmid, together with pSPAX2 and pMD2.G plasmid, is applied to co-transfection of a 293T cell, so that retrovirus-like particles are obtained. A supernatant-infected MDBK cell is cultivated by virtue of cells containing the retrovirus-like particles, and puromycin screening and purifying are conducted, so that the MDBK cell for stable expression of Nectin-4 protein is obtained, and the MDBK cell is named as MDBK-Nectin-4. Through the construction ofthe cell line, an experimental material is provided for deep research of an interaction between PPRV and the receptor Nectin-4 and for the clinical rapid isolation of the PPRV is provided.

The invention provides a saliva preservation solution, and a preparation method and application thereof. The saliva preservation solution comprises a DNA stabilizer including disodium edentate, tartaric acid and at least one selected from a group consisting of nitrilotriacetic acid and dihydroxyethylglycine; a microbial inhibitor including penicillin, streptomycin, geneticin and at least one selected from a group consisting of potassium sorbate, sodium benzoate, ethylparaben sodium, ketoconazole and puromycin; and a buffer solution including a TE buffer solution and at least one selected from a group consisting of a PBS buffer solution and a sodium chloride glucose buffer solution. According to the invention, through preparation of the saliva preservation solution, the morphology of cells in a saliva swab is maintained and prevented from disruption, and the growth of microorganisms in the saliva swab and the saliva preservation solution is inhibited.

The invention relates to building and application of a chronic virus transformation vector with CMV-CBh double promoters. The chronic virus vector pLenti-CMV-3FLAG-EGFP-tCBh-mCherry-T2A-Puro is obtained by replacing PGK in the pLenti-CMV-3FLAG-EGFP-tCBh-mCherry-T2A-Puro with tCBh fragments obtained by performing PCR (Polymerase Chain Reaction) amplification. The tCBh promoter is used for driving expression of mCherry gene and Puromycin resistance gene, and cells for stabilizing over-expression of target gene can be screened by using Puromycin; the mCherry gene is beneficial for judging the virus packaging efficiency and the infection efficiency, and is also beneficial for stabilizing the screening of the cells. Transfection fluorescence observation proves that the chronic virus vector expression system with the double promoters is successfully built, and a basic vector is provided for further researching target genes of interest.

The invention belongs to the technical field of zoobiology, and relates to an establishment method of a positive screening system based on gene knockout cells; on the basis of the established system,p53 and MAD2 genes are knocked out in NIH / 3T3 cells, and the cells, which undergo gene knockout, are subjected to positive screening. According to the establishment method, target sequences of the sixth exon of the p53 gene and the first exon of the MAD2 gene are subjected to site-directed knockout in an NIH / 3T3 gene line, and in the combination with a green fluorescent protein gene and a puromycin gene, the cells, which undergo p53 and MAD2 gene knockout, are subjected to positive screening; and by virtue of the established positive screening system, the p53 and MAD2 genes are simultaneouslyedited in living mouse liver, so that a mouse liver cancer model is finally obtained. The method provided by the invention lays a foundation for related researches on editing the p53 and MAD2 genes ina living mouse.

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 discloses an Luc-GFP marked high-transitivity human hepatoma cell line. Hepatoma cells are taken as mother cells, a virus solution packaged with pCDH-CMV-Luc-EF1-GFP-Puro is transfectedthrough slow viruses, and then through puromycin screening, the high-transitivity human hepatoma cell line with stable GFP expression and luciferase activity is obtained. By using the Luc-GFP marked high-transitivity human hepatoma cell line, an in situ hepatoma model for a naked mouse is established through a cell suspension method and a tumor tissue block conversion method respectively, the tumor formation rate of the hepatoma model is 100% respectively, the rates of lung metastasis are 100% and 80% respectively, a foundation is laid for monitoring the growth of tumor in the level of livinganimals and evaluating the curative effect of hepatocellular carcinoma metastasis resistant medicine, and the cell line has greater application prospect.

The invention discloses a DLD1 stable cell strain efficiently expressing human PKM1 protein and a construction method thereof, and belongs to the field of animal cell strains. The construction method comprises the following steps of: constructing a recombined human PKM1 gene lentiviral expression vector; packaging and harvesting lentivirus; transfecting DLD1 cell; and obtaining the stable cell strain through puromycin screening, RT-PCR and Western Blot technological detection. The stable cell strain constructed according to the method provides a powerful tool for searching colon cancer Wnt signal path and tumor energy metabolism.

The invention relates to a novel human pancreatic cancer nude mouse model construction method for living imaging research and a use thereof. A human pancreatic cancer PANC-1 cell is stably transfected with a plasmid carrying a luc gene through a Lonza nuclear transfection system, a cell strain PANC-1-LUC for stable luciferase expression is screened through puromycin and PANC-1-LUC nude mouse transplant subcutaneous sarcoma is further constructed. An in vitro and vivo bioluminescence detection result proves that the PANC-1-LUC cell strain can stably express luciferase for a long time. Compared with a lentiviral vector-mediated bioluminescence pancreatic cancer nude mouse model, the built pancreatic cancer nude mouse model has a short tumor formation incubation period and a high tumor formation rate, satisfies PANC-1 transplantation tumor growth features and is suitable for living imaging research.

The invention discloses a method for easily screening and obtaining a target gene knock-out cell line by using a CRISPR / Cas9 technology, and a product of the method. According to the method, small guide ribonucleic acid (sgRNA) of a target exon is designed aiming at an exon of a target gene-Nedd 4 gene, a stable knock-out Nedd 4 gene can be obtained by using the CRISPR / Cas9 technology, puromycin or blasticidin screening and Western blot identification, and the cell line which can be screened by using puromycin or blasticidin is obtained. The method combines a PX330 carrier (a CRISPR / Cas9 carrier) and a Tiall carrier for use so as to obtain the target gene knock-out cell line; therefore, an effective tool is provided for researching the functions and action mechanisms of genes.

The invention discloses a vector for genetic drug carrying of a targeting exosome. The vector comprises a CMV7 promoter, a selective marker Puromycin and a resistance gene. An EGFP-F5 / 8 type C sequence shown as SEQ ID NO.1 is added at a multiple cloning site between the CMV7 promoter, a selective marker Puromycin polyclonal site as is added between the CMV7 promoter and the selective Puromycin marker. In addition, the invention also discloses an establishment method and application of the vector. The vector makes the exosome carry exogenous genes to the maximum extent for delivery of gene drugs. The gene sequence of the vector is smaller, the carrying capacity of the gene can be improved, and the selection range of exogenous genes is widened. The fluorescence effect of the vector is stronger, and obvious and intuitive observation can be achieved.

The invention discloses a construction method of a human APP promoter-containing Luci cell line, and the specific method is as follows: construction of an expression vector containing a human APP promoter region-luciferase chimeric gene; cotransfection of constructed pGL3-APPP Luc-Puro plasmids and pRL-SV40 plasmids into a human embryonic kidney cell line HEK-293T cell line; laying and planting of transfected HEK-293T cells after trypsinization into a 10cm culture dish, puromycin pressurization screening to obtain five cell lines with genomes capable of stably integrating the human APP promoter region-luciferase chimeric gene, and detection of activity of firefly luciferase and renilla luciferase in the five cell lines to obtain the cell line containing the human APP promoter region-luciferase chimeric gene. The cell line prepared by the cell line construction method can be used for screening of new anti senile dementia compounds and therapeutic drugs for inhibiting the expression of human APP.

Disclosed is a class of reagents for examining protein expression in vivo that does not require transfection, radiolabeling, or the prior choice of a candidate gene. Further, a series of puromycin conjugates was constructed bearing various labeling moieties. These conjugates were readily incorporated into expressed protein products in cell lysates in vitro and efficiently cross cell membranes to function in protein synthesis in vivo as indicated by flow cytometry, selective enrichment studies, and western analysis. The present invention demonstrates that labeled-puromycin conjugates offer a general means to examine protein expression in vivo.

The invention discloses a CHO cell line with GS gene knockout and a preparation method and application thereof. The CHO cell line has no GS protein expression, no puromycin resistance and no integration of a Cas9 gene and an sgRNA sequence into a cell genome. A GS gene is knocked out by means of a CRISPR / Cas9 method, and the CHO cell line is obtained by optimizing a screening method. The providedpreparation method can rapidly realize GS- / - editing of CHO cells, and the CHO GS- / - cell line with GS gene knockout and no puromycin resistance and no integration of the Cas9 gene and the sgRNA sequence is screened out. The cell line is applied to recombinant protein expression without an MSX-mediated gene amplification process, so that the efficiency of screening high-yield cell lines is greatlyimproved.

The present invention discloses a shRNA inhibiting SRSF6 gene expression, and a lentivirus expression vector and a construction method thereof, wherein the shRNA sequence is F:5'-CCGGCGTACAGAATACAGGCTTATTCTCGAGAATAAGCCTGTATTCTGTACGTTTTTG-3' and R:5'-AATTCAAAAACGTACAGAATACAGGCTTATTCTCGAGAATAAGCCTGTATTCTGTACG-3'. According to the lentivirus expression vector, the shRNA is inserted into vector plasmid PLKO.1 to construct PLKO.1-SRSF6-shRNA, the PLKO.1-SRSF6-shRNA and packaging plasmids PSPAX2 and PMD2. G are transfected into HEK293T cells after vector sequencing verifying is correct to obtain a lentivirus suspension, the lentivirus suspension is used to infect colorectal cancer cells, and then puromycin is used to screen and verify whether the virus packaging is successful. According to the present invention, the Western Blot results verify that the expression level of SRSF6 can be significantly reduced; the proliferation ability and the migration invasion ability of the colorectal cancer cells can be significantly reduced; and the research on the effect of SRSF6 on tumor cell migration and invasion ability is firstly provided.