18 results about "Puromycine" patented technology

The invention provides a construction method of a non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model. The construction method comprises the following steps: designing mutation primers H1.3 R80A-F and H1.3 R80A-R; the method comprises the following steps: by taking a pCDH-HA-H1.3-Flag plasmid as a template, carrying out PCR (Polymerase Chain Reaction) amplification by adopting a mutation primer, digesting a product by DMT enzyme, and converting a competent cell to obtain a mutant plasmid; and co-transfecting the mutant plasmid and a helper plasmid to a packaging cell, collecting a virus solution, filtering, infecting an A549 cell, adding Polybrene to assist infection, culturing, and screening a stably transfected cell strain by using puromycin to obtain the recombinant plasmid. The invention also provides application of the mutant cell model obtained by the construction method. According to the invention, the blank of histone H1.3 methylation research is filled, the 80th arginine is clear as a core modification site, and the established model provides a new tool for lung cancer mechanism research and drug research and development.

The invention belongs to the technical field of gene engineering, and particularly relates to a construction method and application of a recombinant vector based on bCoro1a gene 3 'UTR editing. The recombinant vector consists of an upstream homologous arm of a targeting site, 3 'UTR inserted into a target sequence, 200bp of a nucleotide tailing signal of bCoro1a, a selection marker and a downstream homologous arm of the targeting site; the targeting site is the 29 or 70 site of the 3 'UTR of the bCoro1a gene; the sequence of the 3 'UTR of the bCoro1a gene is as shown in SEQ ID NO. 48; the selection marker comprises enhanced green fluorescent protein and puromycin, the bCoro1a 3 'UTR edited gene expression regulation and control system is based on bCoro1a 3' UTR edited gene expression regulation and control system, and the expression level of bCoronin-1A is reduced in a targeted manner by inserting a specific bta-miR-27b target sequence into 3 'UTR, so that M.tb immune escape is inhibited, and the M.tb infection resistance of a host is improved.

The invention discloses a homologous recombination system, a transposition system, a fruit fly animal gene high-throughput screening method and application. The screening method comprises the following steps: firstly, separating and establishing a stable cell line from a fruit fly animal; then co-transfecting a CRISPR / Cas9 mediated homologous recombination system, and obtaining a cell line for stably expressing Cas9 protein through puromycin screening and a limited dilution method; the sgRNAs plasmid library constructed by combining a PiggyBac transposon system can realize efficient screening and functional analysis of a target gene in the Cas9 cell line. The high-throughput screening method provided by the invention is widely applicable to gene function research and target gene screening, is simple in preparation process and convenient to operate, has good universality, and provides effective technical support and solution for related fields.

The invention belongs to the field of gene engineering, and particularly relates to construction of an RNF31 gene knockout cell line and application of the RNF31 gene knockout cell line as a foot-and-mouth disease virus vaccine production cell line. The method comprises the following steps: firstly, designing two sgRNA sequences at an RNF31 exon by utilizing a CRISPR / Cas9 technology, and connecting with a pX459-puro vector to construct a recombinant plasmid; the method comprises the following steps: transfecting a PK-15 cell with pX459-RNF31-sgRNA, and screening under the action of puromycin, so as to obtain the RNF31 gene knockout cell line. Compared with a wild type cell, the RNF31 gene knockout cell line remarkably promotes FMDV replication and can be used as a production cell line of a foot and mouth disease virus vaccine; and data support is provided for further researching a mechanism of inhibiting FMDV replication by RNF31.

The invention relates to the field of biomedical research, and provides a lung cancer cell strain (H1299) capable of stably and low-expressing Integrin alpha6 protein and a construction method of the lung cancer cell strain (H1299). The preparation method comprises the following steps: designing and synthesizing an shRNA (short hairpin Ribonucleic Acid) sequence for specifically knocking down gene expression aiming at a human Integrin alpha6 gene, forming a double-chain fragment through annealing, and inserting the shRNA sequence into a pLKO.1-puro lentiviral vector by adopting a connection independent cloning (LIC) method, so as to construct a pLKO.1-ITG alpha6-shRNA recombinant plasmid. Furthermore, the recombinant plasmid and lentivirus packaging plasmids psPAX2 and pVSVG are co-transfected to an HEK293T cell by utilizing a lentivirus packaging system, and the lentivirus is produced by packaging. And infecting a target lung cancer cell strain H1299 by using the obtained lentivirus, and then carrying out puromycin resistance screening and continuous subculture to finally obtain the stable and continuous low-expression Integrin alpha6 protein H1299 cell strain. A fluorescence microscope, a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) and an immunoblotting technology are used for verifying that the gene silencing efficiency of the Integrin alpha6 in the cell strain can reach more than 80%. The stable cell strain constructed by the invention provides a reliable and efficient tool cell model for researching the molecular mechanism of the Integrin alpha6 in the processes of occurrence and development, invasion and metastasis, energy metabolism, immune escape and the like of lung cancer, and also provides a new experimental material for screening antitumor drugs targeting the Integrin alpha6.

The invention provides a low-autophagy Vero cell line based on a DDIT3 inhibition gene as well as a construction method and application of the low-autophagy Vero cell line. The cell line stably knocks down DDIT3 expression through lentivirus-mediated shRNA, the autophagy level under the condition of nutrition deficiency is remarkably reduced, the cell proliferation capacity is enhanced, and the yield of influenza A H1N1 viruses is remarkably increased. The construction method comprises the following steps: cloning shRNA (short hairpin ribonucleic acid) to a GV493 lentiviral vector, co-transfecting 293T cell packaging virus with psPAX2 / pMD2. G helper plasmid, infecting Vero cells, and screening through puromycin to obtain a stable cell line. The cell line solves the problem of growth stagnation caused by over-activation of autophagy in serum-free suspension culture of traditional Vero cells, and is suitable for large-scale vaccine production.

The invention relates to the technical field of gene engineering technology, in particular to a preparation method of an efficient and stable gene knockout cell line. According to the AKAP11 gene knockout cell line provided by the invention, the 6th exon of the AKAP11 gene in the cell line is subjected to 3395bp frameshift deletion mutation, so that a coding frame is shifted, and protein translation is terminated in advance; after continuous subculture, the homozygous knockout phenotype is still maintained through PCR sequencing verification. A CRISPR / Cas9 system is combined with puromycin screening, the positive cloning rate reaches 30% or above, the construction period is shortened to 45 days, the constructed cell line can be directly used for AKAP11 related cell signal path research, disease model construction and targeted drug screening, and a reliable tool is provided for basic research and clinical transformation.

There are described compositions comprising an effective amount of a combination of two or more components, said components selected from acacetin, ACTI peptide, alpha-lipolic acid, alprostadil, anisomycin, apigenin, ascorbic acid, astragalus, berberine, β-lapachone, β-hydroxy-beta-methyl-butyrate, Bacopa monnieri, catechin, catechol, chamomile, chrysin, coumestrol, curcumin, dinitrophenol, dinoprost, ellagic acid, (−)-epigallocatechin gallate, green tea extract, fisetin, genistein, ginsenoside RE, glabridin, 18-α-glycyrrhetinic acid, 18-β-glycyrrhetinic acid, glycyrrhizin, hydroquinone, isoquercitrin (EMIQ), kaempferol, kuromanin, leucine, lithium, luteolin, luteolin, luteolinidin, melatonin, menadione, 1-methylnicotinamide (MNA), methyl salicylate, myricetin, nadide, niacin (vitamin B3), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid adenine dinucleotide (NaAD), nicotinic acid mononucleotide (NaMN), parsley (Petroselinium crispum), phenylephrine, pokeweed mitogen, 15-Δ prostaglandin J2, puromycin, quercetin, quinolinic acid, retinoic acid, trichostatin A, troxrutin, rutin, tryptophan, vitamin D3, withaferin A, wortmannin and zinc (including salts thereof).

The invention belongs to the technical field of biology, and particularly discloses a cell line capable of stably expressing an nsP4 gene and application of the cell line in preparation of a replication-deficient chikungunya virus. The inventor uses retrovirus for expressing CHIKV nsP4 to infect hamster kidney cells (hamster kidney cells), after puromycin screening, verification of passage stability of monoclonal cells, protein expression stability, replication-deficient virus titer and the like is carried out, finally, a cell line which can be used for continuous passage of CHIKV replication-deficient viruses and has high titer is obtained, and the preservation number is CCTCC NO: C2025281. The replication-deficient virus CHIKV-delta nsP4 generated by the RNA of the nsP4 gene deleted from the trans-complementary CHIKV of the cell line has good safety, immunogenicity and immune protection efficacy, can effectively resist the challenge infection of wild viruses, and can be used for preparing a safe and effective CHIKV novel vaccine candidate strain.

The invention discloses a stably transfected cell strain for detecting activity of Wnt and / or R-spondin family cytokines as well as a construction method and application of the stably transfected cell strain, and belongs to the technical field of biological medicines. Comprising the following steps: preparing target plasmids: respectively connecting a SuperTOP sequence and a SuperFOP sequence to two different expression plasmids, and respectively connecting luciferase reporter genes to obtain a target plasmid A and a target plasmid B; respectively transfecting the target plasmid A and the target plasmid B into human kidney epithelial cells, culturing in a puromycin-containing culture medium, and sorting positive cells according to labeled fluorescence GFP (Green Fluorescent Protein) to obtain stably transfected cells A and stably transfected cells B. The method has the advantages that activity detection of Wnt family and R-spondin family cell factors is carried out on stably transfected cells, operation is simple, the detection period is shortened by 25%, sensitivity is improved by at least 50%, and the method has high application prospects and economic value.

The invention discloses an immortalized egg shell gland epithelial cell line and application thereof. The invention belongs to the technical field of biology, and particularly relates to an immortalized egg shell gland epithelial cell line and application thereof. The construction method of the immortalized egg shell gland epithelial cell line comprises the following steps: 1) obtaining SV40 overexpression lentivirus; 2) transfecting the lentivirus obtained in the step 1) to chicken primary eggshell gland epithelial cells; amplifying the transfected cells for 3-4 generations, and screening puromycin to obtain successfully transfected eggshell gland epithelial cells; and (3) carrying out subculture on the successfully transfected eggshell gland epithelial cells obtained in the step (2) to obtain the stably subcultured immortalized eggshell gland epithelial cells. The immortalized egg shell gland epithelial cell provided by the invention can provide a reliable cell model for researching related functions of poultry egg shell glands, and has an important value for developing a novel feed additive capable of improving chicken genital tract health and egg shell quality.

The invention relates to a joint and application thereof. A linker according to the present invention comprises: a binding part having a structure capable of binding to a desired genetic information substance; and at least two or more puromycin-like substances capable of covalently binding to the C-terminal of a desired peptide.

The invention discloses a BHK-21 cell line capable of stably expressing a human ENT1 gene and a construction method and application thereof, the cell line is hamster kidney tissue fibroblast BHK-ENT1, the cell line carries the human ENT1 gene, and the ENT1 provides substances and energy basis necessary for replication for viruses by maintaining the steady state of nucleotide and energy in the cell, so that the human ENT1 gene can stably express the human ENT1 gene. The virus titer generated after the Newcastle disease virus infects host cells is improved. The construction method of the cell line comprises the following steps: amplifying a human ENT1 gene and constructing a stable expression vector, co-transfecting the stable expression vector and packaging plasmids psPAX2 and pMD2. G to an HEK-293T cell, culturing, and collecting a supernatant rich in lentiviral particles; and infecting the BHK-21 cell with the collected lentivirus, screening through a puromycin drug, and carrying out enlarged culture, so as to obtain the BHK-ENT1 cell.

This invention discloses a stable A549 expression cell line constructed based on a dCas9-VP64 transcriptional activation system and its applications. Based on the dCas9-VP64 transcriptional activation system, this invention yielded a stable A549 expression cell line with the highest relative expression level of dCas9 protein after puromycin selection. Subsequently, by introducing sgRNAs targeting one or more genes into this cell line, the expression of endogenous genes in lung cancer cells can be activated, and related functional gene studies can be conducted. Furthermore, this invention prepares an A549 cell line with significantly reduced proliferation rate by introducing sgRNA targeting the TP53 binding site in the LTR5Hs sequence into this cell line. This invention is the first to discover a novel target for lung cancer treatment, which is of great significance for the treatment and mechanistic research of lung cancer.

In one aspect, the present invention features a method of inhibiting proliferation and / or reducing survival of a cell comprising a GNAQ polynucleotide or polypeptide having a R183Q or Q209L mutation, comprising contacting the cell with puromycin or a puromycin analog, thereby inhibiting proliferation and / or reducing survival of the cell. In another aspect, a method of treating a vascular malformation or related condition in a subject, comprising administering to the subject an effective amount of puromycin or a puromycin analog is featured. In another aspect, the present invention features a method of identifying a candidate agent that modulates a GNAQ R183Q or Q209L mutation-associated disease, comprising contacting a cell comprising a GNAQ polynucleotide or polypeptide having a R183Q or Q209L mutation with puromycin and a candidate agent and comparing viability of the contacted cell with a reference level of viability, wherein an alteration in viability indicates that the candidate agent modulates the GNAQ R183Q or Q209L mutation-associated disease.

The invention belongs to the technical field of gene engineering, and discloses a plasmid targeting a ZDHHC5 gene in order to solve the problems of transient effect, low knock-down efficiency and high off-target rate of the existing siRNA technology. The plasmid comprises a DNA sequence for expressing shRNA, preferably a SEQ ID NO: 1 site, adopts a stem-loop structure 'CTCGAG' to improve stability, and integrates green fluorescent protein and puromycin resistance genes on the basis of a lentiviral vector to realize visual screening and long-term expression after transfection. And by optimizing the shRNA design and the special transfection process, the specificity and repeatability are remarkably enhanced. Experiments show that the plasmid can efficiently inhibit ZDHHC5 expression, effectively inhibit proliferation in Huh-7 cells and induce apoptosis.

The invention relates to the technical field of gene therapy, in particular to a method for knocking out a Bax gene on the basis of an HEK293 cell line by utilizing a CRISPR / Cas9 technology, constructing a stable cell line expressed by foreign protein and improving the AAV packaging efficiency. The invention discloses a Bax2.6 cell strain. A pro-apoptosis factor Bax gene is knocked out from an HEK293T cell. Compared with a wild type HEK293T cell, the Bax2.6 cell strain provided by the invention shows higher cell activity under various stress conditions, the cell survival rate of the Bax2.6 cell strain is improved by at least 20% under the treatment conditions of small molecule inhibitors ZV-FMK, Nec-1, puromycin (Puro), cis-platinum (CDDP) and rotenone, and the Bax2.6 cell strain shows excellent environmental tolerance and production robustness. When the Bax2.6 cell strain provided by the invention is used for transient transfection and packaging of an adeno-associated virus (AAV) vector, the copy number of the obtained AAV virus can be up to 2 * 10 copies / mL, and the AAV packaging efficiency is improved by more than 10 times compared with that of a conventional HEK293T cell line.

The invention discloses an overexpression vector of an Fos gene, an overexpression Fos CHO cell line and a construction method of the overexpression Fos CHO cell line, and relates to the technical field of genetic engineering, the overexpression vector of the Fos gene comprises a PB513B-Fos-Myc-T2A-Puro plasmid vector constructed by Fos and a CHO-Fos cell line prepared by overexpression Fos; the overexpression vector comprises a codon-optimized Fos gene coding sequence as shown in SEQ ID NO.2, and the codon-optimized Fos gene coding sequence is inserted into a eukaryotic expression vector. The CHO cell line is obtained by transfecting a carrier to CHO cells and carrying out puromycin screening and monoclonal treatment on the CHO cells. According to the method, global transcription is activated through Fos overexpression, the position effect is overcome, the expression quantity of the recombinant protein such as K6-mAb is remarkably improved, and an efficient and stable system is provided for recombinant protein production.