167 results about "Stable cell line" patented technology

The present invention relates to methods and compositions for increasing the production of high titre stocks of recombinant AAV (rAAV) through regulation of expression of the AAV REP and CAP proteins. The methods and compositions of the invention are based on the observation that the low level expression of the AAV REP protein increases the production of AAV viral capsid protein and efficiency of packaging resulting in production of higher titre recombinant viral stocks. The invention encompasses recombinant AAV vectors that direct the expression of AAV REP and CAP proteins and the use of such vectors for the production of novel stable cell lines capable of generating high titre rAAV vectors. The invention provides methods for regulating the expression of the AAV REP gene at the transcriptional and post-translational level. The methods and compositions of the invention can be used to produce high titre stocks of rAAV which can be used in gene therapy for the purpose of transferring genetic information into appropriate host cells for the management and correction of human diseases including inherited and acquired disorders.

The present invention relates to methods and compositions for increasing the production of high titre stocks of recombinant AAV (rAAV) through regulation of expression of the AAV REP and CAP proteins. The methods and compositions of the invention are based on the observation that the low level expression of the AAV REP 78/68 protein increases the production of AAV viral capsid protein and efficiency of packaging resulting in production of higher titre recombinant viral stocks. The invention encompasses recombinant AAV vectors that direct the expression of AAV REP and CAP proteins and the use of such vectors for the production of novel stable cell lines capable of generating high titre rAAV vectors. The invention provides methods for regulating the expression of the AAV REP 78/68, REP 40/52 and CAP gene at the transcriptional and post-translational level. The methods and compositions of the invention can be used to produce high titre stocks of rAAV which can be used in gene therapy for the purpose of transferring genetic information into appropriate host cells for the management and correction of human diseases including inherited and acquired disorders.

The invention relates to a method for rapidly obtaining a CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats/Cas9) gene knockout stable cell line through monoclonal cell sorting and belongs to the technical field of genetic engineering and genetic modification. According to the method, a CRISPR/Cas9 system, single-cell sorting by a flow cytometer and fluorescent protein screening on an expression vector are combined, positive monoclonal cells can be obtained in short time, and gene knockout work efficiency of the cell line is greatly improved. Compared with a traditional cell line gene knockout method, the flow cytometer is utilized for single-cell sorting, on one hand, tedious work of antibiotic screening is omitted, so that a large quantity of single cells can be obtained in quite short time, and on the other hand, the condition that cells in each culture hole are single cells can be guaranteed and false positive rate is reduced. Sorting is performed 40-80 h after cell transfection, at the time point, the highest survival rate of the cells after sorting can be guaranteed, and the screening efficiency is improved accordingly.

The invention relates to an endogenous protein marking method applied to genome-wide binding spectrum analysis. The method comprises the steps of designing sgRNA for upstream and downstream 50bp regions of an encoding termination site of an NCOA1 gene; verifying cutting efficiency of sgRNA mediated Cas9 by an experiment; building an anti-estrogen therapy tolerant MCF-7 breast cancer cell model; by utilizing a CRISPR/Cas9 homologous recombination technology, selecting and using effective sgRNA and DNA donors, and introducing a Flag tag in the 3' end of the ER alpha co-transcription factor NCOA1 gene; and establishing a stable cell line with the Flag tag. Compared with the prior art, the method has the advantages that gene editing is performed by utilizing the CRISPR/Cas9 technology, so that the tag insertion efficiency of the gene editing is greatly improved; more importantly, chromatin structure parameters are introduced for designing sgRNA, so that effective cutting of Cas9 at a target site of a genome is ensured; and positive clone screening is performed by adopting DNA sequencing and RT-PCR sequencing methods, so that the positive clone identification step is simplified and the manpower and material resources for screening are reduced.

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.

Provided are methods of identifying inhibitors of β-secretase that employ modified β-secretase substrates. The modified β-secretase substrates have β-secretase cleavage sites that are altered from wild type. The amino acid sequences of the altered β-secretase cleavage sites contain different amino acids in at least one of the positions P2-P1-P1′-P2′ of the β-secretase cleavage site. Many of the modified β-secretase substrates are more efficient substrates for β-secretase than are corresponding substrates having wild-type sequences, that is, these modified substrates are more susceptible to enzymatic breakdown by β-secretase. Recombinant polynucleotide molecules encoding the modified β-secretase substrates are provided. Antibodies that recognize cleavage products of the modified β-secretase substrates are provided. Stable cell lines expressing the modified β-secretase substrates are provided. Transgenic animals expressing the modified β-secretase substrates are provided.

A system and method for identifying a botulinum neurotoxin inhibitor employing a botulinum neurotoxin substrate complex having a peptide substrate, preferably SNAP-25, a reporter domain on one side of said peptide substrate and an immobilization domain on the opposite side of said peptide substrate. The botulinum neurotoxin inhibitor is identified by its ability to decrease the relative amount of cleaved complex, detected through measuring a decrease in complex bound to a solid support. The method of the present invention also utilizes novel cells that express a botulinum neurotoxin substrate complex. The methods of the present invention are adapted for cell based screening to monitor the catalytic activity of a BoNT in living cells and to identify molecules that inhibit the catalytic activity of a BoNT in living cells. Also provided are novel stable cell lines that express the botulinum toxin substrate complex and viral vectors capable of efficiently expressing an active light chain of the BoNT within mammalian cells.

Recombinant proteins are of great commercial interest. Yet, most current production methods in mammalian cells involve the time- and labor-consuming step of creating stable cell lines. Production methods based on transient gene expression are advantageous in terms of speed and versatility, yet, thus far, those methods have not shown the specific productivity, batch yield and volumetric yield to be an economic alternative to stable cell lines. The inventors improved on the methodology of transient transfection and achieved commercially relevant yields in terms of specific productivity (exceeding 35 pg per cell per day), batch yield (exceeding 700 mg/l) and volumetric yield.

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.

The invention relates to the field of biomedical research, and in particular to a construction method of an LM3 stable cell line with Midkine low expression. The LM3 cell line with Midkine stable low expression is constructed by virtue of an RNA interference technology. The construction method comprises the following steps: conducting primer design and target fragment amplification, and constructing Midkine shRNA recombinant plasmid; then, collecting a liquid medium containing viruses, filtering the liquid medium, adding the filtered liquid medium to a culture dish inoculated with LM3 cells in advance and conducting co-culture; and finally, conducting Puromycin resistance screening and sub-culturing on the LM3 cells accepting transfection, so that the LM3 stable cell line with Midkine low expression is finally obtained. With the establishment of the cell line, a new experimental material is provided for researching a molecular action mechanism of Midkine in tumors and a regulatory effect of the Midkine in tumor energy metabolism.

The invention discloses a novel vector for raising the content of polyunsaturated fatty acids in an animal body. The expression vector provided by the invention can simultaneously transcribe and express omega-3 fatty acid dehydrogenase and delta12 fatty acid dehydrogenase, and the vector sequence contains gene sequences of Sequence NO.1 and Sequence NO.2. In addition, the sequences of the enhancer SP163 and IRES are added between the sFat and Fat2 genes, and the vector provided by the invention can highly express two types of dehydrogenase at the same time. The vector provided by the invention is also transfected into the CHO cells, followed by the screening of antibiotics to obtain a stable cell line for highly expressing the sFat and Fat2 genes with the gene expression amount being 105 times that of untransfected cells. The invention also discloses a concrete method for raising offspring animals having high content of polyunsaturated fatty acids by using the vector. The vector provided by the invention lays a foundation for producing animal products having high content of omega-3 polyunsaturated fatty acids and improving the quality of meat products or dairy products by producing transgenic livestock.

The present invention provides methods of proteolytically converting a precursor protein (e.g. tau) to a product fragment (e.g., a 12 kd fragment) in a stable cell line, wherein the precursor protein is associated with a disease state in which the precursor protein aggregates pathologically (e.g. a tauopathy), and the methods comprise: (a) providing a stable cell line transfected with nucleic acid encoding: (i) a template fragment of the precursor protein such that the template fragment is constitutively expressed in the cell at a level which is not toxic to the cell; and (ii) the precursor protein, which protein is inducibly expressed in the cell in response to a stimulus, whereby interaction of the template fragment with the precursor protein causes a conformational change in the precursor protein such as to cause aggregation and proteolytic processing of the precursor protein to the product fragment. The method is preferably used to screen for modulators of the aggregation process by monitoring production (or modulation of production) of the product band or bands. Also provided are materials for used in the assays, plus medicaments, and related uses and processes, based on compounds which show high activity in the assay of the invention e.g. reduced diaminophenothiazines.

The invention belongs to the field of construction of stable cell lines, and more specifically, relates to a rabies virus labeling a whole brain area neural network structure, and preparation and an application thereof. A third-generation lentivirus technology is used for completing preparation of a BHK-N2C(G) cell line, that is to say, through construction of an FUGW-H2B-GFP-P2A-N2C(G) lentivirusvector and virus packaging, BHK cells are infected to obtain BHK-N2C(G) cells with nuclear localization fluorescence, and the cells are used for production of the defective recombinant rabies virus.The BHK-N2C(G) cells are infected with an RV-[delta]G-X virus solution, and then an RV-N2C(G)-[delta]G-X product is prepared by amplification and purification. In-vivo test results indicate that the prepared recombinant defective rabies virus RV-N2C(G)-[delta]G-X has no obvious inflammation reaction at an injection site, and has more reverse projection areas and high reverse projection efficiency.

An improved mammalian expression vector system which allows: (1) highly expressing exogenous proteins in host mammalian cells; (2) rapidly and efficiently screening recombinant stable cell lines expressing the gene of interest; (3) maintain sustainable expression and prevent gene silencing; and (4) effectively secreting proteins into media in some of cases. The entire expression vector system includes optimized promoters and core promoters, the use of special internal ribosome entry sites, integration of the bacterial backbone into the mammalian expression unit, multiple choices of selection markers, artificial matrix attachment region elements, effective secreting lead sequences and their 5′ and 3′ UTRs, and proper combinations of these expression elements.

Cells that produce inventive pseudotyped retroviruses having a broad host range have been produced. In one aspect of the invention, the cells produce retroviruses pseudotyped with at least two different viral glycoproteins, such as togaviral glycoproteins. In alternative embodiments, the cells produce retroviruses pseudotyped with filoviral glycoproteins. Methods of producing the above-described cells, as well as the pseudotyped retroviruses thus produced, are also provided. In other embodiments, methods of screening agents effective in blocking viral entry into a cell, including filoviral entry or entry of viruses having at least two different viral glycoproteins disposed in their lipid bilayer, such as togaviruses, are provided. Moreover, methods of using the inventive pseudotyped retroviruses for introducing nucleotide sequences into target cells, and kits for forming the inventive pseudotyped retroviruses, are also provided.

The present invention relates to a method for constructing a stable cell line carrying an orthogonal tRNA/aminoacyl tRNA synthetase, and the method stably integrates an orthogonal tRNA/aminoacyl tRNAsynthetase gene into cell genomes by double lentiviral stable transduction and plasmid stable transfection. The invention further relates to the stable cell line obtained by the method and the use ofthe stable cell line in the expression of a protein containing non-natural amino acids, and by utilizing unique reactive groups on the non-natural amino acids, the purpose of high-efficiency and specific protein modification can be achieved.

In one aspect, there is described a method for determining the effect of a genetic variation or mutation on the integrity of an RNA transcript comprising the steps of: (a) providing a nucleic acid construct comprising two different reporters separated by an in frame splicing unit, wherein said splicing unit comprises at least two exons separated by at least one intron and wherein at least one exon (eg. the exon downstream of said intron) comprises the genetic variation; (b) transfecting said construct into a cell and/or generating a stable cell line; (c) culturing said cell; and (d) determining the effect of said genetic variation on the integrity of the RNA transcript, wherein a difference in reporter activity in comparison to a cell comprising the nucleic acid construct without the genetic variation is indicative that said genetic variation affects the integrity of the RNA transcript. Assays—such as high throughput assays—are also described for identifying agents (nucleic acids, peptides and small molecules) that modulate the integrity of the RNA transcript and/or are involved in modulating RNA metabolism.