73 results about "Stable gene" patented technology

The invention discloses a genetic engineering cell line for producing an unfucosylated protein and an establishment method thereof. The invention utilizes the CRISPR / Cas9 technique to knock out the Slc35cl gene and / or the Fut8 gene in host cells, and thereby the Slc35cl gene-silencing and / or Fut8 gene-silencing stable genetic engineering cell line is obtained. By utilizing the genetic engineering cell line disclosed by the invention, the protein from which fucosylation is completely removed can be produced, moreover, the protein is still stable after 30 generations of passage, and thereby the two major problems of incomplete fucosylation removal and unstable passage in the prior art are solved. An unfucosylated antibody produced by the method disclosed by the invention shows enhanced ADCC (antibody dependent cell-mediated cytotoxicity) activity, and thereby the clinical therapeutic effect of the antibody is enhanced.

The invention discloses a breeding method for a Puan black goat line. The breeding method utilizes foundation Guizhou black nanny goats and Nubia billy goats to bi-cross, first-class first Nubia billy goat-black nanny goat hybrid generation nanny goats chosen as the female parent and first-class Guizhou black billy goats chosen as the male parent backcross, first-class backcrossed filial generation billy goats and first-class nanny goats are chosen to cross to be fixed, and therefore the Puan black goat line with more stable genes is bred. By introducing the excellent genes of the Nubia goats, such as big build, high reproduction rate and fast growth, the breeding method ensures that the bred Puan black goat line has the advantages of big build, fast early growth and development, high reproduction rate, high meat productivity and the like besides the characteristics, such as high adaptability, high disease resistance and black clothing hair, of the Guizhou black goats.

The invention provides a method for quickly cultivating Blumea riparia medicinal material, which comprises that collecting Blumea riparia explants in wild, which it contains stem sharp, young stem, old stem, blades, bulbs, and seeds, to be washed and disinfected, and using plant organism quick cultivating method and grafting cultivating method to quickly cultivate the Blumea riparia, cultivating seeds, while the obtained seed can be directly used in field. The invention has high cultivate speed, short period as 2-3 months, with high survival rate more than 90%, simple operation, simple device, low cost, and application in whole year. And the product cultivated by the invention has stable gene character, without variation.

Provided herein are methods for facilitating or inducing stable transgene integration and expression in a proliferating cell, comprising administering to the cell (i) a recombinant AAV (rAAV) vector comprising the transgene flanked by transposon-derived inverted terminal repeat sequences, which sequences are in turn flanked by AAV-derived inverted terminal repeat regions, and (ii) a source of a transposase that recognises said transposon-derived inverted terminal repeat sequences and directs the genomic integration of the transgene into the genome of the proliferating cell. Also provided are methods and transgene delivery systems for the treatment or prevention of diseases affecting, associated with or characterised by proliferating cells, including paediatric liver diseases, bone marrow diseases and cancer.

Methods of constructing a cell comprising in its chromosome one or more copies of an open reading frame (ORF) or operon encoding at least one polypeptide of interest, each copy being under the transcriptional control of a heterologous promoter using a site specific recombinase and in vivo integration by recombination; means for Promoter carrying out the methods, resulting cells, and methods for producing a polypeptide of interest using the resulting cells.

The present invention relates to expression vectors capable of promoting transgene expression. The expression vectors include site specific recombination elements, insulator elements, and recombinase elements. In particular, the present invention provides methods for obtaining specific and stable integration of nucleic acids into eukaryotic cells through site specific recombination.

In certain aspects, the present invention provides methods for inducing a stable gene modification of a target nucleic acid via homologous recombination in a primary cell, such as a primary blood cell and / or a primary mesenchymal cell. In certain other aspects, the present invention provides methods for enriching a population of genetically modified primary cells having targeted integration at a target nucleic acid. The methods of the present invention rely on the introduction of a DNA nuclease such as a Cas polypeptide and a homologous donor adeno-associated viral (AAV) vector into the primary cell to mediate targeted integration of the target nucleic acid. Also provided herein are methods for preventing or treating a disease in a subject in need thereof by administering to the subject any of the genetically modified primary cells or pharmaceutical compositions described herein to prevent the disease or ameliorate one or more symptoms of the disease.

A bacterial host cell comprising at least two copies of an amplification unit in its genome, said amplification unit comprising: i) at least one copy of a gene of interest, and ii) an expressible conditionally essential gene, wherein the conditionally essential gene is either promoterless or transcribed from a heterologous promoter having an activity substantially lower than the endogenous promoter of said conditionally essential gene, and wherein the conditionally essential gene if not functional would render the cell auxotrophic for at least one specific substance or unable to utilize one or more specific sole carbon source; methods for producing a protein using the cell of the invention, and methods for constructing the cell of the invention.

The method disclosed herein describes a novel technology offering unparalleled efficiency, flexibility, utility and speed for the stable integration of transgenes into lymphocytes and other mammalian cells. The novel method is based on the use of an mRNA-encoded transposase (e.g. sleeping beauty transposase) in combination with a minicircle DNA-encoded transposable element. The novel method enables higher gene-transfer rates and is at the same time less toxic than the conventional approach, which is the use of plasmid DNA-encoded transposase in combination with a plasmid DNA-encoded transposable element. Applications of the invention include but are not limited to the stable integration of a transgene encoding an immune receptor (e.g. a T-cell receptor or synthetic chimeric antigen receptor) into human T lymphocytes, with the immune receptor conferring specificity for a molecule expressed by a tumor cell. The transposase mRNA and transposon minicircle DNA may be introduced into lymphocytes by methods including but not limited to electrotransfer such as electroporation and nucleofection.

The invention relates to a pTerm-SC plasmid as well as a construction method and application thereof. The pTerm-SC plasmid comprises a repE gene, a sopA gene, a sopB gene, a sopC gene, an ori2 replicon, an oriV replicon, prokaryote transcription terminators and antibiotics resistance genes. The pTerm-SC plasmid has the characteristics of very low copy number, high volume and stability and the like, can be applied to gene engineering fields such as the cloning, the screening, sequencing and the genome construction of complex-structure genes including repetitive-sequence genes, instable genes and long-fragment genes and has important role in the high-efficiency and high-quality synthesis of genes.

The invention discloses a selection method of reference genes in quantitative real-time PCR analysis of jerusalem artichoke, and relates to the field of quantitative PCR. The method comprises the following steps: taking different tissues (radicles, immature stems, leaves, stem blocks and petals) of the jerusalem artichoke as materials, and carrying out expression analysis of the reference genes of18S ribosomal RNA gene (18S rRNA), transcription elongation factor gene (Ef-1a), actin gene (Actin and beta-actin), 3-glyceraldehyde phosphate dehydrogenase (GAPDH), 25S ribosomal RNA gene (25S rRNA)and poly-ubiquitin enzyme gene (UBQ)7 by using a q PCR technology; carrying out statistical assessment on the obtained data and analyzing expression change of all housekeeping genes by utilizing GeNorm and NormFinder software, so as to screen out relatively-stable genes as the reference genes of the jerusalem artichoke, which are used for studying the gene dosage changes of the jerusalem artichoke.

A method for the stable expression of an introduced exogenous gene in a plant or plant cell is provided. Stable expression of an exogenous gene that was introduced was achieved by operably linking an upstream sequence of sea urchin arylsulfatase gene as an insulator.

The present invention is related to a new method for interrupting multiple DNA sequences in Clostridia, even in genes recognized to be essential for the optimal growth of Clostridii by using a counter-selectable marker that would pinpoint the cells that have lost the plasmid and acquired a modified function that permits survival without the interrupted gene. This method is easy to perform and applicable at an industrial level. This method is useful to modify several genetic loci in Clostridia in a routine manner. This method is based on a replicative vector carrying at least two marker genes.

The invention discloses an STR molecule marker based on sika deer whole genome developing and application of the STR molecule marker. Based on a sika deer whole genome sequence, screening for STR loci is conducted, primer is designed and the application effect is verified, and the sika deer STR molecule marker with high amplification efficiency and the high recognition rate is finally obtained and used for amplifying primer sequences of the STR molecule marker as shown in SEQ ID NO:1-62. According to the STR molecule marker based on sika deer whole genome developing and application of the STR molecule marker, a four-base repeated unit is contained in the sika deer STR locus, compared with routine two-base to three-base repeated units, the distinguishing degree is higher and more stable, gene type judging is easier, and the convenient and efficient molecule marker can be supplied for genetic research on sika deer groups.

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 high-copy pTerm-series plasmid as well as a construction method and application thereof. The pTerm-series plasmid comprises a colE1 replicon gene, a procaryotic transcription terminator and an antibiotic resistance gene. The high-copy pTerm-series plasmid disclosed by the invention has the characteristics of high copy number, high capacity, high stability and the like, can be applied in the field of genetic engineering, such as cloning, screening and sequencing of complex-structure genes including repetitive sequences, instable genes and long-segment genes, constructing a genome and the like and has an important effect on high-efficiency and high-quality synthesis of genes.

A method of creating a human pluripotent transgenic stem cell, wherein heterologous DNA is inserted into specific “hot-spots” in the genome where stable and high gene expression may occur, is disclosed. In one embodiment, the method comprises the steps of: (a) selecting a pluripotent stem cell line, and (b) inserting heterologous DNA at an insertion site selected from the group consisting of insertion site one and insertion site two to form a transgenic cell line. In another embodiment, the heterologous DNA is an exchange cassette and the transgenic cell line formed is a master cell line.

The invention discloses a PRRSV attenuated strain which is capable of inducing pig body to relatively early generate interferon and a neutralizing antibody and possesses wide-spectrum immunogenicity. The porcine reproductive and respiratory syndrome virus (PRRSV) attenuated strain obtained through separation, passage and attenuation is named as HP-PRRSV-LZ-F65, and is prepared by subculturing PRRSV isolated virus in primary porcine alveolar macrophage for 5 generations and in MARC-145 cell for 60 generations and then performing attenuation, and the homologous rates of HP-PRRSV-LZ-F65 with PRRSV VR-2332 and VR-2385 are respectively 97.50% and 98.2%. Experiments prove that PRRSV-LZ-F65 is capable of relatively early inducing a pig body to generate an anti-PRRSV neutralizing antibody with the level substantially higher than that of other same-kind vaccines, and also is capable of preventing challenge infection of homologous type and heterogenous type PRRSV, and possesses stable gene after being subcultured in vitro for 50 generations. The attenuated strain HP-PRRSV-LZ-F65 and passage strains thereof possess wide application prospect to prevent porcine reproductive and respiratory syndrome as safe efficient wide-spectrum vaccine strain candidates.

The invention discloses a method for increasing a germination rate of stored wheat and a white degree of flour through endosperm specific silencing of expression of a wheat lipoxygenase gene. The method can reduce an activity of lipoxygenase (LOX) in wheat endosperm by specifically inhibiting the expression of the lipoxygenase gene in the wheat endosperm through an RNA interference (RANi) technology. An obtained LOX-RNAi wheat strain high-generation material (T6) is stable in a copy number of transgenosis. The expression of the LOX gene in the endosperm is significantly reduced and the activity of the LOX is significantly reduced. A field experiment proves that important growth indexes of the LOX-RNAi wheat strain, compared with the important growth indexes of a wild strain (Longchun 23), are free of any significant change. A germination experiment is carried out to seeds stored for 30 days after being harvested and proves that the germination rate of three transgenic strains is free from being obviously different from the germination rate of the wild strain. A flour-milling experiment proves that of the flour produced from the transgenic strain, compared with the white degree of the flour produced from the wild strain, is significantly improved. The germination rate of the wild strain, compared with the germination rate of the transgenic strain, is significantly reduced after an artificial aging process, and meanwhile the flour produced from the wild strain, compared with the white degree of the flour produced from the transgenic strain, is significantly reduced.

The present invention relates to the generation and production of novel thermostable, organic solvent stable and pH tolerant lipase gene variants. The invention also relates to methods of selecting lipase variants for high temperatures and for their purification.

The invention provides a CRISPR / Cas9 technology based preparation method and applications of a PI3K[gamma] whole body knockout mouse and a kit. The preparation method comprises following steps: designing sgRNA for identify the gene PI3K[gamma]; connecting the sgRNA to a plasmid carrier, carrying out in-vitro transcription to obtain RNA that can be applied to microinjection; injecting the RNA intoa mouse fertilized egg, after injection, giving birth to mice; identifying the genotype of the mice, selecting mice, whose PI3K[gamma] whole body knockout genotype detection result is positive, and mating picked mice with wild mice to obtain PI3K[gamma] whole body knockout mice with a stable genotype. Compared with a conventional gene knockout technology, the operation is easier, the efficiency ishigher, homozygote mutation is obtained more easily, and good foundation is provided for the research on functions of PI3K[gamma].

The invention discloses construction and application of a bacillus subtilis linear plasmid system, and belongs to the field of bacillus subtilis gene engineering. According to the invention, bacillussubtilis is used as an expression host, and a genome replication-related gene cluster derived from bacillus subtilis phage phi29 is expressed in the bacillus subtilis, so that replication and expression of linear plasmids in cells are realized. Through change of different promoters, the copy number and expression level of the linear plasmids can be controlled. According to verification, when the linear plasmid system is used for expressing the green fluorescent protein, a condition of plasmid loss does not occur after shaking-flask culture for 70 hours, and thus the novel expression tool laysa foundation for realizing efficient and stable gene expression, gene editing, metabolic engineering transformation and the like of bacillus subtilis.

Various embodiments perform stable gene analysis of transcriptome sequencing data. In one embodiment, a plurality of datasets each including transcriptome sequencing data are received by a processor. Each of the plurality of datasets includes a plurality of genes and a respective ranking value for each of the plurality of genes. A plurality of rank normalized input datasets is generated based on assigning, for each of the plurality of datasets, a rank to each of the plurality of genes. One or more longest increasing subsequence (LIS) of ranks are identified between each pair of the plurality of rank normalized input datasets. A set of stable genes from the plurality of genes is identified based on each of the one or more LIS of ranks across the plurality of rank normalized input datasets.

The invention discloses a cultivate method for making mouse with spontaneous faint sensitive pathological character, comprising the tested animals and operation method. The invention is characterized in that the invention uses KunMing mouse as resource and the operation method comprises that the mature mouse have equal male and female numbers, to be selected according to the faint index to select 8 couples with maximum faint index as F1 sub generation to cultivate next generation, and selects the sub generation via same method, and uses consanguinity copulation method to cultivate sub generation or the like, while the invention selects 8 couples of minimum faint index as reference, repeats the selection and cultivates more than 12 generations, to obtain the mouse with spontaneous faint sensitive pathological character and stable gene character and normal ones. The inventive mouse can be used to research motion sickness or the like.

An inducible expression cassette for controlled expression of multiple genes includes the DNA sequences of a bidirectional promoter inserted between two DNA sequences. Transfection into a host cell and the two DNA sequences encode genes of interest, provides one DNA sequence expressed constitutively and manipulation of the expression of the other DNA sequence. A regulatory DNA cassette containing another bidirectional promoter and two DNA sequences that encode a marker and a regulatory expression product is also disclosed. Both cassettes can be incorporated in a non-viral vector, like the Sleeping Beauty transposon, or a viral vector to induce controlled expression of multiple genes into host cells. A kit containing a package of each above vector type is also disclosed, as is a method of transforming a host cell.

The present invention relates to novel selection marker vectors, and methods for using these vectors to generate stable gene expression systems in eukaryotic cells utilizing any enzyme useful in the eukaryotic sterol / cholesterol biosynthetic pathway, such as a 3-ketosteroid reductase, as a metabolic selection marker to select transfected cells. In one embodiment, the method comprises transfecting cells that are auxotrophic for cholesterol with a vector encoding 3-ketosteroid reductase and at least one heterologous protein, and selecting cells that have the ability to survive in medium lacking cholesterol and / or producing the heterologous protein in these cells in chemically defined and / or serum-free media.