89 results about "Gene cassette" patented technology

The present invention relates to production of polyketides and other natural products and to libraries of compounds and individual novel compounds. One important area is the isolation and potential use of novel FKBP-ligand analogues and host cells that produce these compounds. The invention is particularly concerned with methods for the efficient transformation of strains that produce FKBP analogues and recombinant cells in which cloned genes or gene cassettes are expressed to generate novel compounds such as polyketide (especially rapamycin) FKBP-ligand analogues, and to processes for their,preparation, and to means employed therein (e.g. nucleic acids, vectors, gene cassettes and genetically modified strains).

The present invention provides a genetic engineering material for insects that enables a target protein to be purified easily, without requiring the use of recombinant baculovirus, while simultaneously providing a process for producing exogenous protein using that genetic engineering material. A gene recombinant silkworm is obtained by inserting an exogenous protein gene such as a cytokine gene coupled to a promoter that functions in silk glands into a silkworm chromosome. An exogenous protein such as a cytokine is then extracted and purified from the silk glands or cocoon of that silkworm or its offspring. A large amount of exogenous protein can be produced within silk gland cells, outside silk gland cells or in silk thread or a cocoon by inserting an expression gene cassette, in which the DNA sequence of the 3′ terminal portion and the DNA sequence of the 5′ terminal portion of fibroin H chain gene are fused to the exogenous protein gene, into silk gland cells and so forth.

The present inventors conducted dedicated studies and successfully constructed expression vectors that enable high-level production of foreign gene-derived proteins in mammalian host cells, which comprise a translation-impaired dihydrofolate reductase gene cistron whose expression has been attenuated by altering the codons to the least frequently used codons in mammals; and a gene cassette which has a cloning site for incorporation of a foreign gene between a highly transcriptionally active promoter and a highly stable polyadenylation signal.

The invention relates to devices and methods that utilize immobilized bacterial bioreporters genetically engineered to emit light visible to the naked eye in the presence of selected analytes. An exemplary bioreporter is an E. coli that has been modified to respond to mercury II as a result of incorporation of a merRop / lux gene cassette into its genome. Systems employing analagously engineered microorganisms can detect selected toxins quickly without need for expensive instruments or highly trained technicians.

This invention relates to a universal transfer vector of poxvirus avium, recombinant poxvirus avium and its preparation. Duplicate nonessential region fragment of Poxvirus avium is used to amplificate and clone for pPCI carrier with PCR. Gene cassette of green fluorescent protein(GFP) is constructed with early or late composed promotor of avian molluscum roup virus, and loxP sequence is transferred into its two sides, and then inserting into pPCI together to obtain universal transfer plasmid vector pHBP, which together with Poxvirus avium genome DNA cotransfecting CEF to obtain recombinant virus rFPVGFP.

The invention discloses a rice wine yeast metabolic engineering bacterium low in urea yield and a construction method thereof. The construction method includes: placing an encoding frame of a carbamido amidase gene DUR 1,2 in control of brewing yeast group forming high-expression thermoplastic polyimide 1 (TPI 1) transcription promoters and TPI 1 transcription terminators, and stably integrating TPI 1p-DUR1,2-TPI1t gene cassette in a bacterial strain rice wine yeast bacterial strain genome through homologous recombination to obtain the rice wine yeast metabolic engineering bacterium. The rice wine yeast metabolic engineering bacterium has the remarkable advantage of being low in urea production level, rice wine produced through fermentation of the rice wine yeast metabolic engineering bacterium is low in ethyl cellulose (EC) content and keeps flavor basically unchanged, and therefore the rice wine yeast metabolic engineering bacterium has wide application prospect.

The invention relates to gene cassette constructs for expression of a nucleic acid sequence of interest. The nucleic acid sequence of interest produces an RNA transcript that is an anti-sense RNA molecule complementary to an endogenously expressed gene of the host cell. Also included are transgenic plants expressing the nucleic acid sequence of interest, and transgenic plant cells, tissues and plants having altered phenotypes resulting from the expression of a nucleic acid sequence of interest in an anti-sense orientation.

The present invention provides a genetic engineering material for insects that enables a target protein to be purified easily, without requiring the use of recombinant baculovirus, while simultaneously providing a process for producing exogenous protein using that genetic engineering material. A gene recombinant silkworm is obtained by inserting an exogenous protein gene such as a cytokine gene coupled to a promoter that functions in silk glands into a silkworm chromosome. An exogenous protein such as a cytokine is then extracted and purified from the silk glands or cocoon of that silkworm or its offspring. A large amount of exogenous protein can be produced within silk gland cells, outside silk gland cells or in silk thread or a cocoon by inserting an expression gene cassette, in which the DNA sequence of the 3′ terminal portion and the DNA sequence of the 5′ terminal portion of fibroin H chain gene are fused to the exogenous protein gene, into silk gland cells and so forth.

The invention discloses an integron In1069 with a sequence as shown in SEQ ID NO.1. The integron is found in a genome of drug-resistant Escherichia coli EC6335. The integron contains a plurality of drug-resistant gene cassettes. Therefore, due to the discovery of the integron disclosed by the invention, a certain guiding effect is provided for clinical medication, some clinically-used antibacterial agents can be favorably reduced, the selective pressure for horizontal transfer of the drug-resistant gene cassettes is reduced, and the outbreak of drug-resistant bacteria is avoided.

The invention belongs to the field of animal genetic engineering, and relates to a ZB (zebrafish) transposon system of and a gene transfer method mediated by the ZB transposon system. The ZB transposon system is derived from zebra fish, and comprises two transgenic donor plasmids with terminal repetition sequences and capable of being inserted into a target gene box, and transposase auxiliary plasmids for providing activity of transposition; the transgenic donor plasmids comprise terminal repetition sequences and Msc1 insertion cloning sites at two sides of ZB transposon. The invention further discloses a gene transfer method based on the ZB transposon system, which is characterized that target genes are guided into a receptor genome through the ZB transposon system. The system and the method can be applied to multiple field of biotechnology: (1) the method can be used for effectively inserting the target gene box into a host cell genome, so that the gene transfer efficiency is improved; (2) by combining with the gene trapping technology, the researches on functions of animal genes can be effectively developed; (3) human gene therapy can be realized through mediation.

The present invention provides a process for producing a target fermentation product. This process includes providing a fermentation medium containing a recombinantly-produced microorganism that over-produces a fermentation product and contains a mutation which causes auxotrophic growth of the microorganism wherein the auxotrophy within the microorganism does not compromise the ability of the microorganism to produce the fermentation product. The medium is then supplied in excess with all substrates required for production of the fermentation product and in growth limiting amounts with a substrate complementing the auxotrophy. Host cells, vectors, and polynucleotide sequences used in the process are also provided. The polynucleotide sequences of the present invention include sequences derived from the biotin operon of B. subtilis and in particular the bioFDB gene cassette.

The present invention discloses biosynthesis process of preparing shikimic acid and the recombinant colibacillus therefor. The biosynthesis process of preparing shikimic acid includes constituting vector including the polygene kit of shikimic acid metabolizing pathway rate-limiting enzyme, replacing the polygene kit for shiA gene, knocking out shikimate kinase isozyme with Red recombinant system, fermenting recombinant engineering bacteria to obtain shikimic acid, purifying shikimic acid and other steps. The process has high shikimic acid expressing yield and lowered shikimic acid producing cost, and can provide material for preparing Oseltamivir phosphate as influenza preventing and treating medicine.

The invention discloses a plant expression vector without antibiotic marker genes and a construction method thereof. The T-DNA region of the vector contains a gene cassette which comprises CaMV 35S promoter, Xba I, Bam HI and XmaI restriction endonuclease enzyme sites, isopentenyl transferase (ipt) gene and nos terminator sequence. The construction method of the vector includes: using a binary vector pBI121 as a base, replacing gus gene with ipt gene, deleting npt II gene through Bsu 36 I and Xba I enzyme sites, reconnecting the CaMV 35S promoter to the T-DNA region. With the technical scheme provided by the invention, transgenic plants can be induced and regenerated on a culture medium with a proper amount of low-concentration cytokinin growth regulators, and the obtained transgenic plants are the transgenic plants expressed by the target genes, which can reduce steps for identifying whether the target gene is expressed, and can avoid potential risks of antibiotic marker genes.

Discloses the formation of carotenoid compounds containing 4-keto-β-ionene rings such as astaxanthin or canthaxanthin in higher plant flowers, and particularly in the corolla and reproductive parts of higher plant flowers, wherein said Petals of higher plants produce carotenoid compounds containing β-ionene rings such as β-carotene or zeaxanthin, but otherwise do not produce astaxanthin or canthaxanthin. One or more genes regulated by a promoter are inserted (transformed) into higher plants. The inserted gene encodes a chimeric enzyme comprising (a) a carotenoid-forming enzyme that is at least a ketolase. The gene is operably linked to (b) a plastid-directed transit peptide. Some of the higher plants to be transformed produce at least zeaxanthin or beta-carotene in their pre-transformation flowers, while other plants produce only minimal, if any, colored carotenoid pigments prior to transformation, and use carotenoid Gene forming cassettes for transformation. Transformation methods and uses of transformed plants are described.

The invention belongs to the field of microorganism animal cell line, and in particular relates to an engineering strain containing an integron. The engineering strain is eschrichia.coli, with a preservation number of C6MCC No.2353. The strain is named DHS, and the chromosome DNA of the strain has a structure of a sequence 1. In the invention, a transposon is used as a tool to insert an integron having a known sequence into the chromosome DNA of a colon bacillus. Tests show that the strain can capture an addA2 drug-resistance gene cassette in the presence of a high-expression integrase. The establishment of the strain lays a foundation for deeply researching a mechanism for the integron to capture the drug-resistance gene cassette in strains with known genetic background, and the strain can be used as a tool strain in other researches about the integron.

The invention relates to a method for carrying out gene knock-out or point mutation on a colibacillary genome by an oligonucleotide mediated recombineering measure. The method comprises the following steps: firstly, integrating a gene box of a cane sugar 6-fructosyltransferase gene and a kanamycin resistance gene containing isogenous arms into a target gene through recombineering; and then, carrying out isogenous recombination on oligonucleotide containing the isogenous arms and the isogenous sequences on the genome through the recombineering to remove the cane sugar 6-fructosyltransferase gene and the kanamycin resistance gene. Thereby, the gene knock-out and point mutation without any basic group redundance are realized. The gene knock-out oligonucleotide design conforms to the basic group sequences of both sides of the target genes, and base groups needing the mutation are introduced in the oligonucleotide by the point mutation oligonucleotide. The invention does not need in-vitro clone and in-vitro realization of some basic group bit mutation or mutation gene transplanting into germ bodies. The method of the invention can provide an effective early-stage operation platform for the industrial production and the research such as genetics, molecular biology, biochemistry and the like.

A process of producing an organic compound and / or an intermediary compound as defined herein by feeding carbon dioxide to a culture of a cyanobacterial cell and subjecting the culture to light, wherein the cell is capable of expressing a nucleic acid molecule, wherein the expression of the nucleic acid molecule confer on the cell the ability to convert a glycolytic intermediate into the organic compound and / or into the intermediary compound and wherein the nucleic acid molecule is under the control of a regulatory system which responds to a change in the concentration of a nutrient in the culture.

The present inventors successfully constructed expression vectors that enable high-level production of foreign gene-derived proteins in mammalian host cells, which comprise a translation-impaired drug resistance gene cistron whose expression has been attenuated by altering the codons to the least frequently used codons in mammals; and a gene cassette which has a cloning site for incorporation of a foreign gene between a highly transcriptionally active promoter and a highly stable polyadenylation signal.

The invention relates to an adenovirus vector for gene therapy on hemophilia B and application thereof, and the adenovirus vector can simultaneously express hFIX and red fluorescent protein; both sides of a tandem gene cassette of the hFIX and the red fluorescent protein are respectively provided with a loxp site of a Cre integrase action site in the same direction, and an action site attB of site-specific integrase PhiC31 is arranged in the gene cassette. The invention obtains a recombinant adenovirus vector which can be cyclized by DNA marked by the red fluorescent protein, and lays the foundation for the recombinant adenovirus vector to be further integrated into a main genome through attb sequences to exert the lasting function for treating hemophilia B. The adenovirus vector or adenovirus particles packaged outside the adenovirus vector is applied in the treatment of hemophilia B.

A kit for constructing a transposon is provided. The kit comprises five plasmids, each of which consists essentially of a gene cassette linked or not linked to a terminal repeat. The kit is useful in selecting the type of piggyBac that exhibits the least enhancer activity in the host cells.

The invention relates to a negative selection marker for escherichia coli gene knock-in, namely, the virulent gene ccdB driven by a plac promoter, and plac-ccdB and the gentamicin resistance gene aacC1 form a gene cassette for gene knock-in. In the first step, DNA fragments containing homologous arms plac-ccdB and aacC1 are obtained through amplification, and electro-transformed into escherichia coli for expression of recombinase. Under gentamicin screening, recombinase catalyzes homologous recombination and the DNA fragments are integrated into escherichia coli genome. In the second step, same homologous arms are utilized for amplification of foreign DNA fragments, ccdB is expressed in the bacterial strains obtained in the first step during the electrotransformation for expression of recombinase under the screening of isopropyl-beta-d-isopropylthiogalactoside, the toxicity serves as the feature of the negative selection marker, recombinase catalyzes homologous recombination among homologous arms, and foreign DNA fragments replace a plac-ccdB-aacC1 gene cassette to realize gene knock-in.

The application discloses a GAMYB gene based cultivation method for a transgenic rice sterile line. The GAMYB gene based cultivation method comprises the steps as follows: A, acquisition of a rice gene cassette; B, acquisition of a maize lethal gene ZMAA1; C, amplification of a maize pollen specific promoter Pg47; D, acquisition of an EGFP gene cassette; and E, connection of each gene expression element. The step E of connection of each gene expression element comprises: a first step of introducing a complete rice GAMYB gene into a pCAMBIA1300 vector, and then connecting the maize lethal geneZMAA1, the maize pollen specific promoter pPg47 and the EGFP gene expression element onto the pCAMBIA1300 vector. According to the invention, a F1-generation heterozygote follows the Mendel segregation law in the self fruitfulness process, and generated progenies not only contain heterozygotes which can keep a triple linked gene, but also contain the sterile line without fertility.

The invention discloses aureobasidium pullulan alb1 gene knockout mutant strain. According to the mutant strain, all or part of coding genes in alb1 gene in aureobasidium pullulan strain are knocked out and replaced by a hygromycin B resistance gene cassette. The hygromycin B resistance gene cassette comprises aureobasidium pullulan transcription elongation factor promoter PTEF and hygromycin B resistance gene HrgR; the sequence of the aureobasidium pullulan transcription elongation factor promoter PTEF is as shown in SEQ ID NO.2 in a sequence table; the sequence of the hygromycin B resistance gene HrgR is as shown in SEQ ID NO.3 in the sequence table. The mutant strain is used for the fermentation production of microbial polysaccharide, does not produce melanin, and can obviously the yield and quality of polysaccharide during the purification process.

The invention relates to a gene combination for producing betaine by expression in rice seed endosperm and an application of thereof, the gene combination comprises a meloS gene, a BvDODA1S gene and aBvCYP76AD1S gene, and further, a nucleotide sequence of the meloS gene is shown as SEQ ID NO.1; the nucleotide sequence of the BvDODA1S gene is as shown in SEQ ID NO.2, and the nucleotide sequence ofthe BvCYP76AD1S gene is as shown in SEQ ID NO. 3, the ORF of the three genes is connected between a rice endosperm specific globulin gene promoter and a terminator to construct a gene box, the gene box is inserted in a plant binary vector, and the betaine-rich rice is obtained, and the obtained rice seeds can be directly used for eating, can also be used as the betaine production raw material seeds, and provide important guiding significance and production application value for the breeding of novel functional crops.

The invention discloses a method for detecting a gene cassette array in a bacterium integron by using an EcoRII enzyme digestion map library, which comprises the following steps of: firstly, carrying out enzyme digestion on the gene cassette array by adopting the restriction enzyme EcoRII when a new screened gene cassette array is determined; comparing the gene cassette array with the constructed EcoRII enzyme digestion map library so as to determine the novelty of the gene cassette array; and selecting connection, transformation and sequencing analysis so as to realize purposeful sequencing if maps in the library are not matched with the gene cassette array. The method is accurate, rapid and economic and is convenient for information sharing. The method is more suitable for relevant departments such as hospitals, quarantine inspection, forecast, preventive treatment and the like to realize the effective monitoring and prevent the outbreak prevalent of drug-resistant bacteria caused by the horizontal transferring and spreading of the drug-resistant bacteria containing the integron under the condition that antibiotic misuse is not well restrained.

The invention discloses a PS transposon system and a mediated gene transfer method thereof, wherein the PS transposon system includes a transgenic donor plasmid capable of being inserted into a target gene cassette and having two terminal repeat sequences and a transposase assisted plasmid providing transposition activity. A target gene is introduced into a receptor genome through the PS transposon system. The gene transfer method based on the PS transposon system can efficiently mediate gene transfer through cell and embryo level verification, and can be applied in many biotechnological fields: 1) the target gene cassette can be inserted into the host genome effectively by using the method provided by the invention, and the efficiency of gene transfer is improved; 2) the study on animal gene functions can be effectively developed by combination of a gene capture technology; and 3) human gene therapy can be medicated to carry out.

The invention provides a construction method for a single copy cell strain of stably and highly expressed protein and application of the single copy cell strain. The method is based on target genes, which can be rapidly and accurately inserted and can be stably expressed a at a high level, of a 293T cell. According to the method, a 293T cell strain which is stably and efficiently expressed and canreplace the target genes is established through random insertion combined with a high-throughput screening strategy. Based on the cell strain, adenosine A2A receptor protein (ADORA2A) belonging to one of GPCR families is successfully expressed. By utilizing such method, repeated cassette-type recombination replacement can be carried out, an antibody or other protein genes to be expressed or optimized can be rapidly and accurately inserted into a cell gene cassette, and the integrity of the gene cassette cannot be broken.

The present disclosure provides nucleic acid molecules comprising a first inverted terminal repeat (ITR), a second ITR, and a genetic cassette encoding a miRNA and / or a therapeutic protein. In certainembodiments, the therapeutic protein comprises a clotting factor, e.g., a FVIII polypeptide, a FIX polypeptide, or a fragment thereof. In some embodiments, the first ITR and / or the second ITR is an ITR of a non-adeno-associated virus (AAV). The present disclosure also provides methods of treating bleeding disorders such as hemophilia comprising administering to the subject the nucleic acid molecule or a polypeptide encoded thereby.

The present invention relates to production of polyketides and other natural products and to libraries of compounds and individual novel compounds. One important area is the isolation and potential use of novel FKBP-ligand analogues and host cells that produce these compounds. The invention is particularly concerned with methods for the efficient transformation of strains that produce FKBP analogues and recombinant cells in which cloned genes or gene cassettes are expressed to generate novel compounds such as polyketide (especially rapamycin) FKBP-ligand analogues, and to processes for their preparation, and to means employed therein (e.g. nucleic acids, vectors, gene cassettes and genetically modified strains).

The invention discloses an integron In1085 of which the sequence is shown in SEQ ID NO.1. The integron In1085 is discovered in the genome of a drug-resistant escherichia coli Eco336. The discovery of the integron In1085 has important theoretical and practical significance in study on the molecular mechanism of bacterial drug resistance transfer at a gene level, further provides a certain guidance effect for clinical medication and is beneficial to reduction of clinical use of some antibacterial medicines, reduction of the selection pressure of horizontal transfer of drug resistance gene cassettes and prevention of outbreak of drug-resistant bacteria.