290 results about "Exogenous DNA" patented technology

The invention discloses a specie limitation-free eucaryote gene targeting method having no bio-safety influence and a helical-structure DNA sequence, and belongs to the field of gene engineering. The specie limitation-free eucaryote gene targeting method comprises the following steps of 1, designing and constructing CRISPR/Cas9 and chimeric RNA, and 2, carrying out Cas9mRNA internal translation so that Cas9 nuclease and the chimeric RNA are bonded, carrying out fixed point clipping so that DNA double-chain cleavage is realized after the clipping, and introducing an exogenous DNA by induction of a natural DNA restoration process which is a non-homologous end bonding process of cells so that cell endogenous gene modification is realized. The specie limitation-free eucaryote gene targeting method has simple processes, realizes flexible site recognition and has low energy consumption.

The invention relates to a gene targeting system, which comprises two parts such as a site-specific cleavage nuclease expression vector and a targeting vector, wherein the targeting vector contains 2-10 donor DNA fragments, 5' ends and 3' ends of every donor DNA fragment are respectively inserted into recognition sequences of the site-specific cleavage nuclease, the donor DNA comprises an upstream homologous arm, a downstream homologous arm and an exogenous DNA sequence positioned between the upstream homologous arm and the downstream homologous arm, and the site-specific cleavage nuclease expression vector is any one selected from an expression vector carrying zinc finger nuclease, a transcription activator-like effector nuclease expression vector, and a RNA-mediated nuclease RNA:Cas9 expression vector.

Described are recombinant yeast which ferment xylose to ethanol and which maintain their ability to do so when cultured for numerous generations in non-selective media. The preferred yeast contain multiple copies of integrated genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase fused to promoters which are non-glucose inhibited and which do not require xylose for induction. Also described are preferred methods for integrating multiple copies of exogenous DNA into host cells by transforming cells with replicative/integrative vectors, and then replicating the cells a number of times under selective pressure to promote retention of the vector in subsequent generations. The replicated vectors thus serve to integrate multiple copies of the exogenous DNA into the host cells throughout the replication/selection phase. Thereafter the selective pressure can be removed to promote loss of the vector in subsequent generations, leaving stable integrants of the exogenous DNA.

Transgenes encoding exogenous proteins are stably integrated into embryonic stem cells and are present in the somatic tissue of transgenic or chimeric birds. The transgenes encode exogenous proteins and are expressed in any of endodermal, ectodermal, mesodermal, or extra embryonic tissue. Tissue specificity is provided by selecting the content of the transgene accordingly. Transgenic birds whose genome is comprised of trangene derived exogenous DNA express exogenous proteins with tissue specificity, and specifically express exogenous proteins in the tubular gland cells of the oviduct to concentrate exogenous proteins in egg white..

The invention discloses a simple, convenient and efficient fixed-point gene editing method relating to the fields of transgenic animal preparation, gene functional study and biomedicines. A testis injection method is a sperm-mediated gene transferring method developed by utilizing the sperm capacity of actively combining, transferring and integrating an exogenous DNA. By using the method disclosed by the invention, an animal testis injection method is optimized, a carrier constructed by utilizing a clustered regularly interspaced short palindromic repeat system (CRISPR/Cas9) is integrated with a sperm chromosome through multi-point testis injection or seminiferous tubule microinjection, so that the aims of deleting, replacing and inserting genes are achieved. Then, a transgenic animal descendant is obtained through various approaches such as natural mating and artificial insemination. According to the method, the advantages of a CRISPR/Cas9 gene editing system are sufficiently utilized, and the existing transgenic animal system is combined, so that the complexity of in-vitro cell operation and requirement on expensive precise instruments/equipment are avoided while the gene modification animal obtaining efficiency is greatly increased.

The invention provides a method for the expression of exogenous DNA libraries in filamentous fungi. The fungi are capable of processing intron-containing eukaryotic genes, and also can carry out post-translational processing steps such as glyclosylation and protein folding. The invention provides for the use of fungi with altered morphology, which permits high-throughput screening and directed molecular evolution of expressed proteins. The same transformed fungi may be used to produce larger quantities of protein for isolation, characterization, and application testing, and may be suitable for commercial production of the protein as well.

The invention provides methods and compositions for obtaining transplastomic Arabidopsis plants. Specifically, the method provides culturing protocols and compositions that facilitate the regeneration of transformed plants following delivery of exogenous DNA molecules.

Methods and compositions are provided for the persistent modification of cell membranes with exogenous proteins so as to alter the function of the cell to achieve effects similar to those of gene therapy, without the introduction of exogenous DNA. DNA sequences, the proteins and polypeptides embodying these sequences are disclosed for modulating the immune system. The modulations include down-regulation, up-regulation and apoptosis.

PiggyBac transposons and transposases with enhanced transposition activity in cells are provided. Also provided are associated methods and kits for both introducing exogenous DNA inserts into the genomes of host cells as well as for the removal of the inserts from the host cell genomes. Cells obtained by use of the compositions, methods and kits are also provided.

The invention discloses a recombinant pichia pastoris engineering bacterium and a method for using the recombinant pichia pastoris engineering bacterium as a whole-cell catalyst for synthesizing RA (rebaudioside A). The recombinant pichia pastoris engineering bacterium is obtained through conversion of pichia pastoris after linearization of an expression vector containing exogenous DNA as follows: the DNA sequence for coding sucrose synthetase Sus1 is represented by SEQ ID NO.3, and the DNA sequence for coding UDP-glycosyl transferase UGT76G1 is represented by SEQ ID NO.4. The intracellular expression of the Sus1 and the surface expression of the UGT76G1 are realized by the aid of the recombinant pichia pastoris engineering bacterium, and the recombinant pichia pastoris engineering bacterium is used as the whole-cell catalyst and can be effectively used for synthesizing the RA, so that the synthetic efficiency of the RA is improved, the raw material utilization rate is increased, the production cost is reduced, and a new industrial way is opened for production and processing of the RA.

The invention provides an in vitro protein synthesis system, a kit and a preparation method. The in vitro protein synthesis system includes: (a) a cell extract; (b) an adsorbent used for enhancing protein synthesis efficiency; (c) amino acids; (d) nothing or NTPs; and (e) exogenous DNA molecules. A method for enhancing in vitro protein synthesis efficiency is provided. The method adopts the synthesis system and includes the following steps: (1) performing mixing; (2) adding the exogenous DNA molecules; (3) performing incubation synthesis; and (4) performing post-processing; and the method alsoincludes a step of adding the adsorbent after the step (1) or (2). In addition, the kit of the in vitro protein synthesis system is also provided. Therefore, the synthesis efficiency of foreign protein can be significantly enhanced.

The invention relates to a gene transfer system binding to a growth factor receptor, comprising 4-element complex gene transfer system consisting of ligand oligopeptide/polycationic polypeptide/endosome release oligopeptide/exogenous DNA or 3-element complex consisting of ligand oligopeptide/polycationic polypeptide/exogenous DNA. The invention exemplifies E5, GE7, GV1 and GV2 systems and they can be targeted for the introduction of exogenous genes into malignant tumor cells or tumor vascular endotheliocytes. They are also able to highly inhibit the growth of tumor cells in animals while p15, p16 or p21WAF-1 was used as exogenous genes. The system according to the invention is a new introduction system in tumor gene therapy.

The invention provides a traceless modification method of a chromosome, comprising the following steps: firstly building a recombinant box composed of a positive selection marker, a counterselection marker, an endonuclease I-SceI, an induced promoter, an I-SceI recognition site and a target nucleic acid displacement sequence which is homologous with a target chromosome nucleic acid sequence; inputting DNA fragments into a host containing recombinant plasmids by electrotransformation; knocking out the following gene sequences from an expected recombinant through a double-strand break repair phenomenon mediated by I-SceI: the positive selection marker, the induced promoter, an I-SceI gene and the counterselection marker; and finally screening the final recombinant from a sucrose medium. The method can help insert, knock out or replace the target gene of the chromosome without any selection markers or exogenous DNA sequences remained, thus being an effective method for traceless modification of the DNA sequence of a host cell.

The invention discloses agarose microsphere-containing cross-linked sodium hyaluronate gel for injection and a preparation method thereof. The preparation method comprises the following steps of 1, adding agarose microspheres into injection water, carrying out stirring, adding injection-level sodium hyaluronate powder into the mixed solution, and carrying out stirring until complete swelling, 2, adding a sodium hydroxide solution and 1,2,7,8-diepoxyoctane into the solution obtained by the step 1, and carrying out stirring under control of a sodium hydroxide concentration, 3, heating the mixture to obtain water-containing gel, 4, cutting the water-containing gel into small pieces and carrying out washing, 5, sieving the washed gel pieces, draining water in the gel pieces and adding a stroke-physiological saline solution into the gel pieces for balance, and 6, carrying out centrifugation and disinfection to obtain the agarose microsphere-containing cross-linked sodium hyaluronate gel finished product. The agarose microsphere-containing cross-linked sodium hyaluronate gel utilizes medical sodium hyaluronate, medical agarose microspheres, endotoxin, other proteins and exogenous DNA, and has indexes satisfying national and industry relevant standards. Through use of the agarose microspheres, the gel has a substantially slow resolving rate after injection and is an ideal injection filling product.

Transgenes encoding exogenous proteins are stably integrated into embryonic stem cells and are present in the somatic tissue of transgenic or chimeric birds. The transgenes encode exogenous proteins and are expressed in any of endodermal, ectodermal, mesodermal, or extra embryonic tissue. Tissue specificity is provided by selecting the content of the transgene accordingly. Transgenic birds whose genome is comprised of trangene derived exogenous DNA express exogenous proteins with tissue specificity, and specifically express exogenous proteins in the tubular gland cells of the oviduct to concentrate exogenous proteins in egg white.

The rape transgenic method of the present invention adopts the in-situ reproduction organ as transgenic receptor. On the basis of cytological observation of pollinated rape style and ovary, the transforming time is determined. The transformation includes cutting off the style head, dropping exogenous DNA solution, transforming the reproductive cell with the gene carrying exogenous gene and glufosinate resistance marker gene to obtain transgenic T1 generation zygocyte in the transformation period; and screening the T1 generation seedling with low concentration glufosinate solution to obtain transgenic positive rape plant. The present invention has transgenic efficiency of 2-10 %, high transformation flux, low cost, less work, no genotype dependence and other advantages.

The invention discloses a method for constructing fixed-point integrated exogenous DNA transgenic pigs. The method comprises the following steps: S1, performing safety target screening and target binding gRNA cleavage efficiency verification; S2, constructing a homologous arm donor plasmid and obtaining a fixed-point integrated transgenic cell line; S3, constructing the fixed-point integrated exogenous DNA transgenic pigs. The method disclosed by the invention has the benefits that a gRNA target sequence is introduced in the donor plasmid, the donor plasmid is linearized while a Cas9 nucleasecleavage target gene is induced by utilizing intracellular transcription gRNA, the test steps are significantly simplified, the labor is saved, and the co-transfection efficiency is favorably improved; carriers used for the fixed-point integration are less, a homologous arm is moderate in size, and the transgenic cell line is more favorably obtained; an efficient site-directed integration technology, a high-activity site-specific transgenic cell culture technology and a somatic cell cloning technology are combined, so that the efficient preparation of fixed-point integrated transgenic animalsis facilitated, and the breeding speed of new transgenic animal varieties is accelerated.

Disclosed herein are transgenic plants and seed having an exogenous DNA which expresses a GB1 protein that imparts increased glycine-betaine content in plants.

A transgenic carrier with deal targets (fibroblast growth factor receptor and integrant) is composed of the polypeptide CR16 specifically linked with alkaline fibroblast growth factor receptor, the polypeptide CP9 specifically linked with integrant, and the transgenic non-virus carrier system of CR16/CP9/cationic polymer PEI/exogenous DNA. Said carrier can effectively introduce the exogenous DNA to the tumor cell line and tumor tissue with high expression of FGFRs, so suppressing the growth of tumor. It can be used to preparation of the medicine for treating tumor and other diseases.