87 results about "Gene Knockout Techniques" patented technology

The invention relates to a method of constructing an Ifit3-eKO1 gene knockout mouse animal model and belongs to the biotechnical field. The method comprises the following steps: S1, determining specific target sites sgRNA1 and sgRNA2 of a to-be-knocked out gene of an Ifit3-eKO1 mouse, and performing in vitro transcription with Cas9 nuclease to mRNA; and S2, micro-injecting active sgRNA and Cas9RNA into an oosperm of the mouse to obtain the Ifit3-eKO1 gene knockout mouse. The method has the advantages that by using the CRISPR / Cas9 gene knockout technology, the Ifit3-eKO1 gene knockout mouse animal model is constructed for the first time, thereby providing a convenient, reliable and economical animal model for researching action of Ifit3 in tumorigenesis and development.

The invention discloses miR-124 gene knockout murine animal models. The murine animal models refer to mice with miR-124-1, miR-124-2 and miR-124-3 genes knocked out. With the adoption of a Crispr-cas9 gene knockout technology, microRNA (micro ribonucleic acid) miR-124 genes with the highest expression quantity in the brains of the mice are knocked out. The obtained mice show obvious nervous system disease states such as reduction of locomotor activity, deterioration of learning and memorizing abilities, increase of soluble amyloid beta protein and the like. Therefore, the simple, reliable and economical animal models can be provided for research of nervous system disease pathology and screening of drugs for treating nervous system diseases.

The invention discloses a construction method for a Sqstm1 whole-genome knockout mouse animal model and application. The mouse animal model is a mouse of which the Sqstm1 gene is knocked out. On the basis of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 gene knockout technology, the Sqstm1 gene knockout mouse model is constructed. The construction method comprises the following steps: S1: designing sgRNA (Ribonucleic Acid) and Cas9 RNA, carrying out in vitro transcription to obtain mRNA, carrying out microinjection on the sgRNA and the Cas9 RNA with activity into amouse oosperm, to obtain the Sqtm1 gene knockout mouse; S2: identifying the Sqtm1 gene knockout mouse animal model. On the basis of the CRISPR / Cas9 gene knockout technology, the Sqstm1 gene knockout mouse animal model is constructed for the first time, and a convenient, reliable and economic animal model is provided for researching a relationship between Sqstm1 and diseases including autophagy, tumors and the like.

The invention provides a method, primer and plasmid for constructing an EPO gene knockout zebrafish animal model and a preparation method of the primer and the plasmid, and belongs to the field of biomedicine. The method includes the steps of 1) establishment of an EPO gene knockout zebrafish CRISPR oligomer sequence plasmid; and 2) establishment and culture of the EPO gene knockout zebrafish animal model on the basis of CRISPR gene knockout technology; wherein the step 1) comprises 1-1) design and synthesis of the primer aiming at an EPO exon 2 region target sequence and 1-2) synthesis of the Escherichia coli plasmid containing the EPO target sequence. The method for constructing the EPO gene knockout zebrafish animal model can be used to construct the EPO gene knockout zebrafish animal model, and facilitates further understanding and research of EPO.

The invention discloses a method for constructing a mouse model for conditional knockout of CCR5 gene of endothelial cell. The method comprises the following steps of firstly, obtaining a CCR5loxp / loxp mouse, and mating with a Tie-2-cre / ERT2 mouse, so as to obtain heterozygous mice with specific knockout of CCR5 gene of the endothelial cell; mutually mating the heterozygous mice, so as to obtain homozygous mice with specific knockout of CCR5 gene out of the endothelial cell. The constructing method disclosed by the invention has the advantages that the mouse with conditional knockout of endothelial cell gene is constructed via the CRISPR / Cas9 system, the mutation is led into the mouse by a cell specificity method, the missing of the CCR5 target gene occurs at the certain tissue organ of the test animal, the controllability of mechanism study is furthest realized, and the disadvantages that different tissues or cells are not distinguished, and target genes in all tissues or cells of themouse body are all removed in the conventional gene knockout technique is overcome.

The invention relates to a method for acquiring a Nogo-B knockout mode mouse based on CRISPR / Cas9 technology and an application thereof, the method for acquiring the Nogo-B knockout mode mouse comprises the following steps of: designing a high-efficiency sgRNA for identifying EGE-ZXH-007 of a specific gene; connecting the sgRNA to a plasmid vector and then carrying out in-vitro transcription to obtain a Ca9 / sgRNA capable of being subject to microinjection; injecting the Ca9 / sgRNA capable of being subject to microinjection into a mouse fertilized egg, and then carrying out genotype detection and identification on the born mouse, and finally obtaining the Nogo-B knock-out mode mouse. According to the invention, the Nogo-B knockout mode mouse is obtained by using the CRISPR / Case9 technology,and compared with the traditional gene knockout technology, the operation is easy and the efficiency is higher, and the homozygous mutation is more easily obtained, thus laying a good foundation for further researching the function of Nogo-B.

The invention provides a gene engineer strain of Aspergillus niger with high L-malic acid yield and a construction method thereof. The method utilizes gene knockout technique to disrupt the Aspergillus niger oxaloacetic acid hydrolase gene (oahA) so as to obtain the malic acid-producing strain S2. After 7 days of shake flask fermentation, 100 g / L glucose is converted into 120.4 + / - 2. 8 g / L of L-malic acid, the conversion of malic acid and malic acid to glucose is 1.62 mol / mol, which is 81% of the highest conversion in theory. A good strain for the industrialized production of malic acid is provided.

The invention discloses a method for breeding a high-producing strain of cellulase by gene knockout, and belongs to the field of enzyme engineering. The method for breeding the high-producing strain of cellulase by gene knockout comprises the step of knocking out an amylase gene of the cellulase producing strain. A high-producing strain of cellulase is aspergillus niger without the amylase gene. An amylase gene knockout plasmid is obtained by connecting the front half part of the recombinant segment amylase gene-hygromycin B-an expression unit of a resistant gene- the rear half part of the amylase gene to a pCAMBIA1300 plasmid. Then, the plasmid is converted into agrobacterium, and the amylase gene is knocked out by agrobacterium-mediated conversion of the aspergillus niger. By using the gene knockout technology, the amylase gene of the aspergillus niger is knocked out. After the amylase gene is knocked out, the cell load is reduced, thereby facilitating production of a lot of cellulase. The cellulase activity of the high-producing strain of cellulase obtained can reach 21U / g.

The invention relates to a method for preparing an atherosclerosis disease canine model, in particular to a method for preparing an apolipoprotein E (APOE) gene knockout disease canine model according to a gene knockout technology.

The invention discloses recombinant Escherichia coli and application of the recombinant Escherichia coli in synthesizing 3-hydroxypropionic acid. The recombinant Escherichia coli is formed by glycerol dehydratase genes dhaB123, glycerol dehydratase reactivating factor genes gdrA, alpha-oxoglutarate semialdehyde dehydrogenase mutant coding genes TUkgsadh and glycerol-3-phosphate dehydrogenase coding genes gpdl which are guided into host bacteria. By means of the gene knockout technique, the yield of byproduct 1,3-propylene glycol is reduced, and meanwhile, cofactors NAD+ of aldehyde dehydrogenase are regenerated. Thus, the yield of the 3-hydroxypropionic acid is improved by 1.5 times.

The invention discloses a construction method of an Amy1 gene knockout mouse animal model and application. An Amy1 gene knockout mouse model is constructed based on a CRISPR / Cas9 gene knockout technology. The construction method comprises the following steps: step 1, designing sgRNA and Cas9 RNA, performing in-vitro transcription to obtain mRNA, and performing microinjection of the active sgRNA and Cas9 RNA on fertilized eggs of a mouse to obtain an Amy1 gene knockout mouse; and step 2, identifying the Amy1 gene knockout mouse animal model. The method has advantages: the CRISPR / Cas9 gene knockout technology is used and the mouse animal model with the Amy1 gene knocked out is established for the first time; and a convenient and reliable animal model is provided for researching the relationbetween the Amy1 gene and related diseases.

The invention discloses a high permeability baker's yeast for detecting environmental carcinogen and method for making the same which relates to baker's yeast and the reforming of descendiblity gene. The permeability of the baker's yeast used for detecting environmental carcinogen is improved by the reforming of descendiblity gene so that the baker's yeast can detect the environmental carcinogen in more large scale and in higher sensitivity. The gene CWP1 and CWP2 of encoded baker's yeast cell wall important structural protein mannose egg albumen are inactivated by gene out-knocking technology to improve the permeability of baker's yeast cell for environmental carcinogen because the baker's yeast cell wall is the main disorder for chemistry permeability. A RNR3-lacZ system is constructed in CWP1 and CWP2 gene out-knocking baker's yeast saccharomycete of reformed baker's yeast cell wall to improve the sensitivity for detecting environment genotoxic carcinogens.

The invention discloses an attenuated Salmonella pullorum and application thereof. By using gene knockout technology, the in vivo colonization gene and the main virulence gene of Salmonella pullorum are deleted, thus obtaining an dual-gene-deleted attenuated Salmonella pullorum SM091-DED strain; and the microbiological preservation number of the strain is CGMCC NO.4604. The virulence of the attenuated Salmonella pullorum disclosed by the invention is obviously reduced, and the in vivo colonization time is short after the attenuated Salmonella pullorum is inoculated into a host; the herd infection test shows that the attenuated Salmonella pullorum has no horizontal transmission capability; the attenuated Salmonella pullorum provides full cross protection for homotype and allotype high-virulence Salmonella pullorum; and after SPF (Specific Pathogen Free) chicks are immunized, the infection of the high-virulence strain can be effectively eliminated, and the herd infection can not be caused. Thus, the invention provides a safe, fine-immunogenicity and low-virulence Salmonellosis avium attenuated live vaccine for preventing Salmonellosis avium.

The invention relates to a method for establishing a CYP2D1 gene knockout rat model, and belongs to the technical field of transgenosis. The method comprises the steps of firstly determining a target point of a to-be-knocked gene and designing and synthesizing a primer sequence of the gene; inserting the primer sequence into a Cas9-gRNA-Bsal carrier subjected to enzyme digestion and performing amplification to obtain a target point sgRNA with a T7 promoter sequence; after in vitro transcription and purification, performing activity detection; micro-injecting active sgRNA and Cas9 RNA into a rat single-cell embryo to obtain a Founder rat; and after screening out a CYP2D1 gene knockout rat heterozygote, performing mating to obtain a homozygote individual, namely, a CYP2D1 gene knockout rat. The invention provides a preparation method replacing a mouse model with a P450 gene knockout rat model, so that a gene knockout technology is really fused in non-clinical safety evaluation of drugs and early toxicity of candidate drugs can be discovered.

The invention relates to a method for establishing a CYP2C11 gene knockout rat model, and belongs to the technical field of transgenosis. The method comprises the steps of firstly determining a target point of a to-be-knocked gene and designing and synthesizing a primer sequence of the gene; inserting the primer sequence into a Cas9-gRNA-Bsal carrier subjected to enzyme digestion and performing amplification to obtain a target point sgRNA with a T7 promoter sequence; after in vitro transcription and purification, performing activity detection; micro-injecting active sgRNA and Cas9 RNA into a rat single-cell embryo to obtain a Founder rat; and after screening out a CYP2C11 gene knockout rat heterozygote, performing mating to obtain a homozygote individual, namely, a CYP2C11 gene knockout rat. The invention provides a brand-new preparation method replacing a mouse model with a P450 gene knockout rat model, so that a gene knockout technology is really fused in non-clinical safety evaluation of drugs and early toxicity of candidate drugs can be discovered.

The invention provides a construction method of an Ano5 gene knockout mouse model. A CRISPR-Cas9 gene knockout technology is adopted to remove 11th and 12th exons of the Ano5 gene. After Ano5 gene knockout, mice all have clinical symptoms of human GDD patients such as lateral bending of shin bones, hyperplasia of cortical bone, hypertrophy of lower jawbone, increasing of alkaline phosphatase in blood, and the like. The established mouse model is used to simulate the human GDD disease, is stable, has stable heredity and similar symptoms of the human GDD disease, can be applied to research on GDD pathogenesis and GDD gene therapy, and is economic, simple and reliable.

The invention relates to a building method and application of an STCH (stress and chaperone) gene knock-out animal model. The model can function as an animal model for the research of occurrence of depression and / or dementia. The model is prepared by using a gene knock-out technique, animal behavior detections show that a mouse suffers depression-like symptoms (forced swimming test and tail suspension test) and memory dysfunction (water maze test) while it grows older and has depression and dementia-like symptoms, and the model is applicable to screening anti-depression and anti-dementia drugs.

A corynebacterium acetoacidophilum strain and a method for producing succinic acid therefrom belong to the technical field of bioengineering. The invention discloses a corynebacterium acetoacidophilum strain YF / delta ldh and a method for producing succinic acid therefrom. The corynebacterium acetoacidophilum strain is an ldh (L-lactate dehydrogenase) missing bacterium built by gene knockout technology, is preserved in china center for type culture collection on February 29th, 2012, and encoded as CCTCC NO.M2012041. The strain is high in acid yield, highly resistant to sodions and high in foodsafety, can accumulate succinic acid more than 95g / L by being culture for 48 hours in the anaerobic environment using glucose as substrate and sodium bicarbonate as carbon dioxide donor, and can accumulate succinic acid 136g / L by being cultured for 94 hours.

The invention discloses an MCIR (MelanoCortin 1 Receptor) gene carrier and a construction method thereof. The MCIR gene carrier comprises MC1R-neo and MC1R-GFP (Green Fluorescent Protein), wherein the nucleotide sequence of the MC1R-neo is SEQ ID NO:1, and the nucleotide sequence of the MC1R-GFP is SEQ ID NO:2. A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 system is adopted to construct an MC1R gene knock-out vector, and only a sgRNA (Ribonucleic Acid) of which the length is about 20bp needs to be designed by aiming at a gene knock-out point to be connected with a universal Cas9 gene. Therefore, compared with traditional gene knock-out technologies including ZFNs (Zinc finger nuclease), TALENs (Transcription Activator-Like Effector Nucleases) and the like, the construction method disclosed by the invention adopting the CRISPR / Cas9 to construct the gene knock-out vector is simpler and more convenient and is more convenient to be further popularized and applied to subsequent experiments.

The invention discloses a preparation method and application of a glycerol-producing Candida engineering bacterium with high yield of 2-phenylethyl alcohol, belonging to the technical field of genetic engineering. Candida glycerinogenes CCTCC M 93018 is a diploid strain. Compared with haploid strains, it has the advantages of fast growth, strong viability, stable genetics, and prevention of lethal or harmful mutations. At the same time, Candida glycerologenus is an excellent 2-phenylethanol producing strain, and its ability to tolerate high concentrations of 2-phenylethanol (4g / L) provides a favorable basis for the metabolic transformation of the strain. The invention utilizes the gene knockout technique to knock out the acetaldehyde dehydrogenase gene ALD3 of the diploid glycerol-producing Candida strain 2-phenethyl alcohol synthesis competition pathway. Compared with the original glycerologenic Candida strain, the ALD3 knockout mutant strain increased the production of 2‑phenylethanol by 18.5%, reaching 3.65 g / L.

The invention discloses an ultrahigh permeability bread yeast and a preparation method thereof, and relates to a pattern biological bread yeast, which can be used for detecting environmental genotoxic carcinogen. Through reconstruction of genetic gene technology, the yeast has improved permeability, so that the yeast has wider detection range and higher sensitivity to environmental pollutants. Through gene knock-out technology, genes CWP1 and CWP2, which are used for encoding protein mannose protein serving as an important structure of bread yeast cell wall, is inactivated to improve the permeability of the bread yeast cell wall to environmental carcinogen; and genes SNQ2 and PDR5, which are used for encoding an efflux pump protein on a cell membrane of bread yeast, is inactivated to improve the accumulation of the environmental pollutants in the bread yeast cell. Through the integration of two reconstruction strategies, an RNR3-1acZ system is constructed when four genes CWP1, CWP2, SNQ2 and PDR5 knock out bread microzyme, which can improve the sensitivity of the system to detecting the environmental genotoxic carcinogen.

The invention provides a kind of transgenic inhuman mammal by producing klone gene without mouse model. Roll outside self murder gene into the mammal. The expression box contains promoter, self murder gene relation with promoter and termination codon. The natural masculine gene will express in testes organize after transgenic animal builds system. The transgenic animal can provide blastosphere for eliminating gene technic to improve the efficiency of the technic and to solve the problem that mouse genetic background is impure.

The invention relates to the technical field of gene knock out, and particularly discloses a method for selective breeding of Lgr6 gene deletion type zebrafish through gene knock out. The method comprises the steps of through a CRISPR / Cas9 gene editing technique, designing an appropriate targeting site on the Lgr6 gene of zebrafish, performing in vitro synthesis of sgRNA and Cas9-mRNA, performingmicroscopic co-injection into one cell of the zebrafish, performing embryo culturing for 60h, selecting embryos, and performing genotype analysis so as to confirm the validity of the selected site. The method can enable silent specific genes to be efficiently and accurately mutated in organism genomes. The method is simple to make and low in cost, shearing of a plurality of sites on the target gene can be realized at the same time, and any number of individual genes can be silent. The Lgr6 gene expression is subjected to interference, and through a heredity means, the function are researched,so that further revealing of the entire process of the skeleton shape generation and adjusting and controlling the molecular mechanisms of processes are facilitated, and the method has important significance in understanding of medical skeleton disease pathology and research and development of a new treatment scheme.

The invention belongs to the technical field of gene knockout, and discloses a CRISPR Cas9 conditional gene knockout mouse and an establishment method. According to the establishment method, a vectoris designed and constructed, and gRNA and Cas9 protein are prepared; a gRNA sequence and a targeting vector are designed; the targeting vector is constructed by the In-Fusion technology, and the targeting vector is verified by restriction digestion, PCR and sequencing; the gRNA and Cas9 protein are prepared at the same time; microinjection and F0 mouse identification are performed; the obtained Donor vector, gRNA and Cas9 protein are co-injected into a fertilized egg; the fertilized egg after microinjection is returned to the oviduct of a surrogate mouse; PCR and sequencing identification areperformed after a mouse is born, and a positive F0 mouse is obtained; the reproduction and identification of a F1 generation mouse are performed; the sexually mature positive F0 mice are matched withwild-type mice respectively for reproducing the first generation, and a polycystic kidney gene PKD1 knockout mouse is obtained successfully.

The invention discloses an application of a photoperiod gene OsNhd1. Rice (oryza sativa) photoperiod gene OsNhd1 is applied to regulation and control of the growth period, the yield and the nitrogen utilization efficiency of rice; and the rice photoperiod gene OsNhd1 is numbered as Os8g0157600 in an RAP-DB (Rice Annotation Project Database). According to the application, an OsNhd1 mutant plant is obtained through a gene knockout technology. Field tests and analysis prove that the mutant plant has the characteristics of prolonged growth period, increased yield and increased nitrogen utilization efficiency. The application proves that OsNhd1 not only directly regulates heading and flowering of rice, but also participates in nitrogen assimilation and transport processes of rice, which indicates that the gene plays an important role in coordinated regulation of growth period and nitrogen utilization efficiency, and establishes a theoretical basis for improvement of rice varieties in the future.

The invention relates to a controllable genome-modified plasmodium, a recombinant expression vector and the construction method and application of the controllable genome-modified plasmodium and the recombinant expression vector. The recombinant expression vector comprises a gene targeting long homologous arm, a gene targeting short homologous arm, a tetracycline repression protein gene expression cassette, a pyrimethamine resistance gene expression cassette and a target gene expression cassette, wherein the tetracycline repression protein gene expression cassette, the pyrimethamine resistance gene expression cassette and the target gene expression cassette are located between the gene targeting long homologous arm and the gene targeting short homologous arm, and tetracycline operator gene sequences are inserted in multiple transcriptional start sites of a target gene promoter, so that the recombinant expression vector can be used for conditional research of the functions of a certain functional gene in a plasmodium genome. Furthermore, a functional gene expression sequence, corresponding to a target gene, in the plasmodium genome is knocked out by means of the gene knockout technique; meanwhile, the recombinant expression vector is transfected into a plasmodium with genes knocked out, so that the controllable genome-modified plasmodium is obtained; a new technical scheme is provided for further research of the functions of all functional genes in the plasmodium genome, and application prospects are broad.

The invention relates to an alkaline protease gene and a construction method of recombinant bacillus subtilis strain thereof, belonging to the field of genetic engineering. According to the invention,the gene sequence of the alkaline protease BmP is optimized according to bacillus subtilis codon preference to obtain Bmp-opt, and at the same time, the alkaline protease, neutral protease and mediumtemperature amylase of bacillus subtilis 168 are knocked out through a gene knockout technique to obtain a mutant strain Bs1. The optimized alkaline protease gene Bmp-opt is expressed in the mutant strain Bs1, and finally the recombinant bacillus subtilis strain efficiently expressing the alkaline protease BmP is obtained.

The invention discloses application of an OsDSK2a protein or an encoding gene thereof in regulation and control of the resistance of rice to rice blast. The amino acid sequence of the OsDSK2a proteinis shown in SEQ ID NO. 2. The invention, for the first time, proves that the rice DSK2a gene (Os10g0542200) is a functional gene for rice blast infection, and cloning and biological function verification of the gene have important reference significance for studying molecular mechanisms of the resistance of rice to rice blast. The invention provides an Os10g0542200 gene editing vector mediated byCas9. After rice is transformed by the vector, the expression level of Os10g0542200 can be greatly reduced, the susceptibility of the transformed plant to the rice blast is reduced along with the reduction of the expression level, the disease resistance is obviously enhanced, and there is no obvious change in growth state and agronomic traits of a transgenic plant. According to the invention, theCas9-mediated Os10g0542200 gene knockout technology can be applied to genetic engineering breeding of rice, and can be applied to production practice to improve the resistance of rice to the rice blast, so that the rice production safety is guaranteed under the current climatic condition of frequently occured rice diseases.

The invention relates to the technical field of gene knock out, and particularly discloses a method for selective breeding of ppm1g gene mutation zebrafish through gene editing. The method comprises the steps of through a CRISPR / Cas9 gene editing technique, designing an appropriate targeting site on the ppm1g gene of zebrafish, performing in vitro synthesis of sgRNA and Cas9-mRNA, performing microscopic co-injection into one cell of the zebrafish, performing embryo culturing for 50h, selecting embryos, and performing genotype analysis so as to confirm the validity of the selected site. The method can enable specific genes to be efficiently and accurately mutated in organism genomes. The method is simple to make and low in cost, shearing of a plurality of sites on the target gene can be realized at the same time, and any number of individual genes can be mutated. The ppm1g gene expression is subjected to interference, and through a heredity means, the functions are researched, so that further revealing of the entire process of the heart development and adjusting and controlling the molecular mechanisms of processes are facilitated, and the method has important significance in understanding of medical heart disease pathology and research and development of a new treatment scheme.

The invention belongs to the technical field of genetic engineering, and discloses a high-yield bacterium of glutamic acid genetic engineering and application thereof. The bacterium is corynebacterium glutamicum with deleted dtsR1 gene and over-expression of pyc gene. The invention adopts a carrier pK19mobsacB to knock out the gene dtsR1 of the corynebacterium glutamicum, uses kanamycin resistance and sucrose as selective pressure respectively to obtain a knockout gene strain deltad, and uses a carrier pVWEx1 to lead the pyc gen into an over-expressed pyruvic acid carboxylase in the deltad, thereby directionally reforming and optimizing a glutamic acid biosynthetic path to construct a genetic engineering bacterium deltad-pyc through a gene knockout technology and gene over-expression. The bacterium can improve the yield of glutamic acid and can be used for the mass production of the glutamic acid industrially.