51 results about "Gene knockin" patented technology

The invention discloses a construction method of a conditional knock-down dynactin animal model of astrocytes and an application of the animal model of the conditional knock-down dynactin animal model of the astrocytes. According to the method, a Dctn1LoxP gene knock-in mouse is hybridized with an Aldh1l1-Cre / ERT2 transgenic mouse, a target genotype mouse is obtained through three rounds of breeding, 100mg / kg Tamoxifen is continuously injected into the intraperitoneal cavity of the 2-month-old mouse for 5 days, and specific knock-down of dynactin in brain and spinal astrocytes is realized, including knockout of p150Glue and reduction of DCTN4, p50 and Arp1alpha protein levels. The model has the advantages of being high in specificity, permanent in intervention aging and capable of covering multiple life stages, the defects of a traditional model are overcome, the model can be used for researching the influence of dynactin on astrocyte neurobiological functions, a reliable tool is provided for screening related targets of nervous system diseases, and the model has important application value.

This invention provides a method for constructing a fluorescently traced mouse model and its application in the sorting of Mcpt4-positive and negative mast cells. First, the Cre gene is knocked into the Mcpt4 gene sequence of C57BL / 6 mice using CRISPR / Cas9 technology. Then, through animal hybridization and Cre-loxP recombination technology, a fluorescently traced mouse model of Mcpt4-positive mast cells is obtained. The fluorescently traced mouse model possesses a dual-fluorescent reporter gene system consisting of the red fluorescent protein tdTomato and the green fluorescent protein ZsGreen. Mcpt4-positive mast cells exhibit red fluorescence, while other Mcpt4-negative cells exhibit green fluorescence. Using this fluorescently traced mouse model, Mcpt4-positive and negative mast cells can be rapidly, accurately, and effectively sorted, providing a reliable technical basis for exploring the roles and mechanisms of Mcpt4-positive and negative mast cells in immune responses.

Developing vehicles that efficiently deliver genes throughout the human central nervous system (CNS) will broaden the range of treatable genetic diseases. Applicants engineered an AAV capsid, BI-hTFRl, that binds human Transferrin Receptor (TfRl), a protein expressed on the blood-brain barrier (BBB). BI-hTFRl was actively transported across a human brain endothelial cell layer and, relative to AAV9, provided 40-50 times greater reporter expression in the CNS of human TFRC knock-in mice. The enhanced tropism was CNS-specific and absent in wild type mice. When used to deliver GBA1, mutations of which cause Gaucher disease and are linked to Parkinson's disease, BI-hTFRl substantially increased brain and cerebrospinal fluid glucocerebrosidase activity compared to AAV9.

Provided are mice with the human ACE2 gene knocked into their genomes, methods for their production, and their use in evaluating the in vivo efficacy of antiviral agents and vaccines against SARS-CoV-2. Human ACE2 was precisely inserted into the mouse genome at the site of the mACE2 gene, establishing a novel knock-in mouse model for the human ACE2 gene at the site of the mACE2 gene.

The invention provides a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 plasmid taking a Leu2 gene as a selection marker, and belongs to the technical field of gene engineering. According to the CRISPR / Cas9 plasmid developed by the invention, Cas9 protein and components required for transcription of sgRNA are constructed on one plasmid, and the complete CRISPR / Cas9 system assembly can be realized only by one-step construction. And the plasmid is used for knocking out an endogenous gene Yl4HPPD (YALI1B28454g) of the yarrowia lipolytica, and meanwhile, an exogenous gene EcaroGS180F from escherichia coli is introduced, so that the betacyanin production capacity of the strain BE12 is further improved. According to the invention, exogenous gene knock-in is successfully realized, the high-yield betacyanin strain BE26 is obtained, and the total yield of betacyanin is 66.1 mg / L after the strain is cultured in an SC liquid culture medium for 48 hours.

The invention relates to the field of gene vector construction, in particular to construction and application of a gene vector integrating fluorescent screening and self-deletion functions, the gene vector comprises a conditional knock-out (cKO) vector and a gene knock-in (KI) vector which are both integrated with a Dre-Rox mediated self-deletion module and a fluorescent screening module, the construction method comprises the following steps: carrying out HindIII / EcoRI double enzyme digestion on a pUC19 vector, carrying out homologous recombination amplification on a target fragment, carrying out connection transformation and screening verification to obtain a qualified vector; the vector is applied to preparation of cKO / KI gene modified mice, and efficient screening of positive individuals is realized through fluorescence preliminary screening, genotype identification and fluorescence quenching detection. The positive screening workload and cost can be reduced by 70% or above, non-target elements are accurately cut off, interference is avoided, time-space accurate regulation and control are achieved, carrier construction is easy and convenient, repeatability is high, and the method is suitable for gene function research, disease model construction and drug target verification.

The application provides a method for constructing a Bmp4 gene knock-in mouse model, and the method comprises the following steps: obtaining sgRNA12 shown in SEQ ID NO. 3; mixing the sgRNA12, Cas9 mRNA and a targeting vector shown in SEQ ID NO. 1, and then microinjecting into a mouse zygote to obtain F0 generation mice; selecting F0 generation positive mice in genotype identification results of the F0 generation mice, and mating the F0 generation positive mice with wild type mice to obtain F1 generation mice with stable genotypes; and screening gene targeting mice with correct recombination, and mating the gene targeting mice with retinal specific knock-out tool mice Six3-Cre to obtain Bmp4 gene knock-in model mice. The method has high gene editing efficiency, and the transgenic mouse can highly express human Bmp4 protein.

The invention belongs to the technical field of gene engineering, and particularly relates to a neural crest stem cell tracing system based on P75NTR-T2A-eGFP and application. The invention focuses on construction and application of a tracing system for differentiating pluripotent stem cells into neural crest stem cells in vitro. By constructing a Crispr / Cas9 vector, a termination codon region of a P75NTR gene expression cassette is precisely targeted. Meanwhile, a gene knock-in vector containing an (LHA) left homologous arm, T2A-eGFP-right homologous arm (RHA) is constructed. The two vectors are introduced into pluripotent stem cells (hPSCs) by means of electric shock transfection. Through the operation, the P75NTR-T2A-eGFP gene knock-in pluripotent stem cell line is successfully obtained, a foundation is laid for building a follow-up tracing system, and the P75NTR-T2A-eGFP gene knock-in pluripotent stem cell line has important application value in the field of neural crest stem cells.

The invention discloses a method for creating high-anthocyanin tomatoes through a gene knock-in technology. A homologous recombination mediated gene knock-in technology is utilized, the Aft gene of the tomato is knocked into the downstream of a promoter of a fruit ripening specific gene E8 at a fixed point, the E8 promoter is successfully utilized to activate Aft gene expression, and the high-anthocyanin tomato with purple black tomato fruit is obtained; and further performing selfing on the high-anthocyanin tomatoes, and screening out non-transgenic high-anthocyanin tomatoes which are subjected to Aft gene homozygous insertion and do not carry exogenous DNA fragments from selfing filial generations of the high-anthocyanin tomatoes. Therefore, the gene editing method disclosed by the invention can be used for quickly converting the common tomato without accumulation of anthocyanin into a high-nutrition tomato material rich in anthocyanin, and has great breeding application prospect and economic value.

The invention discloses a method for improving gene editing efficiency by using fusion protein, and belongs to the technical field of biological medicine. The invention provides a fusion protein, which is a product obtained by fusing a crispr-cas9 protein and a substrate protein of an APC / C compound. The invention also provides a gene editing method based on the CRISPR-Cas9 system, which comprises the following steps: constructing a Cas9 expression vector containing nucleic acid for coding the fusion protein, and co-transfecting the Cas9 expression vector and a gRNA expression vector to a target cell. According to the invention, the crispr protein and the substrate protein of the APC / C compound are fused, so that the crispr mainly plays a role in the G2 / S period, the efficiency of gene knock-in by a crispr method is further improved, and help is provided for application of the crispr in the field of cell therapy.

The application discloses an sgRNA capable of effectively editing a pig CCR5 gene and application thereof, and is premised on an sgRNA capable of specifically recognizing a pig CCR5 gene in a pig genome, and successfully constructs a pig fetal fibroblast cell, a pig kidney cell line PK15 cell line and a pig 3D4 / 21 cell line with EGFP site-directed integration by using CRISPR / Cas9 mediated gene knock-in technology, and the results show that the EGFP gene can be stably and efficiently expressed in the three kinds of cells or cell lines. The application provides more site selection and reference for gene function research, gene modified pig research and development.

The invention provides a method for efficient site-directed gene knock-in in hepatocytes and application of the method. The invention provides a technical scheme for carrying out gene knock-in operation by taking proliferated human hepatocytes (Pro HHs) as target cells, which is characterized in that the gene knock-in operation is carried out by taking the proliferated human hepatocytes (Pro HHs) as the target cells. Through the optimization strategy of the system, the Pro HHs subjected to gene editing can be effectively recolonized and mature, and the treatment of diseases is successfully realized. According to the invention, powerful concept verification is provided for autologous gene edited proliferative cell treatment of human hereditary liver diseases, and a new way is opened up for treatment of liver diseases.

Provided is a method for constructing a humanized FKBP8 gene knock-in animal model, comprising: (1) constructing a targeting vector for humanized FKBP8 gene knock-in, and transfecting the targeting vector, Cas9, and sgRNA into fertilized eggs of donor animals; (2) transplanting surviving fertilized egg cells into the oviduct of pseudopregnant animals to obtain F0 generation animals and identifying the F0 generation animals, wherein animals showing positive homologous recombination are chimeric animals; and (3) mating the chimeric animals with wild-type animals to obtain F1 generation animals and performing identification, wherein F1 animals showing positive homologous recombination are humanized FKBP8 knock-in animals. In the construction method, an FKBP8 gene sequence in a donor animal is substituted by a human FKBP8 gene sequence, so as to achieve the purpose of humanizing the FKBP8 gene in the donor animal, thus successfully establishing a humanized FKBP8 gene knock-in animal model, so that screening for drugs targeting the FKBP8 gene and a product thereof in the animal model, or studying the pathogenesis and treatment mechanisms of the FKBP8 gene and the product thereof in related clinical diseases can be realized.

A method for constructing a mouse model for rapid degradation of Myostatin (MSTN) protein is provided. The method includes: inserting sequentially a 5′ homologous arm, 3 X (Glu-Ala-Ala-Ala-Lys) (3xEAAAK), an FK506-binding protein (FKBP1A) gene synonymous mutation sequence, 3xEAAAK, a red fluorescent protein monomeric Cherry (mCherry) sequence and a 3′ homologous arm into a Donor vector backbone to obtain a Donor recombinant vector; designing a Guide Ribonucleic Acid (gRNA) based on a third exon of the Mstn gene; mixing the gRNA, CRISPR-associated protein 9 (Cas9) protein, and the Donor recombinant vector and co-injecting a mixture of the above into mouse fertilized eggs, culturing the fertilized eggs in vitro and transplanting to obtain the F0 generation mice; and hybridizing the F0 generation mice with wild-type mice, or further breeding hybridized offspring to obtain a knock-in mouse model.

Compositions and methods are provided for genetically modifying a cell to introduce both a gene knockin and a gene knockout. The subject methods use a donor polynucleotide comprising a gene knockin sequence and a knockout guide RNA sequence. Transcription of the donor polynucleotide, after integration of the donor polynucleotide into the genome, produces a mature mRNA sequence comprising the knockout guide RNA. An RNA-guided nuclease is used to excise the knockout guide RNA from the mRNA transcript, which then guides the RNA-guided nuclease to a second target locus where the RNA-guided nuclease creates a double stranded DNA break, resulting in gene knockout at the second target locus. Only cells with the gene knockin are able to express the mRNA containing the knockout guide RNA so that the gene knockin at the first genomic target locus and the subsequent gene knockout at the second genomic target locus are linked.

The invention discloses a construction method and application of a Nox5 gene knocked-in mouse pulmonary fibrosis animal model, and relates to the field of biomedicine.The construction method comprises the following steps that Cas9mRNA, gRNA and homologous recombinant vectors are constructed and jointly injected into mouse fertilized eggs, and F0-generation mice are obtained; mating the F0-generation mice with the wild type to obtain F1-generation mice, and mating the F1-generation mice with the wild type to obtain F2-generation mice; inbreeding the F2-generation mice to obtain F3-generation mice, and hybridizing the F3-generation mice with CDH5 / cre mice to obtain Nox5 < + > / < + > / cre-CDH5 mice; injecting a bleomycin solution into the trachea to obtain a pulmonary fibrosis animal model; according to the construction method and application of the Nox5 gene knocked-in mouse pulmonary fibrosis animal model, the specificity of genome editing is improved through gRNA, the off-target effect is avoided, the cost is low, the success rate is high, the practicability is high, and convenience is provided for researching the effect of the Nox5 gene.

An amyotrophic lateral sclerosis (ALS)-simulating model and a method for constructing the ALS-simulating model based on a caspase-4 (CASP4) gene are provideds. The method includes: (1) constructing a targeting fragment for knock-in of the CASP4 gene; (2) injecting gRNA, Cas9 mRNA, and the targeting fragment into a mouse zygote, culturing, and passaging to produce a hCASP4flox mouse with the CASP4 gene stably inherited; and (3) crossing the hCASP4flox mouse with a Cre driver mouse to produce a double-positive heterozygous mouse, which is a mouse model in which the CASP4 gene is specifically expressed in a nervous system. An ALS-simulating animal model is constructed based on a humanized CASP4 gene. The method can effectively avoid the mouse death caused by this apoptotic factor, and leads to an ALS-simulating mouse model in which TDP-43 fragments accumulate in the cytoplasm and TDP-43 is deleted in the nucleus.

The invention relates to a myogenic cell rapid proliferation gene knock-in bovine primary cell and a preparation method thereof, and belongs to the technical field of gene editing and biology. Specific sgRNA is designed aiming at a bovine genome safety site H11, and directional insertion of a target gene is realized by taking a gene containing a homologous arm and a muscle-promoting cell proliferation gene Ufc1 and a gene Ufl1 as a donor template through Cas9 protein mediated DNA double-strand cutting. The method has the advantages that the cutting efficiency of the safety site H11 is improved to 90% or above, so that the obtaining rate of the positive monoclonal cell strain can be remarkably improved; the ssDNA is used as a donor fragment to be introduced into bovine fibroblasts, the survival rate of electrotransfected cells is two times that of double-stranded plasmid DNA, and meanwhile, the transfection efficiency is improved by more than four times. The cell proliferation efficiency of the Ufc1 gene knock-in cell strain is improved by 1.6 times compared with that of a wild type, and the Ufc1 gene knock-in cell strain is applied to cattle and provides important technical support for genetic improvement of muscle tissue engineering and animal husbandry.

The invention relates to a Kdm6a gene knock-in mouse model as well as a construction method and application thereof. According to the model, an expression cassette containing a CAG promoter, a Kozak sequence, mouse Kdm6a CDS, P2A peptide, an EGFP reporter gene, a WPRE element and a BGH pA tail is inserted into a mouse H11 site. According to the scheme provided by the invention, the Kdm6a gene expression can be observed in real time, the Kdm6a gene expression in the model is stable, endogenous gene interference is avoided, and the model can be widely applied to preparation of Kabuki syndrome and tumor models and screening of targeted drugs.

The application discloses a method for constructing a fluorescently labeled piwil1 gene knockout sterile zebrafish line, which utilizes a CRISPR / Cas9 system to construct a piwil1 gene knock-in vector carrying P2A-EGFP or P2A-mCherry, respectively, to obtain two hybrid strains through microinjection, and then to screen a homozygote expressing EGFP and mCherry simultaneously by mating the two strains. The homozygote shows male sterility and can be used for reproductive development research and ecological prevention and control of transgenic fish. The application realizes visual labeling of the function of the piwil1 gene and accurate screening of the sterile phenotype, and is simple and efficient to operate.

The application discloses a hepatitis B surface antigen specific B cell receptor gene knock-in mouse model, and establishes a hepatitis B virus surface antigen (HBsAg) specific B cell receptor (BCR) gene knock-in mouse model, wherein 70-90% of B cells of the mouse can specifically recognize HBsAg, and differentiate into germinal center B cells and plasma cells. Therefore, the model can be used for basic research on hepatitis B antiviral humoral immune response and tolerance mechanism, and can also be used for application research on development of drugs and vaccines for breaking HBsAg immune tolerance. In summary, the establishment of the model lays a solid foundation for development of a functional cure immune means for hepatitis B.

The present application relates to a kind of construction method of macrophage specific fluorescent reporter mouse model, belong to genetic engineering and genetic modification technical field.The present application is first by CRISPR / Cas9 genome editing technology to C1qc gene site is edited after obtaining a kind of brand-new C1qc-CreChy gene knock-in mouse model, realizes the expression of Cre and mCherry is directly driven by C1qc promoter, and it is verified by flow cytometry that mCherry only in the microglia of mouse brain and the macrophage in spleen has very high expression, and mCherry expression is not detected in other immune cells, to realize the tracing of the two kinds of macrophages, for studying its function.The present application provides a kind of usable animal model for studying the heterogeneity of macrophage, and provides a kind of new animal model for studying the complex immune function in body.

The invention relates to the technical field of gene engineering and cell sensing, in particular to a fluorescent report cell line based on RSAD2 gene transcriptional activity detection and a preparation method thereof, which are a report strategy capable of converting the RSAD2 gene transcriptional activity into tdTomato fluorescent signals, take the transcriptional activity of an antiviral gene RSAD2 as a core, and utilize a CRISPR / Cas9 mediated gene knock-in technology to detect the RSAD2 gene transcriptional activity. A synchronous expression system of the RSAD2 gene and the tdTomato fluorescent protein is successfully constructed in a pig kidney cell line, and the LLC-PK1 cell line constructed by the report strategy can efficiently and sensitively convert the transcriptional activity of the RSAD2 gene into a tdTomato fluorescent signal, and can be widely applied to the aspects of pathogen detection, environmental monitoring, food safety, drug and poison research and the like.

The invention discloses a gene knock-in mouse model with an eGFP-flpo fusion expression element inserted into a V1aR gene as well as a construction method and application of the gene knock-in mouse model, and belongs to the technical field of bioengineering. A targeting vector containing an eGFP-flpo fusion expression element and a homologous arm is designed, a specific sgRNA-guided CRISPR / Cas9 system is combined to realize precise cutting of a V1aR gene locus, a homologous recombination technology is utilized to insert a bifunctional element into an endogenous V1aR gene in a targeted manner, and a gene knock-in mouse model with functions of fluorescence labeling (eGFP) and recombinase (Flpo) is constructed. The model reserves a V1aR endogenous expression function, meanwhile, the gene expression dynamic state is visualized through fluorescent protein, specific genetic manipulation is achieved through an Flpo-FRT system, and the model can be combined with a Cre tool mouse for use to carry out complex gene regulation and control research. The invention solves the problems of insufficient targeting and single function of the traditional research means, and provides an efficient in-vivo experiment platform for researching the physiological function of the V1aR gene and the action mechanism of the V1aR gene in neuropsychiatric diseases and developing precise therapeutic drugs.

The invention discloses a construction method of a microglial cell conditional knock-down dynactin animal model, which comprises the following steps: selecting a Dctn1LoxP gene knock-in mouse, hybridizing the Dctn1LoxP gene knock-in mouse with a Cx3cr1-Cre transgenic mouse, and carrying out three rounds of breeding to obtain a genotype Dctn1LoxP / LoxP; a mouse of Cx3cr1-Cre; the expression of Cre recombinase is driven by a Cx3cr1 gene promoter, and the 2nd to 4th exons of the Dctn1 gene are cut, so that the knockout of p150Glue and the reduction of the protein levels of other subunits of dynactin are realized. The model is strong in specificity and high in knock-down efficiency, animals show microglial cell morphology abnormity and movement coordination function defects, the model can be used for researching the influence of dynactin on microglial cell neurobiological functions, and a reliable tool is provided for pathogenesis exploration and drug screening of related neurological dysfunction.

The invention discloses a gastric cancer drug-resistant animal model with clinical chemotherapy drug resistance and a construction method of the gastric cancer drug-resistant animal model. The model is characterized in that the model carries Cldn18-CreERT2; apcfl / fl; trp 53fl / fl; the method comprises the following steps: hybridizing a gastric cancer spontaneous tumorigenesis transgenic mouse of KrasG12D (Cldn18-ATK) with a CadD1371A / D1371A gene knock-in mouse, so as to obtain a Cldn18-ATK CadD1371A / D1371A double gene edited mouse; experiments show that the tumor volume reduction degree of the model after 5-FU chemotherapy is obviously lower than that of a control group (Cad + / +), and the proliferative activity (Ki-67 level) and the lifetime prolonging effect are also obviously weakened, so that the model simulates the clinical chemotherapy drug resistance phenotype. The model provides an efficient tool for gastric cancer drug resistance mechanism research and chemotherapy drug screening, and has important application value.

The invention discloses a BGL coding gene of Aspergillus niger AGM01 and a gene editing method and application thereof, the BGL coding gene is an HQM09510 gene, the nucleotide sequence is SEQ ID NO: 1, and the amino acid sequence is SEQ ID NO: 2. According to the gene editing method, a gene knock-in system is constructed by means of a CRISPR / Cas9 system, a genetic engineering aspergillus niger strain O9510 is obtained, efficient expression of BGL is achieved, and a scientific foundation is laid for improving the enzyme production capacity of aspergillus niger AGM01 by improving the aspergillus niger AGM01.

The present invention relates to a humanized cereblon (CRBN) knock-in mouse model and a method for evaluating the efficacy of multiple myeloma chemotherapy by using same. Specifically, the present invention relates to a humanized CRBN knock-in mouse model in which a human CRBN gene has been inserted into a CRBN knock-out mouse, and a method for evaluating the efficacy of multiple myeloma chemotherapy by using same. The humanized CRBN knock-in mouse model and the method for evaluating the efficacy of multiple myeloma chemotherapy by using same, according to the present invention, enable low-cost and high-efficiency evaluation of the efficacy of an anticancer chemotherapeutic agent according to the expression of human CRBN in multiple myeloma, in the mouse model, thus enabling research results to be effectively applied to clinical practice.

The invention relates to a vector for improving gene knock-in efficiency and a method for verifying accurate gene knock-in efficiency. The vector comprises a gene knock-out vector, an auxiliary knock-in vector and a donor template vector, the gene knockout vector comprises sgRNA and a knockout vector skeleton, the sgRNA comprises a sequence complementary to a target gene, and the skeleton comprises a sequence for coding a Cas protein and has cleavage activity; the auxiliary knock-in vector comprises crRNA of a 5'and / or 3 'homologous arm of a targeted donor template and an auxiliary knock-in vector skeleton, the crRNA comprises a sequence complementary to the homologous arm, the skeleton comprises a sequence for coding nickase inactivation type Cas protein, and the crRNA and the sequence form a ribonucleoprotein complex to realize an auxiliary knock-in function; the donor template carrier comprises a donor template and a donor template carrier skeleton. The method realizes breakthrough improvement of the gene accurate knock-in efficiency, can quantitatively evaluate the gene accurate knock-in efficiency, and shows important potential in the field of gene therapy.

The invention discloses a construction method of an IER2-T2A-EGFP gene knock-in mouse model and application of the IER2-T2A-EGFP gene knock-in mouse model in myeloid cell sorting, and the method comprises the following steps: aiming at a knock-in target of a mouse IER2 gene, designing sgRNA with a sequence as shown in SEQ ID No: 2; the constructed PX459-sgRNA recombinant plasmid is used as a template, and a single-chain sgRNA + tracr RNA is obtained through PCR (Polymerase Chain Reaction) amplification and in-vitro transcription; synthesizing a Donor sequence containing the T2A-EGFP gene in an overlapping PCR (Polymerase Chain Reaction) mode; a single-chain sgRNA + tracr RNA, a Donor sequence and Cas9 protein are co-injected into male pronucleus of a mouse fertilized egg, and a female mouse is transplanted for inoculation, so that the F0-generation IER2-T2A-EGFP gene knock-in mouse model is obtained. According to the method, the primary myeloid cells can be quickly, conveniently and accurately sorted.