68 results about "Gene knockin" patented technology

The invention discloses a set of LSR gene recombination system for plants, and belongs to the technical field of biology. The recombinant system comprises Kp03 macroserine recombinase and attP and attB corresponding to the Kp03 macroserine recombinase, and efficient gene knock-in editing can be realized in rice and arabidopsis thaliana cells without additional cofactors. The gene editing protein Kp03 can show efficient plasmid level gene knock-in capability in rice and arabidopsis thaliana cells, a 27kb large DNA fragment can be inserted into an attB target site to the maximum extent, and effective gene knock-in editing can still be performed after an attB sequence is truncated to 15bp, so that the application range of gene editing is expanded. In addition, 3.4 kb exogenous DNA fragments can be successfully inserted into the rice genome, a new gene editor is provided for plant large fragment gene knock-in, and the gene knock-in method has important application value.

The invention discloses a construction method and application of a GSDMB intestinal epithelium specific knock-in mouse model. The construction method comprises the following steps: (1) designing gRNA according to an ROSA26 gene, constructing a Donor vector containing a GSDMB gene, introducing the gRNA and the Donor vector and Cas mRNA into a fertilized egg of a receptor mouse, transplanting the fertilized egg and Cas mRNA into a pseudopregnant mouse body for reproduction, and screening out a GSDMB gene knock-in F0-generation mouse; (2) mating and breeding the F0 generation mouse and a wild type mouse, and screening out an F1 heterozygote mouse with GSDMB knocked in; (3) carrying out inbreeding propagation on the F1 heterozygote mouse, and screening out an F2 homozygote mouse in which GSDMB is knocked; (4) the F2 homozygote mouse and the intestinal epithelium specific Vilin-Cre transgenic mouse are subjected to mating breeding, and a heterozygote mouse with GSDMB intestinal epithelium specific knock-in is screened out; and (5) carrying out inbreeding propagation on the heterozygote mouse with the GSDMB intestinal epithelium specific knock-in, and screening out the homozygote mouse with the GSDMB intestinal epithelium specific knock-in.

The invention discloses an E. coli engineering bacterium capable of efficiently expressing hydroxylated collagen. The E. coli engineering bacterium is prepared by knocking out a gene proA / proB of a proline metabolic pathway in an E. coli strain; meanwhile, the P4H gene is knocked into an E. coli strain body to supplement or regulate hydroxylation modification of proline, and a strain which is expected to realize large-scale stable mass production of high-hydroxylation modified collagen is obtained. The problem that the recombinant collagen cannot be hydroxylated or is insufficient in hydroxylation in a traditional prokaryotic expression system is solved, a stable hydroxylated collagen expression strain and a culture method are constructed, possibility is provided for obtaining more ideal triple-helix stable and functional collagen products, and stable and large-scale production of the recombinant collagen is promoted. Furthermore, rapid development of the medical field, beauty and skin care, food supplements, cosmetics and other industries related to the recombinant collagen can be promoted to a great extent.

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 present invention discloses a method for constructing a Drd3-P2A-CreERT2 gene knock-in mouse model and its application, wherein the construction method is: using the CRISPR / Cas9 system to insert a P2A-CreERT2 element before the translation termination site of the Drd3-201 transcript, and CreERT2 is expressed under the regulation of the endogenous Drd3 gene. The present invention uses CRISPR / Cas technology to insert the Cre-ERT fusion protein into the Drd3 gene sequence, realizes the manipulation of the expression of the Drd3 gene in time windows such as the embryonic stage, the developmental stage, and the mature stage, and provides an important tool for the study of the function of the Drd3 gene and the drug screening of related nervous system diseases.

The invention discloses a construction method and application of an animal model for HPV (human papillomavirus) pathogenic mechanism research, and belongs to the technical field of animal models. According to the construction method of the HPV fixed-point integration animal model, HPV16 gene knock-in is carried out on the Col1a1 gene locus of a C57BL / 6J mouse through CRISPR / Cas mediated genome engineering. The method comprises the following steps: (1) designing specific gRNA; (2) constructing a knock-in vector Donor vector; and (3) uniformly mixing Cas9 protein, gRNA and a targeting vector Donor vector to obtain an injection compound, performing micro-injection on the injection compound into a mouse fertilized egg, and sending the injection compound back to a pregnant mouse fallopian tube to cultivate offspring so as to generate a gene knock-in mouse. The animal model prepared by the method can be used as a contrast of a cervical cancer precancerous lesion animal model for scientific research.

The invention discloses a muscle cell efficient growth gene knock-in cell line construction method based on a CRISPR-Cas9 system, and belongs to the crossing field of gene editing and biotechnology. Through a CRISPR-Cas9 mediated gene targeting insertion technology, fixed-point integration of muscle efficient growth related functional genes is realized in bovine fetal fibroblasts, and a gene overexpression cell line with stable hereditary characteristics is successfully constructed. The cell line can be used as a nuclear donor, and precise creation of high-muscle-production-performance gene editing beef cattle is realized by combining a somatic cell nuclear transfer technology and an embryo transfer technology. The key technical support is provided for beef cattle molecular design breeding by directionally enhancing the cattle muscle development core regulation and control network. The genetic improvement new material cultivated based on the technology is expected to significantly improve the beef cattle muscle growth efficiency and industrial economic benefits, and shows important application value and industrialization prospects in the field of agricultural animal genetic engineering.

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 present invention relates to a mouse model of CT45 gene knock-in and its construction method and application, belonging to the field of bioengineering technology. The present invention constructs a CT45 gene knock-in vector and injects it into a mouse to obtain an F0 mouse; the F0 mouse is hybridized with a Cre-LoxP mouse to obtain a CT45 gene expression mouse; finally, the CT45 gene expression mouse is hybridized with a Sftpc-Cre mouse to obtain a gene knock-in mouse that specifically expresses the CT45 gene in the lung, and a CT45 gene knock-in mouse model is successfully constructed. According to genotype identification and experimental verification, the knock-in and specific expression of the CT45 gene cause spontaneous lesions in the mouse lungs, and lung cells become cancerous, which can cause the occurrence of mouse lung cancer in 3-6 months, so the CT45 gene knock-in mouse is a brand-new lung cancer model animal, which can be applied to the preparation of lung cancer diagnosis products and therapeutic drugs, and the screening and development of new anticancer drugs.

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 provides a method for enhancing gene knock-in efficiency mediated by a CRISPR gene editing system in a cell. The method comprises the step of enabling the cell to be in contact with a DNA-dependent protein kinase (DNA-PK) inhibitor and a cell division cycle protein 7 (Cdc7) inhibitor in the gene knock-in operation process of the cell. The invention also provides a kit for enhancing the gene knock-in efficiency mediated by the CRISPR gene editing system in cells. According to the method and the kit provided by the invention, the DNA-PK inhibitor and the Cdc7 inhibitor are combined for use, so that the gene knock-in efficiency can be remarkably improved under the conditions of different cell lines, different insertion sites and different donor template lengths.

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.