31 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.

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 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 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.

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 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 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 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.

The invention provides a method for improving plant genome editing efficiency based on a transcription factor AtSOG1 and application of the method, and belongs to the technical field of gene editing. The invention finds that the AtSOG1 transcription factor has multiple influences on gene editing, a target plant is co-transformed by using an expression vector constructed by the AtSOG1 transcription factor and a gene editing vector, or an expression element of the AtSOG1 transcription factor is constructed into the gene editing vector, and then the target plant is transformed, so that the plant genome editing efficiency can be improved. The method provided by the invention not only can promote targeted gene knock-in based on a PE editor and HDR mediation, but also can improve the knockout efficiency of long fragments. Meanwhile, in the embodiment of the invention, an experiment is further designed, it is found that the AtSOG1 can promote a T-DNA unconformity knock-in event, and a T-DNA free knock-in plant is directly obtained in a T0 generation.

The invention relates to a hypomyelination leukodystrophy animal model and a construction method thereof, and belongs to the technical field of animal model construction. The invention provides sgRNA (small guide ribonucleic acid) targeting c.224 and c.225 sites of Tmem163 of a mouse, wherein the nucleotide sequence of the sgRNA is as shown in SEQ ID NO. 1. The sgRNA is very high in cutting activity on the c.224 site and the c.225 site of the Tmem163; after the sgRNA, ssODN and Cas9 mRNA are introduced into a mouse fertilized egg together, gene knock-in and knockout can be carried out by targeting c.224 and c.225 loci of mouse Tmem163, and then a Tmem163 mutation related myelin sheath formation low white matter dystrophy animal model with a clearer phenotype is constructed and obtained, is used for related researches such as HLD natural disease course, pathogenesis and preclinical drug tests, and has a great application prospect.

A method for improving the efficiency and accuracy of gene knock-in using the non-residence end of Cpf1, where the free end of a target DNA and the free end of a donor DNA are generated using single or paired Cpf1, and combined with a complementary 5′-sticky end generated by Cpf1, so as to realize a more efficient and more accurate gene knock-in based on c-NHEJ. The method can perform NHEJ repair more efficiently and more accurately, and the accuracy can be increased from 0% to 100%. The free end of Cpf1 binds to and is protected by NHEJ core factor KU70 / KU80, thereby making the ligation via NHEJ more efficient and accurate, which provides a foundation for the use of the free end of Cpf1 to improve the efficiency and accuracy of gene knock-in based on NHEJ, realizes gene knock-in or gene correction, and is suitable for non-dividing cells.

The invention provides a reporter plasmid system for multi-gene fixed-point knock-in cells and a screening method. Specifically, the invention provides a set of multi-gene knock-in reporter plasmid system which comprises sgRNA plasmids of a plurality of target genes, a plurality of genome donor plasmids, a target gene knock-in reporter plasmid, a reporter plasmid donor plasmid and a Cas9 plasmid. Wherein the reporter plasmid comprises a truncated eGFP (enhanced green fluorescent protein) gene, a target site sequence is introduced, and a gene knock-in event is accurately reported. The invention further discloses a screening method which is a method for screening simultaneous knock-in of multiple genes only through one target gene knock-in reporter plasmid during multi-gene knock-in editing, and the efficiency of sorting gene homozygous knock-in cells is improved. The method can be efficiently used for gene knock-in through HDR repair, including accurate point mutation and large fragment insertion or deletion, and a clean gene knock-in cell line without residues in a report system is obtained.

The invention discloses a construction method and application of an IER2-Flag gene knock-in mouse model, and the construction method comprises the following steps: determining a target site of an IER2 gene to be knocked in a mouse, and designing sgRNA with a sequence as shown in SEQ ID No: 2; the method comprises the following steps: carrying out recombination on double-stranded sgRNA formed by annealing and CRISPR-Cas9 plasmid PX459, and constructing a PX459-sgRNA recombinant plasmid of which the sequence is as shown in SEQ ID No: 3-4; the method comprises the following steps: by taking a recombinant plasmid as a template, carrying out PCR (Polymerase Chain Reaction) amplification on a double-stranded DNA (Deoxyribose Nucleic Acid) fragment of a T7 promoter sequence + sgRNA + tracr RNA, and carrying out in-vitro transcription to obtain single-stranded sgRNA + tracr RNA, a single-chain sgRNA + tracr RNA, a Donor sequence containing a 3 * Flag tag and Cas9 Protein are co-injected into a male pronucleus of a mouse fertilized egg, an F0-generation IER2-Flag gene knock-in mouse can be obtained until an F3-generation IER2-Flag gene knock-in mouse is obtained, and the IER2-Flag gene knock-in mouse model is obtained. According to the invention, the expression of the Flag tag is detected for the first time to further identify that the IER2 protein is continuously and strongly expressed in the enamel cell, and an important animal model is provided for the research on revealing a mechanism for regulating and controlling enamel mineralization by the IER2 gene.

This invention discloses a method for constructing a TrpA1-3×FLAG-P2A-iCre gene knock-in mouse. A fragment containing a 3×FLAG tag, a P2A sequence, and an iCre recombinase sequence is inserted into the 27th exon region of the mouse TrpA1 gene using a CRISPR / Cas9 system, achieving genetic labeling and Cre functionalization in TrpA1-expressing cells. The donor vector provided by this invention contains a 5' homologous arm and a 3' homologous arm that are homologous to the TrpA1 genome sequence. Stably inherited knock-in mice are obtained by microinjecting the CRISPR / Cas9 system and the donor vector into fertilized eggs.

The invention relates to an animal model for endogenous Col1a1 + cell lineage tracing and a construction method and application thereof.The construction method comprises the steps that firstly, a gene with the genotype of Col1a1-CreERT2-P2A is constructed and knocked into a mouse, the mouse is subjected to mating breeding with a fluorescent report mouse (such as CAA-LoxP-ZsGreen-Stop-LoxP-tdTomato), and a double-genotype mouse is obtained; the mouse model is used for detecting the expression condition of Col1a1 in tissues such as skull, cranial suture, joint and spine for the first time, and the fate of a cell lineage expressed in real time by using tdTomato fluorescent protein to mark endogenous Col1a1 in the processes of tissue and organ development, regeneration and repair and the like can be realized.

The invention discloses a method for improving gene knock-in efficiency through combination of homologous recombination repair protein and a cell cycle retardant, and belongs to the technical field of gene editing. In order to improve CRISPR / Cas9 mediated homologous orientation / recombination repair efficiency and gene knock-in efficiency, the invention provides a method for improving gene knock-in efficiency by combination of homologous recombination repair protein and a cell cycle blocker, through combination and synergistic interaction of the homologous recombination repair protein and the cell cycle blocker, the gene knock-in efficiency is improved in HEK293T, Hela and HSkMC cell lines, and the gene knock-in efficiency is improved. The KI efficiency is improved by 1.73 times, 1.8 times and 1.9 times respectively; a new strategy is provided for optimizing the gene editing technology, and practical application of the gene editing technology in the fields of disease treatment, medicine research and development and the like is promoted.

The application belongs to the technical field of biology and particularly relates to a construction method of a Cyp11b2-P2A-CreERT2 gene knock-in mouse model and application thereof. The construction method comprises the following steps: introducing a CRISPR / Cas9 system and a homologous recombination donor vector into a mouse zygote, then transplanting the zygote into a pseudopregnant female mouse, and waiting for the female mouse to become pregnant and give birth to a baby; F0 generation of the recipient mouse is born, the genotype is confirmed, and after the positive F0 generation mouse matures, the mouse is mated with a wild-type background mouse to generate F1 generation, the genotype is confirmed, and the obtained heterozygote is the Cyp11b2-P2A-CreERT2 gene knock-in mouse model. The mouse model constructed by the construction method can realize specific expression of Cre in adrenal glomerular zone, is time and space controllable and has no off-target, the P2A peptide guarantees normal expression of the gene independently, the traditional model defects are avoided, the mouse model is stable, repeatable, and can be used for aldosterone-related mechanism research and drug screening.

The present invention relates to use of EPO knock-in pluripotent stem cell–derived mesenchymal stem cells and exosomes isolated therefrom for treating renal diseases. According to the present invention, in order to maximize the expression of EPO, induced pluripotent stem cells having the EPO gene knocked-in therein were prepared using a CRISPR-Cas9 gene editing technology, and then differentiated to establish mesenchymal stem cells in which EPO expression and secretion are significantly increased. It was confirmed that exosomes isolated therefrom contain and secrete a high concentration of EPO to treat nephrotic anemia, improve renal function, inhibit and alleviate renal fibrosis, and provide a renal protective effect in an animal model of chronic kidney disease, and thus the exosomes can be used for the prevention or treatment of chronic kidney disease or nephrotic anemia caused thereby.

Provided in the present invention are a method for efficient site-specific gene knock-in in hepatocytes and the use thereof. Provided in the present invention is a technical solution for performing gene knock-in operation using proliferated human hepatocytes (ProliHHs) as target cells. By means of a systematic optimization strategy, gene-edited ProliHHs can effectively repopulate and mature to realize successful disease treatment. The present invention provides a powerful concept verification for autologous gene-edited proliferative cell therapy for human hereditary liver diseases, and opens up a new way for the treatment of liver diseases.