430results about "Microinjection based" patented technology

The invention provides a full-autonomous cell operation system based on body intelligence, and relates to the field of cell operation, the system comprises a system hardware architecture and a system software architecture; the system hardware architecture comprises a micromanipulation execution system, a precision positioning system, a multi-mode sensing system and an intelligent calculation and control center. The micromanipulation execution system is used for cell environment interaction, the precision positioning system is used for sample bearing and environment control, the multi-modal sensing system is used for providing key information, and the intelligent calculation and control center is used for operation decision making; the system software architecture comprises a sensing layer, a planning layer, an execution layer and an interaction layer. According to the application, cell recognition, tool selection, path planning, operation execution and result feedback can be automatically completed without manual intervention; through tool autonomous switching and strategy dynamic adjustment, various operations such as cell clamping, injection, controllable deformation and micro-assembly can be completed, and the problems that traditional cell operation depends on manual work, efficiency is low, and consistency is poor are solved.

The invention discloses a construction method and application of an NMD escape model of an Alport syndrome mouse. A non-human animal model carrying Col4a5 gene c.4432delG frame shift mutation is prepared on the basis of a CRISPR / Cas9 gene editing technology. The method comprises the following steps: co-injecting gRNA of a 49 exon of a targeted Col4a5 gene, homologous recombinant donor oligonucleotide containing c.4432delG mutation and Cas9 nuclease into a mouse fertilized egg, and carrying out embryo transplantation to obtain an F0-generation mutant mouse; a mutation site is verified by combining PCR (Polymerase Chain Reaction) with sequencing, and a stably inherited mutation line is established through two generations of breeding. Through verification, the model accords with pathological characteristics of the Alport syndrome, can stably simulate typical clinical manifestation and pathological characteristics of the human X-linked Alport syndrome, and can be used as an important tool for research of the Alport syndrome.

The invention provides an application of WNT7B in construction of a myopia animal model. Homologous genes wnt7ba and wnt7bb of the WNT7B in the zebra fish are knocked out or knocked down by applying a CRISPR / Cas9 gene editing technology, an animal model of which the eye axis length is remarkably increased, the eyeball movement frequency is remarkably reduced and the light response is reduced is obtained, and the result shows that the WNT7B is related to the high myopia. The model provides a favorable tool for pathogenesis and drug screening of high myopia, and has a good application prospect.

The invention discloses a construction method and application of a mouse model for specifically tracing heart valve cells, and belongs to the technical field of animal model construction. The model construction method comprises the following steps: (1) constructing a Wid1-Cre hybrid mouse before inserting a Cre recombinase coding gene into a termination codon of a mouse Wid1 gene; and (2) hybridizing the Wid1-Cre hybrid mouse with the Rosa26-tdTomato homozygous report mouse, so as to obtain the double transgenic mouse which simultaneously carries the Wid1-Cre gene and the Rosa26-tdTomato gene. According to the invention, on the basis of the expression characteristic that Wif1 is only limited to a valve area in the heart, Wif1-Cre is constructed; according to the Rosa26-tdTomato mouse model, specific tracing of heart valve cells is achieved, and an important experimental tool is provided for mechanism research of heart valve diseases and development of therapeutic drugs.

The invention relates to the technical field of fish genetic breeding, in particular to a method for improving grass carp reovirus resistance of grass carp. Four specific knockout targets of the herc4 gene are disclosed, the grass carp herc4 gene is specifically knocked out by utilizing a CRISPR / Cas9 gene editing technology, and a herc4 gene knockout individual, namely the high-resistance grass carp reovirus fish, is obtained. According to the invention, the function of the herc4 in the grass carp is verified for the first time, a new grass carp germplasm with improved grass carp reovirus resistance is rapidly obtained by knocking out the single gene of the herc4, and the herc4 has important industrial value.

This invention discloses a protein and gene related to resistance to neonicotinoid acetylcholine receptor competitive regulator insecticides in peach aphids, and their applications. It relates to the field of screening technology for neonicotinoid acetylcholine receptor competitive regulator insecticides in peach aphids. This invention obtains and clarifies an MpeABCC_4G protein and its encoding gene that is closely related to the resistance of peach aphids to neonicotinoid acetylcholine receptor competitive regulator insecticides. By knocking down the expression of MpeABCC_4G in resistant peach aphids using RNAi technology, the control efficacy of neonicotinoid acetylcholine receptor competitive regulator insecticides is significantly improved.

The invention discloses application of nilaparvata lugens NlGRP protein in prevention and treatment of nilaparvata lugens, and belongs to the technical field of prevention and treatment of nilaparvata lugens. By inhibiting the expression of the coding gene of the NlGRP protein in the brown planthopper, the growth and development of the brown planthopper can be obviously inhibited. Wide application prospects are realized; the method has great application value for reducing pesticide use, maintaining ecological balance and realizing sustainable development.

The invention provides a method for overspeed preparation of gene edited goldfish. The method comprises the following steps: S1, obtaining gobiocypris rarus without primordial germ cells as receptor fish; s2, designing a target spot for the goldfish amh gene, introducing a Cas9 / gRNA complex into a goldfish fertilized egg, performing sequencing verification to obtain a mutant individual, and feeding the mutant individual as a donor fish; s3, taking gonads of donor fish, cutting into pieces, performing enzymolysis digestion, filtering, and performing density gradient centrifugation to enrich donor germline stem cells; s4, transplanting the germline stem cells of the donor fish into the receptor fish body, and feeding to obtain the gene edited goldfish sperms and ova. S5, hybridizing and feeding goldfish amh mutant sperms and ova produced by gobiocypris rarus to obtain gene edited goldfish individuals. According to the invention, gobiocypris gobiocypris is used as a receptor, the cross-subfamily gene edited goldfish is used as a donor, and an efficient, stable and easy-to-popularize method for preparing the gene edited goldfish is provided by utilizing a belly borrowing reproduction technology.

The present invention relates to a model mouse with a microglia-specific vitamin D binding protein (VDBP) gene knocked out and a construction method therefor. The construction method comprises the following steps: constructing a VDBPloxp / loxp gene mouse; hybridizing the VDBPloxp / loxp gene mouse with a Cre / ERT2 gene mouse to obtain a Cre+ / --VDBP+ / + gene mouse, wherein the microglia of the Cre / ERT2 gene mouse carry Cre gene; and inducing the expression of the Cre gene in the Cre+ / --VDBP+ / + gene mouse to obtain the model mouse with a microglia-specific VDBP gene knocked out. According to the construction method, a VDBP in the microglia of the model mouse can be specifically knocked out on the basis of Cre-loxP recombination, specific knockdown of the expression of the gene of the VDBP in the microglia is achieved, and the knockdown efficiency reaches 80% or more, facilitating more detailed study of the function of the molecule in the brain.

The invention provides sgRNA for knocking out zebra fish pde6a and application of the sgRNA in visual diseases. According to the sgRNA provided by the invention aiming at a specific position, pde6a of the zebra fish can be knocked out or knocked down in a targeted manner by utilizing a CRISPR / Cas9 system. After pde6a is successfully knocked out or knocked down, a zebra fish model with impaired visual functions and normal retina development is obtained, and the model also has morphological changes such as shorter body length, smaller eyes and shorter inter-ocular distance and also shows a trend of increasing the length of an ocular axis. The zebrafish model provides an experimental tool and a model basis for the research of visual function related diseases.

The invention provides a grouper microinjection method, and belongs to the technical field of aquaculture. According to the method, grouper eggs are injected by utilizing a grouper microinjection needle, the microinjection needle is prepared by calcining a glass capillary tube by utilizing a needle pulling instrument, and the parameters of the needle pulling instrument are set as P = 500mW, HET = 720ms, PULL = 60, VEL = 20mu m / s and TIME = 250ms; the outer diameter of the glass capillary tube is 1.0 mm, the inner diameter of the glass capillary tube is 0.75 mm, and the length of the glass capillary tube is 10 cm. The method for injecting the grouper eggs with the micro-injection needle is simple and easy to operate, exogenous gene transfer and endogenous gene accurate editing of the grouper can be realized by adopting the method disclosed by the invention, the target gene editing efficiency is high, and powerful support is provided for deeply researching gene functions of the grouper and cultivating excellent culture varieties.

This invention provides a molecular breeding method for fish with blunted and thickened intermuscular spines, belonging to the fields of gene editing and aquatic organism breeding technology. This invention utilizes CRISPR / Cas9 gene editing technology to mutate the bmp8a gene in fish, thereby obtaining fish with blunted and thickened intermuscular spines. The method of this invention is simple and easy to implement. Verification has shown that this invention can obtain a large number of stably heritable mutant zebrafish through gene knockout, and also provides an important reference for the precise design breeding of other farmed fish. It can serve as an efficient technical means to obtain new germplasm of fish with blunted and thickened intermuscular spines, possessing significant scientific research and industrial application value.

The invention discloses an RNA (Ribonucleic Acid) interference method for parasitic wasps in lepidoptera larvae, which is characterized in that RNA interference is carried out by using a microinjection method or a nano-material immersion method when the parasitic wasps in the lepidoptera larvae are not separated from hosts or are separated from the hosts to cocoon, so that an RNAi system of the parasitic wasps is successfully constructed, and the defects that the parasitic wasps are small in size in an egg stage, and the parasitic wasps are not damaged in the egg stage can be effectively overcome. And RNAi operation difficulty caused by cocoon wrapping in the pupal stage and short life in the adult stage is avoided. Moreover, the method disclosed by the invention is simple to operate and high in interference efficiency, provides a theoretical basis and a reference effect for further researching gene functions of the parasitic wasps, and has important significance on biological control.

The invention belongs to the technical field of biological genetic engineering, and particularly relates to a mouse model construction scheme capable of reproducing a key phenotype of hemophagocytic syndrome, which comprises the following steps: inserting a loxP-STOP-loxP-LMP1 conditional expression cassette into a Rosa26 safe port site in a fixed point manner to obtain an Lmp1cKI mouse strain; and hybridizing the mouse with an Ncr1-Cre transgenic mouse of NK cell specific expression Cre recombinase. In a double transgenic offspring (Lmp1Ncr1-Cre), STOP fragments are cut off by Cre enzyme, and continuous high expression of EB virus LMP1 protein in NK cells is driven. The model does not need chemical induction or immune intervention, and typical hemophagocytic syndrome phenotypes, namely serum IL-6, TNF-alpha and IFN-gamma, spontaneously appear within 7-9 days after young rats are born; the body weight is reduced and the survival rate is obviously reduced; the proportion of lymphocytes is sharply reduced; and NK cells are depleted. The invention provides a stable and reliable tool for hemophagocytic syndrome pathogenesis research, drug screening and treatment evaluation.

This invention belongs to the fields of locust biotechnology and gene editing technology, specifically relating to a method for preparing locusts. Crz Methods using gene mutants and their application in locust control. This invention discovers that inhibiting gene mutants in locusts... Crz Gene expression can be used to control locusts. Crz The gene sequence is shown in SEQ ID NO:3. This invention successfully established a homozygous Crz mutant strain of the migratory locust using gene editing technology. It was found that the albino migratory locusts obtained after Crz deletion were more easily preyed upon by natural enemies than the wild-type locusts, exhibited significantly lower levels of the aggregation pheromone 4VA, and showed weaker adaptability to low temperatures compared to the wild type. This provides a new strategy for the biological control of migratory locusts and can also serve as a reference for the biological control of other pests.

The invention belongs to the field of animal model construction, and particularly relates to application of an NFATC2IP gene knockout mouse model. Experiments prove that after the NFATC2IP gene is knocked out, the mouse has the characteristic changes of ovarian weight reduction, volume reduction, estrus cycle disorder, fertility reduction, AMH reduction, FSH increase and the like. It is indicated that the NFATC2IP gene is a key factor for maintaining ovarian reserve and endocrine balance, and an NFATC2IP gene knockout mouse can be used for revealing the occurrence mechanism of ovarian dysfunction and can also be used for screening of novel ovarian protection drugs, research of a hormone regulation mechanism and discovery and verification of biomarkers.

The invention discloses construction and application of a Primrose syndrome non-human animal model, and belongs to the technical field of animal models and disease research. According to the invention, through a CRISPR / Cas9 technology, a codon CAC for coding 596th histidine in a No.14 exon of a mouse Zbtb20 gene is subjected to site-directed mutagenesis into CGC for coding arginine, and a hybrid mouse model for simulating pathogenic point mutation (p.H596R) of human Primrose syndrome is constructed. The model can stably reproduce key clinical phenotypes of the Primrose syndrome, including overgrowth after adult, serum IGF-1 rise, memory dysfunction and anxiety behaviors, and shows abnormal hippocampal neuronal development and synaptic transfer related pathways and the like. The invention provides an important experimental tool for deeply revealing the pathological mechanism of Primrose syndrome, developing drug screening and treatment intervention research and the like.

The disclosure is directed to compositions that comprise a plasmid DNA construct having a DNA sequencing encoding a therapeutic protein, or a fragment thereof, in vivo, along with methods of generating and manufacturing the antibody or therapeutic protein, as well as methods for preventing and / or treating a disease in a patient.

The invention relates to the technical field of genes, in particular to preparation of a Trmt61a enzyme activity deleted zebrafish model, and the Trmt61a enzyme activity deleted zebrafish model is used for preparing a zebrafish strain which stably inherits trmt61aD181A-P2A-EGFP knock-in zebrafish strain. According to the invention, trmt61a D181A mutation site specificity is introduced into a zebra fish genome by using a CRISPR / Cas9 technology, and a zebra fish model with Trmt61a catalytic activity deficiency and tRNA m1A modification level significantly reduced is obtained. The zebrafish model fills the existing research blank, provides an available in-vivo research model for biological development regulation mechanism research related to tRNA m1A epigenetic modification and pathological mechanism analysis of diseases (such as malignant tumors and hematopoietic dysfunction), and can be directly used for exploring the core effect of the epigenetic modification in development and diseases.

The invention provides a culter turunx2b gene knockout method and application of the culter turunx2b gene knockout method in creation of intermuscular thorn-free mutants, and belongs to the technical field of gene editing. The invention provides an efficient gene knockout method which comprises the following steps: selecting two targets on a second exon of a culter turunx2b gene as target sites, designing and synthesizing two gRNAs, mixing the gRNAs with Cas9mRNA, introducing the mixture into a fertilized egg of culter turunx2b in an I cell stage in a microinjection manner, hatching, and screening mutated F0-generation culter turunx2b individuals. By utilizing the method disclosed by the invention, the F0-generation knockout efficiency is up to 87.5% under the condition of ensuring that the fertilized eggs are hatched to survive. The method disclosed by the invention is simple and easy to implement and simple to operate, the culter alburnus gene mutation strain can be rapidly obtained by utilizing the gene mutation method, and the method has important scientific significance for developing economic fish gene function research, revealing genetic development law and creating new germplasm.

The application discloses a method for breeding Ptereleotris radiata by using siRNA molecules interfering with melanin tyr gene expression, wherein the sequence of the siRNA molecules is shown as SEQ ID NO. 6-35, and the siRNA molecules can interfere with the expression of melanin tyr gene in the skin tissue of Ptereleotris radiata. The siRNA sequence is designed and synthesized by using the melanin gene of Ptereleotris radiata, and a powder injection preparation containing the siRNA is obtained. The powder injection preparation is dissolved and injected into the body of Ptereleotris radiata. The individuals with faded melanin in the skin tissue of Ptereleotris radiata are screened out for breeding of parent fish and cross breeding, and the offspring of Ptereleotris radiata is obtained, wherein the skin tissue traits of the offspring are the same as those of the parent fish. By using the breeding method provided by the application, the melanin in the skin tissue of Ptereleotris radiata can be reduced and stably inherited, a foundation for obtaining Ptereleotris radiata varieties is laid, and the economic value of Ptereleotris radiata is improved.

The present invention delivers a substance such as a reagent into a cell. A tubular body coated with an electroconductive polymer for introducing a substance into a cell and / or recovering a substance from a cell. The use of a tubular body coated with an electroconductive polymer to introduce a substance into a cell and / or recover a substance from a cell. A device for introducing a substance into a cell and / or recovering a substance from a cell, the device being equipped with a substrate and a tubular body coated with an electroconductive polymer, and the tubular body being positioned on the substrate. A system for introducing a substance into a cell and / or recovering a substance from a cell, the system being equipped with the abovementioned device, a voltage supply unit for supplying a voltage, and, as needed, electrodes.

The invention discloses a bactrocera dorsalis point mutation gene editing method based on CRISPR-Cas9 and homologous directional repair, and relates to the technical field of gene editing. The method comprises the following steps: designing high-specificity sgRNA for a PAM sequence adjacent region of a target gene; extending the length of the homologous arm to 100bp, and designing a single-stranded DNA (deoxyribonucleic acid) containing a mutation site as a homologous repair template; after a mixture containing the high-specificity sgRNA, the homologous repair template and Cas9 protein is injected into a bactrocera dorsalis fertilized egg in a microinjection mode, incubation and incubation are carried out, and G0-generation imagoes are obtained through culture; hybridizing the G0-generation imago with wild bactrocera dorsalis, and screening to obtain a G1-generation hybrid mutant; and carrying out selfing by using the G1-generation heterozygous mutant, and screening to obtain a G2-generation homozygous mutant. By adopting the method, the mutation efficiency and embryo survival rate of bactrocera dorsalis can be effectively improved.

The invention discloses application of an HIF1A inhibitor in preparation of a medicine for preventing and / or treating hypertrophic cardiomyopathy, and belongs to the field of biological medicine. For a rat model of spontaneous hypertrophic cardiomyopathy caused by MYH7B gene knockout, hypertrophic cardiomyopathy can be significantly relieved by intraperitoneal injection of the HIF1A inhibitor LW6, heart remodeling can be relieved, heart functions can be improved, and myocardial hypertrophy caused by silent MYH7B genes can be effectively resisted by using siRNA of mRNA transcribed by the HIF1A gene. The key mechanism of the HIF1A inhibitor for treating the hypertrophic cardiomyopathy is that the hypertrophic cardiomyopathy is prevented and treated by reducing the expression level of glycolysis key enzymes GLUT1, HK2 and the like, inhibiting the formation and accumulation of cardiac lactic acid and improving the ventricular remodeling of the hypertrophic cardiomyopathy. The results show that the HIF1A inhibitor can be used for treating and / or preventing the human hypertrophic cardiomyopathy and has the potential of being developed into the medicine for clinically preventing and treating the hypertrophic cardiomyopathy.

The invention provides a non-alcoholic fatty liver disease zebra fish model and a construction method thereof, and relates to the technical field of genetic engineering and molecular biology. The sgRNA of the targeted zebrafish fads2 gene contains a guide sequence which can be specifically combined with a nucleotide sequence shown as SEQ ID NO.4 in the fads2 gene. The sgRNA targeting the fads2 gene of the zebrafish can accurately and efficiently target and knock out the fads2 gene of the zebrafish, so that a non-alcoholic fatty liver model which is caused by DHA (docosahexaenoic acid) deficiency and is represented by liver lipid accumulation is successfully constructed, and the sgRNA is a key technical tool for constructing a specific disease model.

The invention discloses a method for regulating and controlling migratory locust embryo diapause by a BTB transcription factor LOCMI00260 and application of the method. According to the present invention, the full length of the BTB transcription factor LOCMI00260 is cloned from migratory locust, the specific double-stranded RNA interference sequence fragment dsRNA of the BTB transcription factor LOCMI00260 is developed, and the dsRNA of the BTB transcription factor LOCMI00260 is introduced into the migratory locust egg by using the microinjection method; the result shows that the migratory locust BTB transcription factor LOCMI00260 can improve the diapause rate of migratory locust. The invention provides a new means and thought for migratory locust prevention and treatment.

A method for producing a target protein, comprising: providing a host cell, a vector comprising a nucleic acid sequence encoding the target protein, and a transposase; transfecting the host cell using the vector and the transposase to obtain transfected cells; culturing the transfected cells to form a culture; and harvesting the culture to obtain the target protein from the harvested culture, wherein the cells of the culture are not passaged before the culture is harvested.

The invention belongs to the technical field of GPCR drug research and development, and discloses a ratio type cAMP fluorescent probe RaCamp and a construction method of a transgenic mouse, the probe RaCamp is formed by series fusion of G-Flamp2 fluorescent protein and mCherry fluorescent protein through a flexible linker, and the preparation method of the probe RaCamp comprises the following steps: S1, obtaining an mCherry fragment; s2, a G-Flamp2 linear carrier is obtained; s3, homologous recombination; s4, converting and screening; the construction method of the transgenic mouse comprises the following steps: T1, designing a carrier; t2, microinjection of fertilized eggs; t3, embryo transplantation and reproduction; the invention has the beneficial effects that mCherry is added as an internal reference protein on the premise of keeping the sensitivity of the original G-Flamp2 probe, and the selected connecting peptide (GGGGS) 2 enables the expression quantity of the two proteins in cells to be accurately controlled to be 1: 1, so that the problem of background interference in cell observation of in-vivo imaging is well solved. And the sequence is inserted into a Rosa26-LSL box, so that the construction of a Rosa26-LSL-RaCamp mouse is realized.

The present application relates to animals or cells having a myostatin gene with a 12 base pair deletion in the second exon. The application may also include compositions capable of engineering a 12 base pair deletion in the myostatin gene to construct animals or cells. The application also relates to the use of the compositions to increase muscle mass.

The invention discloses an ADO (adenosine dioxygenase) gene deleted zebrafish model as well as a construction method and application thereof. According to the invention, ADOa gene knockout is carried out on a model organism (zebrafish) to successfully construct an ADO gene deleted zebrafish model. Based on the model, the function of the ADO gene in fish taurine metabolism can be researched more deeply, so that a new scientific basis is provided for understanding the ADO function of zebra fish and even other fishes, and meanwhile, the model can be used for drug screening and provides a theoretical basis and technical support for breeding and genetic improvement of aquaculture industry.