642results 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 preparation method and application of a self-reactive animal model. An autoreactive animal model is constructed based on an SWHEL system, it is found through the model that MTCH2 is a key target for regulating and controlling B cell autoreactivity, deletion of MTCH2 will cause generation of a large number of autoreactive B cells subjected to antibody category conversion, the disease risk is increased, and a basis is provided for research of B cell autoreactivity related mechanisms.

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.

The present application provides a brown planthopper lethal gene Nlpfam and its application in biological control. After the brown planthopper lethal gene Nlpfam is interfered with, it can cause the death of brown planthoppers. Therefore, by transcribing the brown planthopper lethal gene Nlpfam to prepare dsRNA or an interfering reagent, injecting it into brown planthoppers can effectively interfere with the Nlpfam gene, causing the abdomen of brown planthoppers to turn black, effectively killing brown planthoppers, and achieving biological control of brown planthoppers.

The invention relates to an application of a CTHRC1 inhibitor in preparation of a medicine for preventing and / or treating arthritis. Specifically, the invention provides application of an inhibitor or an antagonist of a CTHRC1 gene or a protein thereof, the inhibitor or the antagonist is used for preparing a composition or a preparation, and the composition or the preparation is used for (a) preventing and / or treating arthritis-related diseases; and / or (b) inhibiting cartilage cell senescence.

The invention provides a breeding method for constructing XX / XY sex determination all-female sterile fish and application. The method comprises the following steps: knocking out a catalytic enzyme coding gene of a fish sex steroid hormone synthesis pathway by using a gene editing technology to block the fish sex steroid hormone synthesis pathway, screening to obtain an effectively mutated homozygote XX sex genetic type pseudo male fish, and hybridizing the pseudo male fish with improved allotetraploid crucian and carp female fish to obtain the fish sex steroid hormone synthesis pathway. Therefore, an allotriploid population is obtained, and the population completely develops into female fishes and is sterile, so that the effect of obtaining all-female and sterile populations is achieved at the same time. The method provided by the invention has very strong applicability, and can be widely applied to aquaculture of fishes to obtain female unisexual groups of the fishes so as to improve the aquaculture yield, realize effective control on female fertility and protect ecological safety; the method has a wide application prospect in the fields of genetic breeding and ecological safety of aquaculture fishes.

The invention provides a genome editor enDelIscB and a genome editor enDelIscB-T5E based on an IscB system, and further provides a base editor ICBE and a base editor IABE based on the enDelIscBD60A. According to the invention, a genome editor and a base editor which are more compact in size and higher in cleavage activity and insertion / deletion efficiency are realized by optimizing a single mutation site of the activity of a DelIscB protein cleavage plasmid, an optimized variant of the DelIscB protein and an optimized sgRNA variant of the DelIscB. According to the present invention, it is verified that the albinism model is efficiently constructed by using the enDelIscB editor and the enDelIscB-T5E editor through micro-injection of the mouse embryo, such that the enDelIscB editor and the enDelIscB-T5E editor have great potential in the field of genome editing.

The invention is applicable to the technical field of rabbit breeding, and provides a method for increasing the body weight of a rabbit based on a gene editing technology, which comprises the following steps: designing an action target sgRNA on a rabbit NR6A1 gene aiming at an NR6A1 p.P192S site; uniformly mixing the synthesized sgRNA and SpRY-CBE, injecting fertilized eggs, transplanting the fertilized eggs into fallopian tubes of receptor female rabbits, and feeding the receptor female rabbits until production; dNA is extracted from produced young rabbit ear tissues, PCR and sequencing are carried out, and if C at the fifth site in NR6A1 gene target sites is mutated into T, it is proved that single base mutation is obtained, and the NR6A1 gene edited rabbit is obtained. According to the invention, the rabbit variety with increased weight is successfully cultivated; a new thought of animal genetic breeding is developed by utilizing a gene editing technology, the method has application values in promoting improvement of genetic characters of rabbits and improving production performance and economic value of the rabbits, and powerful support is provided for high-quality development of the animal breeding industry in China.

The invention discloses a trpa1b gene mutant and a construction method of a tilapia low-temperature-resistant strain, and belongs to the technical field of biology. A tilapia mossambica model with a trpa1b gene knocked out is constructed based on a CRISPR / Cas9 gene editing technology, and the method comprises the following steps: determining a specific target sgRNA of the trpa1b gene, introducing the sgRNA and Cas9 protein with activity into a tilapia mossambica embryo through micro-injection, cultivating and screening F0-generation trpa1b knocked-out tilapia mossambica, and obtaining homozygote mutant tilapia mossambica through two-generation mating. The tilapia with the trpa1b gene knocked out shows higher low-temperature resistance, and particularly, the survival time of the tilapia under the low-temperature condition is prolonged. The invention provides an important model for low-temperature-resistant breeding of tilapia mossambica, and also provides a potential gene target for low-temperature-resistant strain breeding of other fishes in view of the conservative property of the function of the trpa1b gene.

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 construction method and application of a dilated cardiomyopathy animal model, belongs to the technical field of biological medicine and animal genetic model construction, and provides a novel dilated cardiomyopathy zebrafish model which is constructed by specifically overexpressing a truncated dnajb6b gene long isomer fragment in zebrafish myocardial cells. The nucleotide sequence of the gene segment is as shown in SEQ ID NO. 1; the invention also provides a construction method and application of the dilated cardiomyopathy zebra fish model, and further discovers application of 4-phenylbutyric acid in preparation of drugs for treating dilated cardiomyopathy.

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 invention provides a construction method and application of a gene Idh1 conditional knockout mouse model and a preeclampsia heart injury animal model, and relates to the technical field of biology. According to the preeclampsia heart injury animal model, flox mice with conditional gene Idh1 knockout and mice with placental trophoblast specific expression Cre recombinase comate, PE-related phenotypes and typical symptoms of heart injury spontaneously appear in the double-positive pregnant mouse model, and the double-positive pregnant mouse model can be used for research of disease causes, diagnosis methods and drug research and development.

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 belongs to the field of biological genetic engineering, and particularly relates to a method for constructing a congenital heart disease mouse model. The invention provides gRNA (guide Ribonucleic Acid) for targeting a mouse Tmed1 gene. Nucleotide sequences of the gRNA are as shown in SEQ ID NO: 1 and SEQ ID NO: 2. Meanwhile, the invention provides a method for knocking out the mouse Tmed1 gene based on a CRISPR / Cas9 technology to obtain the mouse with the congenital heart disease. The Tmed1 mouse model is successfully constructed, a reliable mouse model is provided for congenital heart disease and visceral ectopic syndrome patients, subsequent detection and verification of upstream and downstream target gene functions and subsequent verification of development of related target drugs are facilitated, and great convenience is brought to disease mechanism research and drug research and development verification work.

The invention discloses a method for constructing an Asiatic corn borer GBP gene homozygous mutant, which comprises the following steps: amplifying and cloning an Asiatic corn borer GBP gene sequence by using RACE technology, and the nucleotide sequence of the Asiatic corn borer GBP gene is as shown in SEQ ID No.1; two sgRNA target sequences are designed on a second exon of a GBP gene sequence of the ostrinia furnacalis, target sgRNA is synthesized through in-vitro transcription, the target sgRNA and Cas9 protein are fused in vitro to form an RNP mixture, and the RNP mixture is injected into ovum of the ostrinia furnacalis; the method comprises the following steps: carrying out PCR (Polymerase Chain Reaction) amplification on a target spot by using a target spot detection primer, sequencing to confirm a mutant individual, hybridizing and selfing to obtain a GBP homozygous mutant population. The Asiatic corn borer GBP gene homozygous mutant is constructed, so that the reproduction and immune functions of the mutant are remarkably reduced, and reference and research basis are provided for later development and formation of efficient pest biopesticide.

The invention relates to the technical field of bioengineering, in particular to a coccinella septempunctata forkhead transcription factor Cs-FoxO gene as well as an inhibitor and application thereof. The invention provides a nucleotide sequence of a coccinella septempunctata forked head transcription factor Cs-FoxO gene as shown in SEQ ID NO. 1 and an amino acid sequence as shown in SEQ ID NO. 2; the invention also provides an application of the forkhead transcription factor Cs-FoxO gene inhibitor in relieving biological diapause. After dsRNA expression inhibition, the forked head transcription factor Cs-FoxO gene provided by the invention can relieve the diapause of the coccinella septempunctata, and the dsRNA of the Cs-FoxO gene can be used as a related preparation and product for relieving the reproductive diapause of the coccinella septempunctata, so that the development of the natural enemy industry is promoted, and the application prospect is wide.

The invention discloses a chicken sex identification method based on fluorescence labeling, and belongs to the technical field of gene engineering. The chicken sex identification method comprises the following steps: co-transfecting a CRISPR / Cas9 system and a WPG-EGFP donor plasmid into chicken primordial germ cells PGCs, screening to obtain EGFP positive PGCs, micro-injecting the EGFP positive PGCs into a receptor chicken embryo, hatching to obtain an EGFP reproductive chimera chicken, and identifying the sex through fluorescence labeling, the CRISPR / Cas9 system comprises sgRNA of a targeted chicken W chromosome WPG gene; the WPG-EGFP donor plasmid is constructed by inserting an EGFP gene into a WPG gene locus through a homologous recombination strategy. According to the method, the sex of the chicken can be visually and rapidly identified. The method solves the problems that a traditional sex identification method is complex in operation and depends on sample ages, has the advantages of high efficiency and accuracy, and is suitable for sex control and screening of poultry such as laying hens and broilers.

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 present application discloses a gene point mutation mouse model construction method, comprising: by taking T274M mutation on Kcnq2 Exon6 as a target, designing sgRNA and ssODN; selecting a female mouse of a set week age as an ovum donor, injecting a certain dose of serum gonadotropin and human chorionic gonadotropin into the abdominal cavity of the female mouse, then mating the female mouse with a mating male mouse, and collecting zygotes of the mated female mouse; injecting Cas9 protein, ssODN and sgRNA into the pronuclei of the zygotes by means of a microinjection technique, transplanting the zygotes obtained after injection into the ampulla of the fallopian tube of a surrogate female mouse, and obtaining a neonatal mouse after the pregnant female mouse gives birth; respectively designing a pair of identification primers F1, R1 at two ends of a mutation site on the basis of a Kcnq2 gene point mutation site, identifying the neonatal mouse, and screening out a Kcnq2T274M / + point mutant mouse in which T274M mutation is successfully knocked.

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 relates to a gene point mutation mouse model construction method. The method comprises the following steps: designing sgRNA and ssODN by taking T274M mutation on Kcnq2Exon6 as a target spot; the method comprises the following steps: selecting a female mouse at a set week age as an ovum donor, injecting a certain dose of serum gonadotropin and human chorionic gonadotropin into the abdominal cavity of the female mouse, mating with a breeding male mouse, and collecting fertilized ova of the mated female mouse; injecting Cas9 protein, ssODN and sgRNA into the pronucleus of the fertilized ovum through a micro-injection technology, transplanting the fertilized ovum after injection into the oviduct pot belly of the female rat to be pregnant, and obtaining a newborn rat after the female rat to be pregnant is delivered; according to a Kcnq2 gene point mutation site, a pair of identification primers F1 and R1 are designed at the two ends of the mutation site respectively, and a newborn mouse is identified, so that a Kcnq2T274M / + point mutation mouse with T274M mutation successfully knocked in is screened out. The Kcnq2T274M / + point mutation mouse editing method is simple in preparation and high in repeatability, and a larger number of Kcnq2T274M / + point mutation mice can be obtained while the editing efficiency is improved.

The invention discloses a sialoprotein gene for regulating and controlling the growth and development of brown planthoppers as well as dsRNA and application thereof. The nucleotide sequence of the sialoprotein gene SISP is shown as SEQ ID NO.1; the dsRNA sequence of the sialoprotein gene for regulating and controlling the growth and development of the brown planthopper is as shown in SEQ ID NO.3. According to the dsRNA disclosed by the invention, SISP can be effectively silenced, the dsRNA is prepared into a nucleic acid pesticide to be applied to brown planthopper, the inhibition of the brown planthopper on the insect resistance of rice can be reduced, and the dsRNA has a serious adverse effect on normal feeding, survival and reproduction of the brown planthopper. The method has the advantages of no pollution and reduction of the use amount of chemical pesticides. Besides, the SISP gene is overexpressed in the rice, so that the content of lignin and flavonoid compounds in the rice can be remarkably reduced, and the insect resistance of the rice is reduced.

The invention belongs to the field of fish genetic breeding, and discloses a method for efficiently obtaining homozygous red crucian carp, which comprises the following steps: (1) carrying out artificial false fertilization on eggs of the red crucian carp to obtain fertilized eggs; (2) performing gene editing on the fertilized eggs; (3) incubating the fertilized eggs subjected to gene editing for 18-32 minutes, and then carrying out cold treatment; and (4) incubating the fertilized eggs after cold treatment to obtain the homozygous red crucian carp. According to the method disclosed by the invention, gene editing and gynogenesis methods are comprehensively utilized, the hatching rate, the survival rate and the homozygous rate are high while the high gene editing efficiency is maintained through the early cold shock treatment time and the proper cold shock treatment time, and the gynogenesis red crucian carp mutant with a high proportion can be obtained.

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.