101 results about "Fish embryo" patented technology

A method for breeding stat1a (signal transducer and activator of transcription 1) gene-deleted zebra fish through gene knockout comprises steps as follows: design of a CRISPR / Cas9 gene knockout target site: a gRNA expression carrier is established and gRNA is synthesized in vitro; micro-injection of a zebra fish embryo; detection of effectiveness of the target site with a T7E1 method through Sanger sequencing; tail cutting identification according to the identification steps after two months of injection; TA cloning of a target sequence; Sanger sequencing of plasmids; obtaining of heritable F1 generation of a zebra fish mutant; obtaining of F2 generation homozygote of the zebra fish mutant, F3 generation pure line inheritance of the gene-deleted zebra fish with the above method, and obtaining of a new zebra fish strain.

The invention provides statla gene deletion type zebra fish. After an experiment design region is knocked out, the sequence is represented as SEQ ID No.1; an experiment comprises the following steps: designing a CRISPR / Cas9 gene knockout target point: constructing a gRNA expression vector and synthesizing gRNA in vitro; carrying out micro-injection of zebra fish embryos; detecting the effectiveness of the target point by a T7E1 method and Sanger sequencing; two months after injection, carrying out tail shearing and identification and carrying out identification steps above; carrying out TA cloning of a target sequence; carrying out Sanger sequencing of plasmids; obtaining an F1 generation of descendible zebra fish mutants; obtaining F2 generation homozygotes of the zebra fish mutants; and carrying out F3 generation homozygous heredity of the gene deletion type zebra fish according to the method above to obtain a new zebra fish line.

The invention relates to the activity detection method for the new-type anti or promote angiogenic factor by zebra fish embryo model. Wherein, after hatching, adding former protein constituent to culture for some time at constant temperature; observing its development; then, dyeing blood vessel, observing with cubic microscope, sampling and storing image. This invention is simple and fit to wide application with low cost.

The invention relates to an application of zebra fish to testing water quality and toxicity and a method for applying zebra fish to test water quality and toxicity. The method is characterized by comprising the following steps of: preparing an embryo culture solution, then adding a water source to be tested to the embryo culture solution, using the embryo culture solution to which the water source to be tested is added to culture zebra fish embryos and then observing and recording the developmental aberration rates and mortality of the zebra fish embryos in 12-24 hours to judge the toxicity of the water source to be tested toward the embryos, wherein the ratio of the volume of the added water source to be tested to the volume of the embryo culture solution is 1:3; the embryo culture solution is directly prepared from alkalescent water with pH value being 6.7-8.5; and the embryo culture solution contains 5mM of NaCl, 0.17mM of KCl, 0.43mM of CaCl2 and 0.33mM of MgSO4. The method has the following beneficial effect that the embryo culture solution prepared from the alkalescent water ensures that the zebra fish embryos can quickly test the comprehensive toxicity of the water source to be tested, thus shortening the detection period.

The invention relates to a method for applying zebra fish to test toxicity of an organic solvent. The method comprises the following main steps of: preparing an embryo culture solution, then adding the organic solvent to the embryo culture solution, ensuring that the concentration of the organic solvent in the embryo culture solution reaches the concentration needed by measuring, using the embryo culture solution to which the organic solvent is added to culture zebra fish embryos and then observing and recording the developmental aberration rates and mortality of the zebra fish embryos in 6-12 hours to judge the toxicity of the organic solvent toward the embryos, wherein the embryo culture solution is directly prepared from alkalescent water with pH value being 6.7-8.5; and the embryo culture solution contains 5mM of NaCl, 0.17mM of KCl, 0.43mM of CaCl2 and 0.33mM of MgSO4. The method has the following beneficial effect that the embryo culture solution prepared from the alkalescent water ensures that the zebra fish embryos can quickly test the toxicity of the organic solvent, thus shortening the detection period.

The invention relates to a modified zebra fish drug metabolism modeling method, which specifically comprises the steps of (1) enriching metabolites by virtue of a zebra fish metabolism model; (2) separating and analyzing the enriched metabolic components of the zebra fish in the last step by using modern chromatography or the hyphenated technique thereof, capturing or knocking out target components or metabolites; (3) collecting zebra fish germ cells; (4) putting zebra fish embryos in three days after fertilization in a culture plate which holds an embryo culture medium and administrating a drug; (5) dyeing the bones of the zebra fish; and (6) detecting and analyzing the dyed bones of the zebra fish. According to the invention, an adolescent zebra fish osteoporosis model is applicable to in-vivo effective evaluation of anti-osteoporosis activity of Chinese herbal medicinal ingredients such as trace ingredients; an adult zebra fish metabolism model is simple in metabolites and efficient in enrichment; the modern chromatography or the hyphenated technique thereof provide powerful guarantee for the separation and analysis of the complex Chinese herbal medicinal system and the metabolic components. The model obtained by the method has the outstanding advantage that the separation and analysis of Chinese herbal medicinal original-form ingredients and the metabolic components are integrated with the in-vivo anti-osteoporosis activity evaluation, and simple and efficient.

A method and system for producing large quantities of aquatic animal embryos includes providing a water filled spawning tank adapted for holding the male and female aquatic animals in various configurations. The system can include a spawning platform which includes a porous or perforated element that allows the embryos but not the aquatic animal to pass through and a separator which includes a porous or perforated element that can be used to separate the male aquatic animals from the female aquatic animals during a priming phase. In operation, the spawning platform can be placed in the bottom of the tank in order to provide a bottom collection area where the embryos can be collected and the aquatic animals cannot eat or otherwise harm the embryos. The female aquatic animals can be placed in tank above the spawning platform. The separator can be placed in the tank above the female aquatic animals and the male aquatic animals can be placed in the tank above, remaining separated from the female aquatic animals, beginning the priming phase. When embryos are desired, the separator can be removed allowing the male aquatic animals to mingle with the female aquatic animals and the height of the water above the porous or perforated element of the spawning platform can be changed, by raising the spawning platform or lowering the water level, in the spawning phase. The porous or perforated element of the spawning platform can be undulating or angled with respect to horizontal to create areas of varying depth over the surface of the porous or perforated element of the spawning platform to improve embryo production.

The invention discloses a method for predicating the embryotoxicity of non-steroidal anti-inflammatory drug type novel pollutants on an early-phase life stage of zebra fish. The method comprises the following steps: exposing zebra fish embryos in the non-steroidal anti-inflammatory drug type novel pollutants which have equal logarithm space concentrations; recording the death rate and the aberration rate of the zebra fish embryos 7 days after exposing; and calculating by using SPSS software to obtain corresponding LC50 and teratogenetic EC50, which are used for evaluating the toxicity of the non-steroidal anti-inflammatory drug type novel pollutants. With the adoption of the method, the toxicity feature and the toxicity level of the non-steroidal anti-inflammatory drug type novel pollutants are subjected to an analytical test and quantitative description; and meanwhile, the method also can be used as an index for monitoring and evaluating the wastewater biological toxicity of non-steroidal anti-inflammatory drugs, and reference is provided for risk predication and evaluation of the potential biological toxicity of the pollutants in a water body.

The invention relates to a microfluidic chip applicable to medicine screening of zebra fish embryos. The microfluidic chip is low in price, easy to prepare and suitable for large scale, high flux and automation. The microfluidic chip comprises at least one microfluidic layer, at least one control layer, a connecting layer and at least one micro cell, wherein the control layer is adjacent to the microfluidic layer; the connecting layer is used for connecting the adjacent microfluidic layer and the control layer; the micro cell is used for accommodating the zebra fish embryos and providing the growth environment for the zebra fish embryos; the microfluidic layer is provided with a microfluidic channel; the microfluidic channel is communicated with the internal space of the micro cell; the control layer is provided with at least one control channel; the microfluidic channel and the control channel are provided with at least one intersected point; and the connecting layer at the position of the at least one intersected point consists of an elastomer material and forms an elastomer valve. The communication state of the microfluidic channel can be controlled convenient through the elastomer valve, so that fully-automatic operation of medicine screening of the zebra fish embryos can be realized through an electromagnetic valve controlled by an external computer.

The invention relates to an application of zebra fish to testing safety of a safe substance and a method for applying zebra fish to test safety of the safe substance. The method is characterized by comprising the following steps of: preparing an embryo culture solution, then adding the safe substance to be tested to the embryo culture solution, using the embryo culture solution to which the safe substance to be tested is added to culture zebra fish embryos and then observing and recording the developmental aberration rates and mortality of the zebra fish embryos in 4-72 hours to judge the toxicity of the safe substance to be tested toward development of the zebra fish embryos, wherein the addition of the safe substance to be tested ensures that the concentration of the substance to be tested in the culture solution is distributed in echelon; the embryo culture solution is directly prepared from alkalescent water with pH value being 6.7-8.5; and the embryo culture solution contains 5mM of NaCl, 0.17mM of KCl, 0.43mM of CaCl2 and 0.33mM of MgSO4. The method has the following beneficial effect that the embryo culture solution prepared from the alkalescent water ensures that the zebra fish embryos can quickly test the comprehensive toxicity of the safe substance to be tested, thus shortening the detection period.

A vitrifying freeze storage method for the embryos of fish, especially the bastard halibut, is disclosed, which features that the formula of vitrifying liquid suitable for the embryo of seawater fish is screened out, the embryo development stage suitable for vitrifying it is determined, a particular eluting solution is disclosed, and the step balance and the fast cooling and thawing techniques are used. Its advantages are high safety, easy operation and low cost.

The invention discloses a fish embryo chromosome flaking method, relating to genetic engineering. The method includes the steps of taking out 100-200 roes during the middle-stage gastrula and the late-stage gastrula, washing the roes for twice through normal saline for fish to remove the membranes and the vitellines of the roes, adding the roes into colchicine-containing 1640 culture solution for cell culture, striking the roes to enable the cells to be even, adding fixative to fix the cells, drying and dyeing through staining mother solution. The invention overcomes the defects that the conventional method is time-consuming and labor-consuming and batch production can not carried out; and the fish embryo chromosome flaking method improves the stability of the flakes and the accuracy of the inductivity. Therefore, the fish embryo chromosome flaking method provides a reliable guarantee for genetic breeding and breed improvement of fish.

The invention relates to a method for evaluating kidney toxicity of compounds through detecting the contents of creatinine in zebra fish tissues. The method is applied to kidney toxicity researches. In the researches that utilize a zebra fish embryo model to study the toxic effect of compounds on the kidney, the creatinine component in embryo tissues is extracted, then the creatinine content is measured through a LC-MS (liquid chromatography-mass spectrum) method, the creatinine content can be taken as a detection index for evaluating the embryo kidney functions, and thus a rapid and precise compound kidney toxicity evaluation system with strong specificity is built.

The invention relates to a method for rapidly evaluating the damage effect on the hepatic function of zebra fishes by compounds. The method includes the steps that 1, fertilized normal zebra fish embryos are moved into breeding holes; 2, continuous chemical hatching is performed on the zebra fish embryos in the breeding holes, and the zebra fish embryos are marked to be a compound set to be detected; 3, livers of the zebra fish embryos are observed, and the liver area of the zebra fish embryos and the area of zebra fish embryo bodies are recorded and calculated; 4, according to the range relation between the index of the liver area of the zebra fish embryos of the compound set to be detected and the index of the liver area of normal zebra fish embryos, whether the compound set to be detected has liver toxicity is judged. The index of the liver area is used as a detection index for evaluating the hepatic function of the zebra fishes for the first time, and therefore the method is more scientific and objective and improves the accuracy of results. An established model for evaluating the damage effect on the hepatic function of the zebra fishes by the compounds has the advantages of being easy to manufacture, rapid to use, stable, reliable and good in repeatability.

The invention discloses an exposure device for fish embryos and larva fish for long-term chronic toxicity tests. The exposure device comprises a tank and a tank cover which are arranged externally. The tank cover is provided with a plurality of small container ports. A small container is hung on each small container port so as to be allowed to extend into the inner space of the tank. The bottom of each small container keeps a certain distance from the bottom of the tank. An overflow pipe is arranged in each small container. Each overflow pipe penetrates out of the bottom wall of each small container and the bottom wall of the tank to be connected to a water inlet of a three-way valve. The other water inlet of each three-way valve is connected with a drain pipe, and each drain pipe penetrates out of the bottom wall of the tank and extends into the bottom of the tank. A water outlet of each three-way valve is connected with a protruding drain outlet, and the highest position of each protruding drain outlet is at the same level with an overflow pipe nozzle at the utmost top of each overflow pipe. The tank cover is further provided with a plurality of operating openings, and the lower periphery wall of each small container is provided with a narrow slit capable of leaking water and blocking the fish embryos and the larva fish from passing by.

The invention discloses a cuttle-fish artificial insemination and incubation method and special agent thereof, wherein the eggs in oviduct of the female cuttle-fish are transferred into a container with nutrient solution to perform external insemination and isolated culture and an agarose layer is set between the container bottom and eggs; the nutrient solution contains the fresh or freeze-dry oviduct gland secretion of the female cuttle-fish. The special agent contains culture container bottom liner and nutrient solution and the culture container bottom liner is 0.2% of agarose and the nutrient solution contains 1-10 portions by weight of blood serum and 1000 portions by weight of sterile sea water. Because the molecular structure of agarose is loose, the nutrient material can freely flow and penetrate, the eggs on the agarose liner has little distortion without nutrition deficiency of the contact surface, The nutrition is provided by the oviduct gland secretion and the blood serum of low concentration, the eggs do not distort, thus the whole development process of the cuttle-fish embryo cultured in vitro is greatly accomplished.

The separation and cultivation method for fish embryonic cell comprises: collecting cell; in CM, separating the cell of blastula-archenteron late time with laceration method, and culturing the initial and passage cells; identifying the cell on morphology, proliferation feature and chromosome karyotype. Wherein, using 70% alcohol to dip and sterilize for embryo; culturing cell in common incubator with 24Deg to passage 2-3 times every week. The objective cell is litle, circle or polygon, and growing stably, and has high rate of diploid cell. This invention is simple and convenient, and can be applied into separation and cultivation for almost all fish embryonic cell.

The invention provides a zebra fish embryo incubation device for an experiment. The device comprises an incubation box and a supporting frame arranged in the incubation box. A first heater for heating a water bath is arranged at the bottom in the incubation box, a first cover body is arranged on the top of the incubation box, and a light source is arranged at the bottom of the first cover body. The supporting frame comprises a first installing plate, and a second installing plate parallel to the first installing plate. The edge of the first installing plate and the edge of the second installing plate are connected through a side plate to form a culture dish heating cavity, multiple arrayed installing holes are formed in the first installing plate, multiple water inlets are formed in the first installing plate, and the diameter ratio of the installing holes to the water inlets is 5:1 to 4:1. Due to the adoption of water bath heating, temperature is more constant than temperature of hot air heating adopted, in an illumination incubator, meanwhile, the diameter ratio of the installing holes to the water inlets is 5:1 to 4:1, fluctuation of heat exchange of water in the culture dish heating cavity and a culture solution in a culture dish is small, therefore, the experimental temperature is more constant, and the experimental result is more reliable.

The invention discloses a high-precision instant orthogonal optical projection tomography system. Three-dimensional tracking for neutrophile granulocyte in zebra fish embryo can be realized. The collecting rate can reach camera frame rate and the optical radiation to a biological sample is small. Various image processing and recognition algorithms are used for detecting and recognizing the neutrophile granulocyte in the zebra fish embryo, so as to acquire the position change and morphological features of the cells. A basis is established for data analysis of the next step; the mutual relation of the neutrophile granulocyte structure, morphological feature, motion trail, inflammation and drug effect can be realized; the experimental and theoretical basis is established for researching the disease change and the drug effect mechanism.

The invention belongs to the technical field of biology, and particularly relates to a method for detecting development toxicity of environmental pollutants by a whole-mount in-situ hybridization technique. The method comprises the following step: detecting the development toxicity of environmental pollutants to zebra fish embryos by a whole-mount in-situ hybridization technique. The method can be used for predicting potential toxic actions of the environmental pollutants on zebra fish organs and tissues, thereby further disclosing the action mode of the environmental pollutants, shortening the detection period, and achieving the goal of early warning in deed.

The present invention aims at providing one homologous recombination method for in-situ fish gene modification breeding. The method includes the following steps: 1) separating specific fish gene flanking sequence; promoter regulating sequence DNA segments to constitute homologous recombination vector with actin gene promoter regulating sequence to replace growth hormone gene promoter regulating sequence; 2) microscopic injecting the homologous recombination vector to zebra fish embryo to screen positive homologous recombination replaced fish fry; and 3) analyzing relative gene expression level and genetic phynotype of the endogenous gene modified fish. The present invention is used in breeding fish variety.

The invention relates to a refrigeration method for preserving sea fish embryo which comprises, selecting heartbeat stage embryos, charging into 0-4 deg C. antifreezing liquid, pre-cooling for 90-60 minutes, sectionwise cooling down, placing cores at -12 deg C during the cooling period, preserving in liquid nitrogen at the temperature of -30 deg C, equalizing 10 minutes at -80 deg C when defreezing, defreezing with 43 deg C water till full thawing, eluting with eluent, cultivating in sea water so as to obtain the revival embryos.

A method for freeze storage of fish embryo without damage to it includes such steps as preparing the antifreeze protective liquid from two or more antifreezing agents temp balancing loading the mixture in centrifugal tube, quickly putting it in liquefied nitrogen at -196 deg.C for storage, and thawing which includes temp lalancing at -180 deg.C and thawing by water bath at 38-40 deg.C.

An object of the invention is to effectively prepare a fish embryo with a correct chromosomal ploidy by nuclear transplantation in which an exogenous fish nucleus is transplanted in a cytoplasmic recipient. For this object, the invention comprises a step of preparing a fish embryo by transplanting a fish cell nucleus to an unfertilized egg. The step of preparing a fish embryo comprises a step of imposing physical and / or chemical stress to the unfertilized egg after activation. By imposing such stress, the stage of haplosis in a female nucleus which happens at the early stage of a series of developmental steps occurring in an unfertilized egg is suppressed and the correct ploidy of an obtained embryo is at least secured.

The invention discloses an application of a compound IC-4 with the structural formula represented by formula (I) or its salts in antiangiogenesis, and further discloses an application of the compound IC-4 with the structural formula represented by formula (I) or its salt in preparation of antiangiogenic agents. Researches on the application of the IC-4 or its salt in the antiangiogenesis are based on researches on growth factor induced endothelial cell growth inhibition, angiogenesis inhibition, endothelial cell migration inhibition and endothelial cell transfer inhibition of the IC-4 or its salts, and combine with a scientific and reasonable research system constructed through integral experiments of zebra fish embryo angiogenesis.

The invention relates to a screening system of drugs influencing bone mineralization. The screening system of the drugs influencing the bone mineralization comprises a zebra fish embryo in a test group, a zebra fish embryo in a positive control group, a zebra fish embryo in a negative control group, a calcein dyeing agent, an image acquisition system, and image analysis and statistics software. Compared with a traditional dyeing method using alizarin red, the screening system of the drugs influencing the bone mineralization solves the problem that the traditional dyeing which is judged by the depth of color is influenced easily by external factors such as dyeing conditions, dyeing time and operation, and overcomes the disadvantages that the bone mineralization degree cannot be reflected exactly as the existence of the dye instead of the depth of the dye is utilized to evaluate the variation of the bone formation.

The present invention relates to an analysis method for the activity of mismatch repair of zebra fish embryo DNA, and the application of the method in toxicity evaluation of mismatch repair of environmental compound DNA. The method includes the following steps: preparing single-stranded DNA molecules with plasmids pGEM-EGFP; preparing homoduplex and heteroduplex with the single-stranded DNA molecules and linearized plasmids pGEM-EGFP and plasmids pGEM- (mt) EGFP respectively; microinjecting the homoduplex and heteroduplex to a zebra fish embryo with 1-cell stage; and quantifying the activity of mismatch repair of embryo DNA. The present invention establishes the analysis method for the activity of mismatch repair of DNA on the zebra fish embryo level for the first time, and successfully applies the method to the toxicity evaluation of mismatch repair of environmental compound DNA, which is significant for the evaluation of health risk and cancerization risk for environmental compound people. In addition, by applying the zebra fish to the evaluation of the toxicity of mismatch repair of compound DNA, the method has the advantages of simple operation, direct viewing, fastness, simple administration for tested compounds, small dosage, and the like.

The invention belongs to the technical field of biology, and relates to a method for quantitatively evaluating proliferation of tumor cells transplanted into zebra fish embryos. The method is characterized by comprising the following steps of: performing fluorescence labeling on the tumor cells before injection; selecting the zebra fish embryos carrying the fluorescence tumor cells for further culture one day after injection; and amplifying human tumor cell genes by a quantitative PCR method 3 to 7 days after injection to evaluate the colonization and proliferation conditions of the human tumor cells in the zebra fish embryos. The injection period of the tumor cells subjected to fluorescence labeling in advance is the period of 48 hours after fertilization, and the injection part is a perivitelline space area. The injection volume of each embryo is 10 nL, and the number of the injected tumor cells is 300 to 500. During quantitative evaluation, a plurality of embryos are selected as a group generally, and the content of the human genes is detected by quantitative PCR. The period of 3 to 7 days after injection is an effective time window for evaluating whether the human tumor cells are proliferated in the embryos. The method is beneficial to the practicability of a PDX (patient-derived cell transplantation) model of zebra fish.