111 results about "Transgenic zebrafish" patented technology

The present invention relates to the technical field of biology, particularly to development and applications of a heat shock induced Cas9 enzyme transgene danio rerio. The present invention firstly provides a Cas9 enzyme expression vector, which utilizes a heat shock induced promoter HSP70 to drive the expression of a downstream Cas9 gene. According to the present invention, the Cas9 enzyme expression vector and Tol2 mRNA are co-injected into wild type danio rerio single cell fertilized egg, and selection is performed to obtain the heat shock induced Cas9 enzyme transgene danio rerio, such that the gene editing research of the CRISPR-Cas9 system in the danio rerio is successfully achieved, and the know-out of the MC4R gene in the transgene danio rerio is firstly achieved; and the Cas9 enzyme expression vector is further suitable for heat shock induced gene knockout, gene knock-in, gene expression modification and other applications of other fishes.

The present invention relates to the method and use of reef coral fluorescent proteins in making transgenic red, green and yellow fluorescent zebrafish. Preferably, such fluorescent zebrafish are fertile and used to establish a population of transgenic zebrafish and to provide to the ornamental fish industry for the purpose of marketing. Thus, new varieties of ornamental fish of different fluorescence colors from a novel source are developed.

Four zebrafish gene promoters, which are skin specific, muscle specific, skeletal muscle specific and ubiquitously expressed respectively, were isolated and ligated to the 5' end of the EGFP gene. When the resulting chimeric gene constructs were introduced into zebrafish, the transgenic zebrafish emit green fluorescence under a blue light or ultraviolet light according to the specificity of the promoters used. Thus, new varieties of ornamental fish of different fluorescence patterns, e.g., skin fluorescence, muscle fluorescence, skeletal muscle-specific and / or ubiquitous fluorescence, are developed.

Four zebrafish gene promoters, which are skin specific, muscle specific, skeletal muscle specific and ubiquitously expressed, respectively, were isolated and ligated to the 5′ end of the EGFP gene. When the resulting chimeric gene constructs were introduced into zebrafish, the transgenic zebrafish emit green fluorescence under a blue light or ultra-violet light according to the specificity of the promoters used. Thus, new varieties of ornamental fish of different fluorescence patters, e.g., skin fluorescence, muscle fluorescence, skeletal muscle-specific and / or ubiquitous fluorescence, are developed.

The invention relates to a transgenic zebrafish expressing a gene Cas9 and a construction method and the application of the transgenic zebrafish. According to the transgenic zebrafish expressing the gene Cas9, the gene Cas9 is inserted into a gene Mitfa of the zebrafish. The transgenic zebrafish capable of expressing the gene Cas9 is constructed in a targeting way at the fixed point of the pigmentgene Mitfa through the CRISPR / Cas9 transgenic technology, and pigment fading is taken as a mark for screening the homozygous knockout line. Using the transgenic zebrafish to edit a gene Tyr and a gene ZFERV proves that the transgenic zebrafish is more effective in the gene knockout, and a homozygous knockout individual is obtained on the F0 generation. Compared with the application of the traditional CRISPR / Cas9 technology to the zebrafish, the gene editing efficiency of the Cas9 transgenic zebrafish is higher; and a simple effective tool is provided for the large-scale gene screening and theestablishment of a disease model.

The present invention relates to the method and use of reef coral fluorescent proteins in making transgenic red, green and yellow fluorescent zebrafish. Preferably, such fluorescent zebrafish are fertile and used to establish a population of transgenic zebrafish and to provide to the ornamental fish industry for the purpose of marketing. Thus, new varieties of ornamental fish of different fluorescence colors from a novel source are developed.

The present invention relates to the method and use of reef coral fluorescent proteins in making transgenic red, green and yellow fluorescent zebrafish. Preferably, such fluorescent zebrafish are fertile and used to establish a population of transgenic zebrafish and to provide to the ornamental fish industry for the purpose of marketing. Thus, new varieties of ornamental fish of different fluorescence colors from a novel source are developed.

The present invention relates to zebrafish models for Alzheimer's disease that allow recapitulation of pathologies associated with Alzheimer's disease. This invention also relates to methods for screening of compounds for their ability to modulate a pathology associated with Alzheimer's disease in vivo in a whole vertebrate organism. The present invention further relates to methods of identifying gene targets for compounds that modulate a pathology associated with Alzheimer's disease.

The present invention provides a method for producing systemic red fluorescent zebrafish. The present invention also provides a new gene fragment and a systemic red fluorescent zebrafish.

The invention belongs to the technical field of medicines and particularly relates to a construction method and an application of an osteoporosis zebra fish model. The construction method comprises the following steps: constructing transgenetic zebra fish with green fluorescence of a skeleton report after obtaining a promoter of a specific transcription factor of osteoblast by virtue of applying a tol2-gateway technique; constructing the glucocorticoid induced osteoporosis zebra fish model by using the transgenetic zebra fish; and meanwhile, estimating the model by applying a bone-strengthening and kidney-tonifying traditional Chinese medicine herba epimedii general flavone. The osteoporosis model constructed by the method provided by the invention is convenient, feasible and effective, and is suitable for high throughput screening of anti-osteoporosis drugs. Compared with the conventional chemical staining methods, the construction method is more stable, time-saving and efficient.

The invention discloses a vector for efficiently labeling zebra fish PGC, and a preparation method and a use of transgenic fish. A Gal4 / UAS transcription activation system is utilized and mRFP is used as a report gene to realize an inductive gene expression regulation technology in the primordial germ cells of zebra fish. The preparation method of the transgenic fish comprises the following steps: 1, respectively constructing activation transgenic line Tg (kop:KalTA4) and an effect transgenic line Tg (UAS:mRFP); and 2, hybridizing Tg (kop:KalTA4) male fish with Tg (UAS:mRFP) female fish to obtain the transgenic fish for efficiently labeling the primordial germ cells of zebra fish. The transgenic fish for efficiently labeling the primordial germ cells of zebra fish can be widely applied to the fish bioengineering.

The present invention relates to the method and use of fluorescent proteins in making purple transgenic fluorescent fish. Also disclosed are methods of establishing a population of such transgenic fish and methods of providing them to the ornamental fish industry for the purpose of marketing. Thus, new varieties of ornamental fish of different fluorescence colors from a novel source are developed.

The present invention provides methods and systems that uses transgenic zebrafish with an easily assessable reporter gene under the control of pollutant-inducible DNA response elements. Transgenic zebrafish, carrying pollution-inducible response elements, are placed in the water to be tested, and the contaminants become bioconcentrated (generally 1,000- to 40,000-fold, relative to the water) in the tissues of the fish thereby activating specific response elements, which up-regulate the LUC reporter gene. Fish are then removed from the test water and placed immediately in a luminometer cuvette and incubated with luciferin. Luciferin is rapidly taken up into the tissues of the fish, oxidized by luciferase, and light is produced. The luminescence is proportional to the environmental concentration of the pollutant (to which the fish had been exposed), which drives the expression of the LUC gene by means of the various DNA motifs. The luminescence is quantitated in the luminometer. In each response element-containing construct, a specific class of polluting chemicals, allowing for differential identification of pollutants in a complex mixture activates the expression of the LUC gene. This assay does not require killing the fish and allows for repeated analysis of the same site with the same fish. The sensitivity of the system can be manipulated by varying the sequence of the response element.

The present invention relates to transgenic fluorescent orange ornamental fish, as well as methods of making such fish by in vitro fertilization techniques. Also disclosed are methods of establishing a population of such transgenic fish and methods of providing them to the ornamental fish industry for the purpose of marketing.

The invention discloses transgenic zebrafish with specific myelin sheath removal capacity as well as a preparation method and an application thereof. The preparation method comprises the following steps: recombinant plasmid mbp-nfsB-EGFP and a Tol 2 transposase mRNA (messenger ribonucleic acid) are jointly introduced into wild zebrafish; and a zebrafish strain with stable inheritance is cultured and obtained, and the zebrafish strain with stable inheritance is the transgenic zebrafish with specific myelin sheath removal capacity, wherein the nucleotide sequence of an mbp gene promoter carried in the recombinant plasmid mbp-nfsB-EGFP is represented as SEQ ID NO.1. With adoption of the method, the transgenic zebrafish which can specifically remove myelin sheath, cause myelinoclasis and has stable inheritance capacity can be prepared and obtained effectively, under the action of metronidazole, oligodendrocyte of juvenile transgenic zebrafish can be removed specifically and the juvenile transgenic zebrafish has pathologic change of myelinoclasis together with decreased motor function, therefore, the transgenic zebrafish can provide a forceful animal model for a regulatory mechanism for viviperception of myelin sheath damage and regeneration and related drug screening.

The present invention relates to transgenic blue ornamental fish, as well as methods of making such fish by in vitro fertilization techniques. Also disclosed are methods of establishing a population of such transgenic fish and methods of providing them to the ornamental fish industry for the purpose of marketing.

The invention relates to a method for quickly and quantitatively evaluating a blood vessel generation promotion function of chemical compounds on zebra fish. According to the method, transgenic zebra fish with blood vessels specially expressing green fluorescent protein are adopted, a to-be-detected medicine is directly added to water for culturing the zebra fish, the relative area of tail venous rete of the zebra fish is detected, and the method for quickly and quantitatively evaluating the blood vessel generation promotion function of the chemical compounds on the zebra fish is established and used for medicine screening. In the experiments of the zebra fish, the relative area of the tail venous rete is initially taken as a new evaluation index about the blood vessel generation function, whether the to-be-detected medicine has a blood vessel generation promotion function is analyzed more scientifically and more objectively, and the accuracy of results is improved.

The present invention relates to zebrafish models of thrombosis that allow screening of compounds for anti-thrombotic or thrombotic properties in vivo in a whole vertebrate organism. The present invention also relates to the identification and validation of platelet genes as targets for anti-thrombotic or thrombotic compounds.

The invention discloses a transgene zebra fish system and an establishing method thereof. The transgene zebra fish system is characterized in that at the premises of not changing the expression of gene abcd4 of zebra fish, the change of expression of gene abcd4 is monitored by EGFP (enhanced green fluorescent protein), namely that the transgene zebra fish system is an EGFP-expressed Tg (abcd4: EGFP) transgene zebra fish system which is driven by an abcd4 gene promoter. The transgene zebra fish system has the advantages that at the premises of not changing the expression of gene abcb4 (which is homologous with the gene ABCB1 of human body) of the zebra fish, the change of expression of gene abcd4 is monitored by EGFP, namely that the transgene zebra fish system is the EGFP-expressed Tg (abcd4: EGFP) transgene zebra fish system which is driven by the abcd4 gene promoter; the early experiment basis is provided for gene abcb 4 in the study of multi-drug resistant mechanism.

A disclosed preparation method of transgenetic zebrafish used for detecting environment pollutants comprises: (1) cloning the promoter regulatory sequence of Mdr1 protein from zebrafish genome; (2) fusing the DNA fragment obtained by cloning into the upstream of GFP coding sequence for constructing an expression vector; (3) performing zebrafish embryo microinjection on the expression vector; (4) culturing the embryo subjected to injection to sexual maturity, and enabling cultured zebrafish and wide zebrafish to copulate and oviposit; and (5) screening positive F1 generation under a fluorescence microscope, and performing inbreeding to obtain F2 generation and further to obtain a transgenic line with stable inheritance. Because the expression level of fluorescin can be induced in a concentration dependent way by multiple environment pollutants such as heavy metals, polycyclic aromatic hydrocarbons and the like, the transgenic zebrafish prepared by employing the preparation method can be used to detecting the existence and the concentration of the above chemical pollutants in environment; and the preparation method has the advantages of being economic, rapid, convenient and accurate.

The invention relates to a preparation method of transgenic zebrafish, and relates to gene engineering. The preparation method comprises the following steps of (1) modifying a CYPIA starter sequence; (2) establishing a Tol2 transposon system knocking plasmid, and preparing a microinjection sample; (3) performing microinjection and subculture on zebrafish embryos. The invention provides an establishing method of a CYP1A starter containing twelve DREs (dehydration-responsive element), and a recombination Tol2 transposon knocking plasmid which contains the starter and is connected with a green fluorescent protein gene, a plasmid linear purifying method, and a microinjection sample method. The Tg(pCYP1A-6*DRE-EGFP) and Tg(pCYP1A-12*DRE-EGFP) transgenic zebrafish prepared by the microinjection technique can be used for comparing the activities of the starters respectively containing six DREs and twelve DREs.

The present invention belongs to the field of life science. Said invention provides a regulatory controlling sequence expressed by zebra fish vitellogenin 1, (vtg 1) gene of sequence 1. Said invention utilizes the transgenic zebra fish experiment to show that said sequence has promoter activity in the zebra fish body interior, and can make downstream gene implement specific expression in liver under the induction of female sex hormone. By adopting the connection of zfvtg 1-1.7 sequence provided by said invention and reporter gene (fluorescin) the fluorescence can be observed in the living body interior of transgenic zebra fish so as to quickly and visually reflect expression of vtg 1.

The invention discloses a zebra fish P-selectin gene and a protein. The nucleotide sequence of the zebra fish P-selectin gene is shown as SEQ ID NO.1, and the encoded protein sequence is shown as SEQ ID NO.2. The zebra fish P-selectin gene can be used for constructing transgenic zebra fishes. The constructed transgenic zebra fishes can be used for researching the generation mechanism of thrombotic diseases and sieving the antithrombotics. The zebra fish P-selectin gene and the encoded gene can be used for preparing drugs for preventing and treating the thrombotic diseases.

The invention relates to an incubation method for transgenic zebrafishes, and aims to provide a method for incubating transgenic zebrafishes with intestinal-specific expression of red fluorescence. According to the invention, by observing the expression of embryonic red fluorescent protein gene, zebrafish embryos with intestinal-specific fluorescence are selected, and hybrid offsprings are obtained by artificial feeding. The stably genetic zebrafishes with intestinal expression of red fluorescent protein are obtained by selfing of the hybrid offsprings, and then parent zebrafishes corresponding to embryos with 100% intestinal expression of red fluorescent protein are screened as double integration pure lines for seeding and preservation after test cross with wild type zebrafishes. The method of the present invention is simple and easy-to-control in implementation process, and can obtain a promoter with intestinal-specific expression and zebrafish strains with red fluorescent protein highly expressed in the intestine. The obtained zebrafish strains are used for the study of zebrafish intestinal function and can be observed for expression characteristics of intestinal red fluorescentprotein at any time, thereby meeting the integrity and completeness requirements on study of zebrafish intestinal function.

Disclosed are systems for expression of an autonomous lux reporter system in a vertebrate cell, such as mammalian or fish cell. In some examples the lux reporter system is operably connected to a pollutant-inducible DNA response element. Also disclosed are transgenic zebrafish, carrying pollution-inducible response elements, and methods of using such zebrafish to monitor pollutants.

The invention, belonging to the technical field of medicament, particularly relates to a screening method of medicines for inducing the formation of myocardial cells by using a model organism zebrafish, comprising the following steps: first, letting 25-30 pairs of Tg (cmlc2: EGFP) transgenic zebrafish homozygotes mate to obtain embryons, then transferring the embryons into a culture plate, adding a proper amount of zebrafish embryons at the stage of 50% epiboly in every pore, using an embryon culture solution containing 1-30 mu M small molecular compounds in an incubator for culture; then carefully checking the embryon heart size, heart shape and the number of myocardial cells; in addition, checking the whole shape (such as dorsal-ventral body axis, anterior-posterior body axis, etc.) and whether the formation of other organs of the embryons is influenced; and finally preliminarily judging whether the small molecular compounds promote the formation of the myocardial cells without influencing the growth of the other organs according to the experiment results. The new medicines which are screened out by the method has strong specificity and low toxic and side effect.

The invention discloses a transgenic zebrafish model with macrophages aggregated specifically in liver. The zebrafish refers to the Tg (fabp10a: il34) transgenic zebrafish. The transgenic zebrafish model with macrophages aggregated specifically in liver is realized by the following steps: (1) zebrafish culture; (2) whole-mount in situ hybridization and antibody staining of zebrafish embryos; and (3) laser scanning confocal fluorescence microscopy in vivo imaging. The transgenic zebrafish model with macrophages aggregated specifically in liver proves for the first time that heterotopically expressed and overexpressed interleukin 34 in the liver can induce the macrophages to enrich into the liver, and provides solid and direct evidence by using the in vivo imaging technology. In addition, the Tg (fabp10a: il34; mpeg1: GFP) adult zebrafish has the following phenotypes: (1) the number of the macrophages increases only specifically in the liver region; (2) the number of the macrophages remains unchanged in other tissue regions such as tail hematopoietic tissue (CHT); and (3) the number of granulocytes is unchanged.

The invention relates to the technical field of medicines, and discloses sponge-derived meroterpenoid compound Septosone A. The Septosone A has a chemical structural formula shown in the description.The invention also discloses a preparation method and application of the Septosone A. The compound Septosone A provided by the invention has very strong inhibitory activity on a NF-kappaB signal channel, and can inhibit formation and diffusion of transgenic zebrafish inflammation in vivo and be used for preparing anti-inflammatory and anti-tumor drugs.

The invention belongs to the field of biological medicine research, and particularly relates to a zebrafish embryo alcoholic liver detecting model and a construction method and application of the zebrafish embryo alcoholic liver detecting model. The transgenic zebrafish with liver specific expression fluorescent proteins is adopted by the method to construct a model of the alcoholic liver disease; the zebrafish embryo alcoholic liver detecting model is constructed by analyzing the information of swelling liver appearance, reduction of embryo yolk absorption amount, fluorescent reading and the like of the zebrafish. The model has the characteristics of simple observation index, easiness in observation, capability of being qualitative and quantitative and the like, and is especially suitable for being applied to high throughput screening of medicaments for preventing and treating the alcoholic liver.

The present invention relates to transgenic red ornamental fish, as well as methods of making such fish by in vitro fertilization techniques. Also disclosed are methods of establishing a population of such transgenic fish and methods of providing them to the ornamental fish industry for the purpose of marketing.