31 results about "Zygote" patented technology

The present invention addresses the problem of specifically clarifying the effects on a fertilized egg when 5-ALA is administered to a human female suffering from infertility, and constructing a more effective means for treating infertility through the suppression of chromosomal abnormalities. It has been confirmed that, when 5-ALA is ingested in combination with a drug therapy that is generally performed in the treatment of female infertility, the rate of chromosome aneuploidy in a fertilized egg or the like is significantly reduced, and the qualitative improvement of an embryo through improvement of the stability of the chromosomes is expected.

The application discloses a submerged Cinnamomum micranthum in-vitro rapid propagation and brown-preventing seedling raising method based on zygote embryos, directly induces adventitious buds of the submerged Cinnamomum micranthum for the first time, significantly improves a propagation coefficient, and overcomes problems such as vitrification and browning of the adventitious buds of the submerged Cinnamomum micranthum. The application establishes an efficient and stable in-vitro regeneration system of the submerged Cinnamomum micranthum, and provides a basis for large-scale seedling raising of the submerged Cinnamomum micranthum.

This invention discloses a gRNA combination and its application in the preparation of drugs for the prevention of metabolic-associated fatty liver disease (MASLD). The gRNA combination, when mixed with Cas9 protein, yields an RNP complex, which, when microinjected into mouse zygotes, can breed a stable and heritable mouse strain with TMEM68 gene knockout. Combining lipidomics, transcriptomics, and primary hepatocyte functional verification, the core regulatory role of TMEM68 in hepatic lipid metabolism is revealed for the first time systematically. Experiments demonstrate that TMEM68 deficiency significantly reduces the storage of triglycerides (TAG) in the liver and hepatocytes, decreases lipid droplet formation, and reshapes the metabolic homeostasis of various lipids such as glycerophospholipids, cholesterol esters, and bile acids. Therefore, the gRNA combination of this invention, or the RNP complex obtained by mixing the gRNA combination with Cas9 protein, can be used to prepare drugs for the prevention of MASLD, providing a novel intervention strategy for MASLD prevention with broad application prospects.

The present invention relates to the technical field of prevention and control of aquatic animal diseases, and specifically relates to a nanocomposite for a nervous necrosis virus, a preparation method therefor, and the use thereof. Provided in the present invention is a nanocomposite consisting of poly(lactic-co-glycolic acid), polyethyleneimine and a siRNA. Also provided in the present invention are a preparation method for the nanocomposite and the use thereof in the preparation of a drug for improving the survival rate of fish eggs infected with a nervous necrosis virus. The present invention constructs a vector for a capsid protein of the nervous necrosis virus by means of simulating the nervous necrosis virus and expresses same in cells, and screens out a siRNA that can effectively inhibit the expression of the viral capsid protein, thus constructing the nanocomposite for the nervous necrosis virus. The nanocomposite can block NNVs at the embryonic phase (fertilized eggs), thereby improving the survival rate of fish fry, and further opening up new possibilities for the treatment of early stage diseases in fish fry. The method is innovative in the field of artificial breeding of aquatic animals and provides new ideas for virus prevention and control in the field of aquaculture.

This invention provides a method for transient transformation of oil palm zygote embryos based on Agrobacterium-mediated transformation, which employs the Agrobacterium-mediated approach and is based on the optimal Agrobacterium concentration (OD) for stable transformation of oil palm. 600 The optimal Agrobacterium infection time for zygotic embryos was determined by measuring the concentration of acetylsuccinone and co-culture time. The transient GUS expression staining rate was compared between zygotic embryos of different oil palm varieties and at different growth stages. The method described in this invention optimizes infection conditions based on stable transformation parameters, clarifies the optimal explant materials and treatment process, and effectively improves transient transformation efficiency and experimental repeatability. This study establishes for the first time an Agrobacterium-mediated transient transformation system for oil palm zygotic embryos and obtains zygotic embryos most suitable for transient transformation, achieving efficient transient expression of exogenous genes in oil palm zygotic embryos. This provides a rapid and low-cost technical means for studying oil palm gene function.

This invention discloses an artificial breeding method for the Oriental blackfish, belonging to the field of fish artificial breeding technology. It includes six core steps: broodstock selection and domestication, enhanced maturation cultivation, broodstock selection and anesthesia, spawning induction, artificial insemination, fertilized egg hatching, and fry rearing. One year in advance, suitable-aged wild Oriental blackfish are selected and domesticated in a flowing water pond. Before breeding, tocopherol is mixed into their feed to enhance gonadal development. High-quality broodstock are selected at a female-to-male ratio of 1:2 and anesthetized. A specific ratio of spawning inducing agent is injected using a two-injection method. Artificial insemination is performed in a standardized fiberglass circular fishpond under controlled water flow conditions. Fertilized eggs are incubated at 26±1℃ for 22 hours, and after hatching, they are transferred to a sterilized micro-flow water pond for rearing. This invention achieves a 100% spawning efficiency, a stable fertilization rate of over 95%, and a hatching rate exceeding 82%. It is simple to operate, cost-effective, and allows for large-scale breeding, providing key technical support for the conservation and population expansion of the Oriental blackfish.

This invention relates to a medaka hatching enzyme, its preparation method, and its application. The preparation method of the medaka hatching enzyme includes the following steps: 1) collecting fertilized medaka eggs; 2) incubating the fertilized medaka eggs with embryo culture medium in a 26℃ incubator for 6 days, and collecting mature embryos; 3) adding NaCl solution and Tris-HCl buffer (pH=8.0) to the mature embryos, grinding them, collecting the filtrate, and allowing it to stand overnight at 4℃; 4) centrifuging the filtrate at 15000 rpm for 15 minutes, and extracting the supernatant to obtain the medaka hatching enzyme solution. The preparation method is simple to operate, and the obtained medaka hatching enzyme has high activity, which can help to quickly remove the medaka eggshell artificially. The naked medaka embryos obtained by this method can be directly used for in vivo imaging and in-situ quantitative analysis, and embryo model construction, providing support for subsequent genetic, developmental biological, and toxicological studies of medaka embryos.

The present application relates to the field of aquatic organism breeding technology, and particularly relates to a method for preparing rainbow trout tetraploid fry. The method provided by the present application comprises the following steps: obtaining fertilized eggs by dry fertilization of naturally matured rainbow trout parents, inducing the rainbow trout fertilized eggs to obtain tetraploid rainbow trout after 6.2-6.4 hours, and the duration of the induction is 10-18 minutes; after the tetraploid rainbow trout fry is obtained by induction, the chromosome ploidy of the fish is determined by using techniques such as chromosome karyotype analysis and flow cytometry DNA content determination, and the tetraploid rainbow trout fry is identified and screened for cultivation. The present application adopts a drug induction method, and controls the starting time and duration of the induction to inhibit the first mitosis time of the fertilized eggs, thereby improving the induction rate and survival rate of the tetraploid rainbow trout, and the induction rate of the tetraploid rainbow trout can reach 25-30%, and the survival rate of the fry can reach more than 90%.

This invention relates to a method for constructing a mouse model of autosomal dominant polycystic kidney disease and its applications, particularly to a method for preparing a non-human animal model carrying a nonsense mutation in the Pkd1 gene c.616_618GAG>TAG based on CRISPR / Cas9 gene editing technology. The method involves co-injecting mouse zygotes with gRNA targeting exon 5 of the Pkd1 gene, homologous recombinant donor oligonucleotides containing the c.616_618GAG>TAG mutation, and Cas9 nuclease. Mutant mice are obtained via embryo transfer. The mutation site is verified by PCR combined with sequencing, and a stable mutant line is established through breeding. This invention also includes the application of this model animal as a research tool for autosomal dominant polycystic kidney disease, particularly in the areas of pathogenesis analysis, drug screening, and therapeutic target validation. This model can stably simulate the typical clinical manifestations and pathological features of human autosomal dominant polycystic kidney disease, providing an important tool for basic research and translational medicine of this disease.

This invention relates to the field of biotechnology, specifically disclosing a method for constructing and applying a rare gudgeon strain susceptible to GCRV based on ISG58 knockout. The key technical points are as follows: This invention employs CRISPR / Cas9 gene editing technology to design dual-target sgRNAs targeting the ISG58 gene of rare gudgeon (target 1 induces a -2 bp deletion, and target 2 mediates an -8 bp deletion). The sgRNA sequence is optimized using ZiFiT target design software, and target specificity is ensured by NCBI BLAST alignment. The in vitro transcribed sgRNA and Cas9 protein are co-microinjected into rare gudgeon fertilized eggs to obtain F0 generation chimeras; after self-crossing of F1 generation heterozygotes, homozygous mutants conforming to Mendelian inheritance laws are successfully screened in the F2 generation. This invention, based on ISG58 knockout, obtains a rare gudgeon strain more sensitive to GCRV infection, which not only provides an important model for subsequent research on antiviral immune mechanisms in fish but also serves as an indicator organism for GCRV in aquaculture environments.

The application provides a construction method and application of a CYP1A1 double-site mutant mouse model, and belongs to the technical field of genetic engineering. Specifically, the double-site mutant mouse model provided by the application is a CYP1A1 p.L118R and CYP1A1 p.R245P double-site mutant mouse model, which is obtained by the following method: S1: designing gRNA and a donor vector; S2: microinjecting Cas9 mRNA, gRNA and the donor vector into a fertilized egg; S3: obtaining F0 generation mice; S4: mating the positive F0 generation mice with wild-type mice to obtain F1 generation mice; S5: interbreeding the positive F1 generation mice to obtain F2 generation mice; and S6: screening the F2 generation mice to obtain the double-site mutant mouse model. The application can solve the technical problems of a complex preparation process and a low success rate of a double-site mutant mouse model far apart.

The application relates to a construction method of a conditional overexpression ZFP595 transgenic mouse model and application thereof, and relates to the technical field of transgenesis.The construction method comprises the following steps: constructing a homologous recombination vector containing a ZFP595 gene, wherein the vector comprises a 3.3 kb 5' homologous arm, CAG-LSL-Zfp595-HA-IRES-tdTomato-Wpre-pA and a 3.3 kb 3' homologous arm; microinjecting Cas9 mRNA, gRNA and the homologous recombination vector into a zygote of a recipient mouse to obtain F0 generation mice, and performing identification; mating the F0 generation mice identified as positive with wild type mice and backcrossing to obtain a mouse model for conditionally overexpressing ZFP595.The conditional overexpression ZFP595 mouse model constructed by the construction method can be crossed with various Cre mice to realize specific overexpression in different tissues, organs and cells.

This invention discloses a method for constructing and applying an anti-obesity animal model based on ISCA1 / 2 gene regulation, belonging to the field of biomedical technology. The method utilizes CRISPR-Cas9 gene editing technology to design specific single-stranded guide RNAs targeting the ISCA1 and ISCA2 genes in mice. Cas9 mRNA and sgRNA are introduced into fertilized eggs of C57BL / 6J strain mice via microinjection. The injected embryos are then transferred into pseudopregnant mice to obtain F0 generation chimeric mice. By mating F0 generation mice with wild-type C57BL / 6J mice, F1 generation heterozygous mice carrying the target mutation in the germline are selected, thus obtaining the anti-obesity animal model. This model is not only a phenotypic model but also a mechanistic research model, suitable for drug target validation, energy metabolism pathway exploration, and personalized treatment strategy evaluation.

This invention discloses a highly efficient artificial breeding and intermediate rearing method for *Sinocyclocheilus rubra*. Gonadally mature *Sinocyclocheilus rubra* are first air-dried, then transferred to seawater, where sodium sulfite is added to induce oxygen deprivation and stimulate spawning. Based on the physiological characteristics of *Sinocyclocheilus rubra*, this invention uses sodium sulfite to induce oxygen deprivation and asphyxiation, achieving excellent spawning-inducing results. Compared to existing traditional artificial spawning-inducing methods for tropical sea cucumbers, such as flowing water stimulation and air-drying, which have a maximum spawning rate of 13.3-18.0%, this method is significantly more effective (spawning rate as high as 75-81%). Sodium sulfite has no significant toxicity to parent sea cucumbers and is inexpensive; traditional spawning-inducing methods must be performed at night, while this invention has no time requirement, is highly operable, easy to promote, reduces the amount of parent sea cucumbers, and can provide a stable supply of fertilized eggs for large-scale sea cucumber breeding.

This application discloses a method and application for screening fish embryos based on 1,6-hexanediol-induced intracellular phase changes, belonging to the field of fish breeding technology. This method addresses the lack of non-destructive screening methods for early embryos in existing technologies. During the maternal zygote transition period, which includes fish embryonic development, 0.1% 1,6-hexanediol is used for stress treatment. Utilizing its ability to interfere with intracellular biomolecule condensation, it induces intracellular phase changes, thereby eliminating embryos with poor internal environmental stability and low robustness, while retaining high-quality embryos with high developmental potential. The screened embryos show a significantly reduced rate of deformities in the current generation, and superior traits are heritable to offspring. The fertilization rate and hatching rate of offspring are significantly improved, and the deformity rate is significantly reduced. This application is simple to operate, cost-effective, and suitable for large-scale seedling production, providing a new technical pathway for the breeding of superior fish breeds.

This invention discloses a AIPL1 A gene mutation-related congenital amaurosis animal model, its construction method, and its application. The method includes: S1, constructing a humanized... AIPL1 S2, a homologous recombination vector with a point mutation of c.421 C>T (p.Q141X) knocked in; S3, Cas9 mRNA, gRNA1, gRNA2 and the homologous recombination vector are injected into animal zygotes to perform homologous recombination, resulting in transfected zygotes; S4, the transfected zygotes are transplanted into the uterus of pseudopregnant animals to reproduce offspring, and offspring are selected from the offspring. AIPL1 Homozygous animals with the c.421 C>T (p.Q141X) point mutation were obtained, thus yielding the aforementioned... AIPL1 A gene mutation-related congenital amaurosis animal model. The animal model of this invention exhibits the clinical phenotype of Leber congenital amaurosis type 4 (LCA4) from 12 days after birth; while heterozygous mice do not show a significant phenotype, consistent with... AIPL1 The clinical phenotype and inheritance pattern of LCA caused by the c.421 C>T (p.Q141X) gene mutation demonstrate the effectiveness and reliability of the animal model of this invention, providing a basis for understanding the genetic model of LCA. AIPL1 Animal models provide a reliable basis for studying the disease mechanisms and drug screening of mutation-related LCA.

The present disclosure relates to the technical field of prevention and control of diseases in aquatic animals, and particularly relates to a nanocomplex targeting a nervous necrosis virus as well as a preparation method and use thereof. The present disclosure provides a nanocomplex composed of poly(lactic-co-glycolic acid), polyethyleneimine and siRNA. The present disclosure further provides a preparation method of the nanocomplex and use of the nanocomplex in the preparation of a drug for improving the survival rate of fish eggs infected with NNV. In the present disclosure, a vector for an NNV capsid protein is constructed by simulating the NNV; the constructed NNV capsid protein is expressed in cells; the siRNA capable of effectively inhibiting the expression of the viral capsid protein is screened, and then the nanocomplex of the NNV is constructed; and the NNV is blocked in the embryonic stage (fertilized eggs).

This invention discloses a heavy ion mutagenesis breeding method for fish, belonging to the field of fish breeding technology. To address the technical problem of how to efficiently apply mutagenesis breeding technology in fish genetic improvement, this method first breeds the target fish species to obtain fertilized eggs in the single-cell stage; then, it uses a heavy ion beam to induce mutagenesis in the single-cell stage fertilized eggs, hatches and raises the mutagenized eggs to obtain an M1 generation population; next, it performs molecular screening to identify M1 generation individuals carrying one or more gene mutations; finally, it uses breeding methods to obtain offspring strains that can stably inherit the stated gene mutations. This invention shortens the breeding cycle from several generations to as short as 1-2 generations, significantly improving screening efficiency and providing an efficient, precise, and universally applicable pathway for rapidly creating new germplasm and strains of various fish species.

This invention discloses a batch centralized hatching device for swamp eels, comprising an installation frame, with multiple support plates equidistantly connected inside the frame. Each support plate has a support frame directly below it, and several limiting sleeves are connected within the support frames. The limiting sleeves have an upward-opening trumpet shape in cross-section. A rotating shaft is rotatably connected to the top center of each support plate, with a sleeve coaxially and securely connected to the outside of the shaft. A motor is securely fastened to the top of the shaft. Symmetrically symmetrical through holes are provided on the top of each support plate, and guide wheels are rotatably connected to the side of the through holes closest to the motor. Traction ropes are symmetrically connected to the top of the support frames, with the ends of the ropes passing through the through holes and winding around to the top of the guide wheels. Driven by a motor, this invention allows for the raising and lowering of the support frames, thereby enabling continuous suspension and settling of fertilized eggs in the water. This ensures sufficient dissolved oxygen, prevents fertilized eggs from clumping and molding, promotes embryonic development, and effectively improves the hatching rate of swamp eels.

The application discloses a kind of Australian freshwater lobster egg anti-mucus and application thereof, it is related to lobster breeding field, including albumin 0.5%~1.5%, soybean lecithin 5%~15% and plant oil acid 5%~15%, and the rest is water.The application provides a kind of shrimp egg anti-mucus, can be soaked by the way, the female shrimp's pedipalpus cannot be adhered with the discharged spermatozoon, so that the spermatozoon of female shrimp is separated from female shrimp without mechanical or artificial strong intervention, by collecting spermatozoon and centralized hatching, the hatching rate of spermatozoon can be effectively avoided in the centralized feeding of Australian freshwater lobster, because the situation of spermatozoon membrane damage due to territoriality, so as to improve the hatching rate of spermatozoon.The application also provides a breeding device, under the cooperation of water flow, gravity and anti-mucus, can realize the spermatozoon and female shrimp separation, automatic centralized collection of separated spermatozoon, save manpower, and can shorten the time of parent shrimp in temporary pond, unified centralized hatching also reduces the workload of hatching process.

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

This invention discloses a method for constructing a zebrafish model of fatty liver based on glucokinase overexpression and its application. The method includes the following steps: using cDNA as a template, a zebrafish GCK gene fragment is obtained by PCR amplification; the vector fragment and the GCK gene fragment are homologously recombined using a homologous recombinase to obtain a recombinant plasmid; the recombinant plasmid is transformed into competent cells, and an endotoxin-free plasmid is extracted as a zebrafish GCK expression vector; a microinjection system is used to inject the plasmid into one-cell-stage fertilized eggs; after the fertilized eggs hatch into juveniles, fluorescent juveniles are selected for further culture until sexual maturity; sexually mature males and females are paired to spawn, and a stably heritable GCK-overexpressing zebrafish strain is selected from the offspring. This invention can obtain a zebrafish strain with stable high GCK expression, which can be used as a zebrafish model of fatty liver based on GCK overexpression.

This invention discloses a method for interspecific hybridization and seedling cultivation of *Pterocarya spp.* and *Pterocarya martensii*, comprising: selecting mature, live male *Pterocarya spp.*, cleaning and drying them, placing them in seawater, continuously aerating and artificially circulating the water to induce natural sperm release; dissecting mature female *Pterocarya martensii* to extract gonads, collecting mature eggs, cleaning them, and allowing them to settle to obtain an egg mixture; adding ammonia to the egg mixture to activate the eggs; adding the activated egg mixture to a sperm-containing water body for fertilization in the dark; after successful fertilization, removing the male *Pterocarya spp.* and transferring the fertilized eggs to a seedling pond; and cultivating the fertilized eggs in seawater to develop into D-shaped larvae. This invention is the first of its kind for distant hybridization and seedling cultivation of *Pterocarya spp.* and *Pterocarya martensii*, breaking through the reproductive isolation between the two species, achieving stable and efficient hybridization and fertilization. The process is simple and highly stable, and can cultivate new hybrid pearl oyster seedlings that combine the excellent traits of both parents. It has significant implications and promotional value for the innovation of marine pearl oyster germplasm and industrial upgrading.

An experimental method for low-high temperature hatching stimulation and dual-high temperature tolerance threshold determination for schizothoracine fish eggs in high diurnal temperature variation waters includes: step 1, determining daily extreme water temperatures and a daily water temperature variation during natural incubation of the schizothoracine fish eggs; step 2, implementing an incubation water temperature with the daily water temperature variation; step 3, determining a key hatching water temperature; step 4, inducing mass hatching of the schizothoracine embryos; and step 5, determining temperature tolerance thresholds. Through indoor water temperature gradient experiments on the schizothoracine fish eggs, a hatching mechanism under low-high temperature stimulation for fertilized eggs of Schizopygopsis microcephalus in Yangtze River source, and dual-high temperature tolerance ranges and thresholds. This provides scientific support for the protection and restoration of fishery resources in the Yangtze River source under the background of global warming.

The present application relates to the field of aquaculture technology, and more particularly to a method for improving the attachment rate and survival rate of sea urchin larvae, which comprises the following steps: culturing sea urchin fertilized eggs in a culture water body; when the larvae develop to the late prism larvae stage, synchronously adding a chemical induction system and an attachment substrate material to the culture water body; and promoting the sea urchin larvae to complete stable attachment and metamorphic development by maintaining a chemical microenvironment during the attachment and post-attachment stages. In the present application, the chemical induction system and the physical attachment substrate are synchronously added and synergistically act, effectively overcoming the defects of scattered induction signals and unstable microenvironment; the microcapsules can realize the directional slow release of induction components such as gamma-aminobutyric acid, ions and amino acids on the surface area of the attachment base, thereby establishing and maintaining a long-term, stable and concentration-suitable high-efficiency induction microenvironment around the larvae, and realizing the synchronous and synergistic improvement of the attachment rate and survival rate of the sea urchin larvae.

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

This invention provides a zebrafish model of hypertrophic cardiomyopathy characterized by ventricular cavity shrinkage and reduced mobility, and a method for constructing the model. Using zebrafish tmem260 as the target gene, CRISPR / Cas9 technology is used to microinject in vitro designed and synthesized tmem260-specific sgRNA and Cas9 protein into wild-type zebrafish fertilized eggs. After successive generations of screening, tmem260 gene-specific knockout zebrafish mutants are obtained. The phenotype of hypertrophic cardiomyopathy characterized by ventricular cavity shrinkage and reduced mobility in the mutants is observed and passaged for conservation.