29 results about "Microinjections" patented technology

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

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

The application provides a construction method of a humanized keloid mouse model, comprising the following steps: BAC plasmid construction and preparation; superovulation of experimental mice and collection of zygotes; pronuclear microinjection of zygotes; post-injection embryo transplantation; genotype identification; breeding and genetic analysis; and verification of construction results of the humanized immune system by immunohistochemistry and immunofluorescence. In the application, peripheral blood mononuclear cells treated by sCD27 and skin around keloids (homologous cells and tissues) are transplanted into NSG-MHC-DKO immune-deficient mice with overexpression of CD70 genes, so that HLA rejection of different homologous immune cells and tissues can be avoided, the internal microenvironment of keloids and the interaction between the internal microenvironment and the immune system can be restored to the maximum extent, and the influence of immune factors on the occurrence and development of keloids can be realized in vitro. The application first discovers and verifies that the activation of the CD27-CD70 axis can promote the occurrence and development of keloids, and provides a suitable animal model for the research and development of anti-keloid drugs, especially immunotherapy.

This utility model relates to the field of fish egg microinjection and discloses a detachable and adjustable fish egg fixing device, including a tray. The tray is divided into several sub-chambers by several partitions, and several first egg holder assemblies, second egg holder assemblies, third egg holder assemblies and fourth egg holder assemblies are fixedly installed on the tray in the vertical direction. Several supporting grooves are opened on both sides of the tray, which are engaged with one end of each first egg holder assembly, second egg holder assembly, third egg holder assembly and fourth egg holder assembly. The device of this utility model is convenient for fixing fish eggs of different sizes, thereby facilitating the subsequent injection process.

The invention belongs to the technical field of biological model construction, and particularly relates to a method for constructing a vasculitis model in zebra fish. The compound is obtained by inducing a Candida albicans water soluble substance (CAWS), and the specific method is micro-injection or soaking. According to the method, whether modeling succeeds or not can be judged in the living body state of the model animal, the method is more suitable for visually observing the inflammation occurrence position, a research method for vasculitis (including Kawasaki disease) is developed, follow-up scientific research data can be richer and more accurate, and a more solid theoretical basis is laid for clinical transformation of targeted therapy of vasculitis (including Kawasaki disease).

The invention relates to the technical field of assisted reproduction, and discloses a microinjection device and an operation method, the microinjection device comprises a handle, a rotary vibration shaft, a piezoelectric vibrator and a micromanipulation needle, the piezoelectric vibrator is tangentially arranged between the handle and the rotary vibration shaft, and electric energy is converted into circumferential high-frequency micro vibration of the rotary vibration shaft. The circumferential vibration is transmitted to the micromanipulation needle connected with the front end, so that the unique fish-mouth-shaped end face of the micromanipulation needle penetrates through the cells in a high-frequency rotary cutting mode, and extrusion damage to the oocytes caused by traditional axial puncture is remarkably reduced. The invention further discloses a gallium-based alloy which is high in density and better in biocompatibility and serves as an operation medium, and the transmission efficiency of vibration energy is improved. The invention also provides a method for pre-filling the microinjection needle by utilizing the physical characteristics of low-temperature curing and normal-temperature liquefaction of the gallium-based alloy and adopting cold chain transportation, so that the product can be used immediately after being opened, the clinical operation steps are obviously simplified, the operation threshold is reduced, and the microinjection efficiency and success rate are improved.

The invention discloses a method for improving the normal fertilization rate of an in-vitro ICSI embryo of a dairy cow and reducing the abortion rate, which comprises the following steps: preparing an ICSI egg from X-sex controlled sperm and an oocyte which is cultured by a mature oocyte and is used as a donor by adopting a microinjection mode, the ICSI eggs are incubated in an oocyte maturation culture solution (OM) for 3-5 h and then transferred to ETH to be activated for 4-6 min so as to promote normal fertilization of the ICSI eggs, then embryo culture is conducted, and then sexual control embryos are obtained; the ETH refers to an oocyte maturation culture solution containing 6 to 8 percent of ethanol. The normal fertilization rate of the ICSI embryos produced by the method is remarkably improved, and the early abortion rate is remarkably reduced in inspection after transplantation.

According to the microinjection device and the microinjection control method, the problems that in microinjection membrane breaking operation, a membrane structure is high in deformation damage, solid particle injection is not facilitated, and the membrane breaking efficiency is low are solved, and the microinjection device and the microinjection control method belong to the technical field of microoperation and microinjection. The microinjection device comprises a linear motion module, a rotating module and a microneedle, the linear motion module is used for driving the microneedle to do linear motion, the rotating module is used for driving the microneedle to do rotating motion, and a driving signal of the rotating module is a periodic alternating rotating signal. A periodic alternating rotary shearing mode is adopted to replace a traditional one-way rotation or fixed vibration mode, the deformation process of the membrane is homogenized through dynamic shearing force, the deformation quantity of the membrane is reduced, the cell activity is remarkably improved, membrane penetration is accelerated through the shearing fatigue effect and the crack expansion effect generated by alternating rotation, and the cell quality is improved. And the membrane breaking efficiency is further improved compared with one-way rotation.

The application discloses a method for introducing in-vitro cultured intestinal symbiotic microorganisms into Spodoptera exigua, and comprises the following steps: isolating, purifying and shake-culturing the intestinal symbiotic microorganisms from the Spodoptera exigua, starving the larvae of the Spodoptera exigua, cutting corn leaves into small pieces, soaking the small pieces in the prepared bacterial liquid, and feeding the larvae of the Spodoptera exigua with the dried small pieces, so that the in-vitro cultured intestinal symbiotic microorganisms are introduced into the Spodoptera exigua. The method does not need a microinjector, is simple to operate, does not need continuous feeding of multiple generations, and has a short experimental period. The method can change the bacterial structure in the insects, so as to achieve the purpose of studying the influence of the symbiotic bacteria on the growth and development of the insects and the like.

The utility model belongs to the technical field of microinjection, and particularly relates to a single cell microinjection device which comprises a rotating shaft, a mounting frame is mounted at one end of the rotating shaft, rotating shafts are rotatably mounted on two sides of the mounting frame in a penetrating manner, a locking screw is rotatably mounted in one end of a side plate, and a gear groove is mounted at the other end of the rotating shaft. A clamping block is assembled at the bottom end of the limiting rod, and an extension spring is fixedly connected between the limiting block and the supporting plate. A limiting block is lifted upwards, a limiting rod moves upwards, an extension spring is stretched, a clamping block is separated from a gear groove, a rotating shaft drives a mounting frame to rotate, the angle of the injector in the X-axis direction is changed, the angle of the injector in the Y-axis direction is adjusted, a locking screw releases fixation of a rotating shaft, and the injector is rotated through rotation of the rotating shaft; therefore, the angle of the injector can be adjusted at will, the injector can be aligned with target cells more flexibly in the micro-injection process, and the injection effect is improved.

The invention provides a microinjection method of scleroderma guani eggs. The method mainly comprises the following steps: firstly, inducing Scleroderma guani to lay eggs and collecting the Scleroderma guani; long-horned beetle larvae are provided for female scleroderma guani which is mated after eclosion, and eggs on the surfaces of the long-horned beetle larvae are collected after the female scleroderma guani spawns. Secondly, the collected scleroderma guani eggs are arranged on the edge of the cover glass, and the rear ends of the eggs are uniformly arranged in the direction where the injection needle is located; then, the injection needle is drawn, microinjection is conducted on the egg granules, and the injection position is the rear ends of the egg granules; and finally, transferring the injected egg granules to the surface of the frozen long-horned beetle larva for culturing. The method is reasonable and efficient in design, conforms to the egg growth characteristics of the scleroderma guani, and provides a brand new way for gene function research of the scleroderma guani.

An automated drug microinjection device for administering a controlled amount of drug, and a method of operating the device is provided. The device comprises a container storing the drug, a microneedle assembly, a pump, a motor, and a controller. The microneedle assembly comprises a spring-loaded microneedle and a shape memory locking member. The spring-loaded microneedle is biased to be in an extended position and the shape memory locking member is configured to retain the microneedle in a locked and retracted position until a release signal is applied. The pump comprises a pump cavity and a pump actuator. The pump actuator is operable to fill the pump cavity with drug from the container and dispense the controlled amount of the drug from the pump cavity through the microneedle. The motor is configured to drive the pump actuator through a gear assembly comprising one or more gears integrated into the pump actuator.

The invention belongs to the technical field of gene editing, and particularly relates to an efficient grouper gene editing method based on fertilized egg injection. The method comprises the following steps: soaking fertilized eggs of groupers fertilized within 10 minutes in a weakening chorion culture medium buffer solution at 26 DEG C for 30-60 minutes, then taking out the fertilized eggs, and injecting a target CRISPR / Cas9 mixture into the fertilized eggs of the groupers before embryos develop to a four-cell stage; the attenuated chorion culture medium buffer solution is prepared by using sterilized seawater and comprises 102.8 mM of sodium chloride, 10mM of trihydroxymethyl aminomethane, 20mM of glycine and 1mM of reduced glutathione. According to the application, the rapid hardening of the chorion of the fertilized egg of the grouper can be effectively weakened, after the hardening time is prolonged to a four-cell stage, a relatively long operation window stage is obtained for micro-injection, the injection efficiency and the injection success rate can be improved, and the injection problem of the fertilized egg of the grouper is solved.

The invention discloses a construction method and application of a multi-organ development injury animal model based on non-coding long-chain RNA (Ribonucleic Acid). According to the present invention, it is found that the expression level of LncRNA URS0000A7E830 is significantly increased compared to the normal control group in the embryonic period amoxicillin exposed zebra fish model, and the URS0000A7E830 siRNA microinjection intervention is performed on the fertilized one-cell period zebra fish embryo based on the embryonic period amoxicillin exposed zebra fish model so as to effectively improve the multi-organ differentiation damage. The model established by the invention is novel, reliable, simple and convenient, and has important significance for illuminating the occurrence mechanism of the development injury of multiple organs and determining clinical early warning and intervention targets.

The utility model discloses an auxiliary device for microinjection, which comprises an injection table serving as a mounting carrier, a cylinder bin is mounted in the center of the top of the injection table, six groups of screw rods are arranged in the injection table in a central symmetry manner, and inner threaded blocks are arranged on the outer sides of the screw rods in a threaded manner. An outer sleeve is mounted at the top of the internal thread block, and a fixing pipe is arranged in the outer sleeve. According to the device, the driving device is controlled to drive the six groups of screw rods to synchronously rotate, so that the internal thread blocks are forced to drive the outer sleeves to move towards the central position of the cylindrical bin, and a culture dish positioned in the cylindrical bin is jointly pushed to the central position in the cylindrical bin through one ends of the six groups of outer sleeves and is clamped and fixed; as the six groups of outer sleeves can freely and synchronously move, culture dishes with different sizes can be fixed and automatically pushed to the central position of the bottom in the cylindrical bin, so that the convenience of the microinjection equipment in the use process is improved.

The invention discloses a construction method and application of an animal model for expressing human ATP7B and a high-frequency mutant transgenic mouse of the human ATP7B, and belongs to the technical field of animal model construction. The invention provides a construction method of a human ATP7B high-frequency mutant transgenic mouse model. The method comprises the following steps: firstly, transforming a BAC plasmid, knocking out a non-target gene, and accurately introducing specific p.R778L, p.P992L and p.T935M point mutation of East Asian population by utilizing a CRISPR / Cas9 technology, so as to obtain mutant BAC DNA (Deoxyribose Nucleic Acid); and then, microinjection is carried out on the mouse fertilized eggs, and transgenic positive F0-generation mice are obtained through cultivation. And finally, hybridizing and screening with Atp7b gene knockout mice in multiple generations to obtain a target strain of which the endogenous Atp7b is completely deleted and which only expresses the human mutant ATP7B. The model can accurately simulate the genetic background and pathological phenotype of East Asia WD patients, and can be used for screening targeted drugs aiming at human ATP7B mutation and evaluating the improvement effect of the drugs on liver and nerve injury.

The application discloses a construction method of a mouse model with increased transcription factor FOXG1 copy number and application thereof, belongs to the field of animal models, and is characterized in that FOXG1 syndrome is caused by mutation of a risk gene FOXG1, and the increased copy number makes FOXG1 expression up-regulated as one of the mutation forms. FOXG1 up-regulation is also found in some autism patients. The construction method of the mouse model with increased FOXG1 copy number comprises plasmid construction and microinjection of CAG-loxp-stop-loxp-Foxg1-IRES-EGFP, F0 generation Founder mouse identification, and F1 generation mouse stable genetic genotype identification. The application over-expresses a mouse Foxg1 gene, simulates the increase of the FOXG1 syndrome clinical case copy number and the up-regulation of FOXG1 in autism patients. The application can provide a model for autism research and be used for developing precise diagnosis and intervention strategies for the FOXG1 syndrome.

The application relates to the technical field of animal model construction, and particularly discloses a Sypl1 gene knockout colorectal cancer mouse model construction method and application, which comprises the following steps: synthesizing a specific target site gRNA of a Sypl1 gene, mixing Cas9 protein and the target site gRNA to obtain an injection compound, microinjecting the injection compound into mouse zygotes, and transplanting the surviving zygotes into the oviducts of pseudopregnant female mice to obtain F0 generation mice, and the mice born after 20 days are Sypl1 gene knockout mouse animal models; and a chemical induction modeling method is used to construct a colorectal cancer model in wild type mice and gene knockout mice. The Sypl1 gene knockout mouse is constructed by using a CRISPR / Cas mediated genome engineering technology Sypl1 combined with the most widely used colorectal cancer chemical inducer azoxymethane / dextran sodium sulfate, a new colorectal cancer mouse model is constructed, compared with a traditional chemical induction model, the tumor formation rate can be significantly improved, and the tumor formation time can be shortened, and a more efficient animal model is provided for colorectal cancer research.

The utility model belongs to medical storage technical field, concretely speaking is a kind of microinjection needle storage device, including lower box and upper box, the upper box one side is hinged on the one side of lower box, and is opened and closed by opening and closing piece to carry out opening and closing operation, the lower box both sides wall all are fixed with multiple sets of locating plate, the locating plate between adjacent is equipped with placing plate by inserting piece, the placing plate top end is equipped with oval slot, the oval slot in fixed attachment has foam pad, the utility model provides a kind of microinjection needle storage device, to the problem of existing microinjection needle needle body storage inconvenience.

The application discloses a robot cell microinjection operation method based on dynamic balance game mechanism reinforcement learning. The method comprises the following steps: training an injection operation decision model using a total loss function; the total loss function comprises a loss of a PPO reinforcement learning network, an entropy regularization term and a state prediction loss of a state prediction network; during the robot cell microinjection operation process, injection environment images are collected in real time, input into an instance segmentation network model, and environment mask images are obtained; the injection needle tip real-time coordinates and cell mask images are obtained according to the environment mask images; a feature extraction network receives the cell mask images, a PPO reinforcement learning network receives the feature extraction results and the injection needle tip real-time coordinates, and the expected position of the injection needle tip is obtained as feedback information to control the robot. The application can perform safe and high-precision zebrafish embryo cell microinjection operation in a real scene, and provides technical support for the fields of gene editing and cell breeding.

The invention discloses an application of a CEBPD gene in improving the development efficiency of a pig cloned embryo and a method for improving the pig cloning efficiency. By microinjection of CEBPD gene overexpression plasmids into pig cloned reconstructed embryos, the blastocyst rate and the blastocyst cell number of the pig cloned embryos can be increased, the development efficiency of the cloned embryos can be improved, and after the injected cloned embryos are transferred into uterus of a pregnant sow and a piglet is born, the cloning efficiency can be increased from 1.16% to 3.93% and increased by 239%. Compared with the prior art, the gene regulation strategy has better stability, controllability and high efficiency, and a new technical path and method are provided for improving the development efficiency of pig cloned embryos and improving the pig cloning efficiency.

The invention relates to the technical field of animal model construction, and particularly discloses a construction method and application of a Sypl1 gene knockout colorectal cancer mouse model, and the construction method comprises the following steps: synthesizing a specific target site gRNA of a Sypl1 gene, uniformly mixing Cas9 protein and the target site gRNA to obtain an injection compound, microinjecting the injection compound into a mouse fertilized egg to obtain a Sypl1 gene knockout colorectal cancer mouse model. And transplanting the survival fertilized ova into the salpingtube of a pseudopregnant female mouse for 20 days to obtain a mouse which is an F0-generation mouse, obtaining a Sypl1 gene knockout mouse animal model, and constructing a colorectal cancer model in a wild type mouse and a gene knockout mouse by adopting a chemical induction modeling method. A Sypl1 gene knockout mouse is constructed through a CRISPR / Cas mediated genome engineering technology, a novel colorectal cancer mouse model is constructed in combination with a colorectal cancer chemical inducer azomethane / dextran sodium sulfate which is most widely applied at present, and compared with a traditional chemical induction model, the novel colorectal cancer mouse model has the advantages that the tumor formation rate can be remarkably increased, the tumor formation time can be shortened, and the tumor formation cost can be reduced. And a more efficient animal model is provided for colorectal cancer research.

The invention belongs to the field of gene engineering, particularly relates to a construction method of a Kcnk4 gene A245P point mutation mouse model, and designs the construction method of the Kcnk4 gene A245P point mutation mouse model. The cutting specificity is improved by systematically optimizing a gRNA design algorithm, an efficient oligo donor DNA synthesis strategy is developed to improve the recombination efficiency, and a multi-level genotype identification system is established to ensure the mutation accuracy. The method comprises the following specific steps: designing gRNA of targeted Kcnk4 gene exon6, synthesizing oligo donor DNA containing A245P mutation, carrying out microinjection to Balb / c mouse fertilized eggs, transplanting, screening positive F0-generation mice through PCR amplification and sequencing peak map analysis, and mating with a wild type to obtain an F1-generation heterozygote with stable heredity. The mutation efficiency reaches 90% or above, the genotype identification method is simplified, heterozygotes and homozygotes can be distinguished through a sequencing peak graph, and false positive of a traditional enzyme digestion method is avoided. The model provides a reliable tool for nervous system disease research and drug screening.

The invention discloses a construction method and application of an H11 site humanized ACE2 gene overexpression golden hamster model, and belongs to the technical field of genetic engineering animal model construction. According to the method, hormone-induced superovulation female golden hamsters and male endosomes are fertilized to obtain two-cell stage embryos, an exogenic CRISPR-Cas9 gene modified sample is microinjected into a single two-cell stage embryo cleavage bulb, and after culture recovery, the sample is transferred into a real pregnancy replacement receptor body for development. And the construction method of the golden hamster model for overexpressing the ACE2 gene at the H11 site is further obtained. The golden hamster is used as a model background animal, the humanized ACE2 overexpressed golden hamster model is constructed at the H11 site, and the golden hamster model can be used for researching an ACE2 gene mechanism and SARS-CoV-2 infection.

The invention relates to the field of molecular biology, in particular to sgRNA for preparing an animal model of which a GJB2 gene is systematically knocked out. The technical problem to be solved by the invention is to provide a technology for establishing a systematic GJB2 gene knockout animal model for the field. According to the technical scheme for solving the technical problem, the sgRNA capable of preparing the animal model of which the GJB2 gene is systematically knocked out is provided. The sgRNA and Cas9mRNA disclosed by the invention are simultaneously introduced into a pig fertilized egg or an early development embryo through microinjection, so that an animal model of which the GJB2 gene is systematically knocked out can be prepared. The method can more accurately simulate a genetic mutation mode of GJB2 gene function deletion non-syndromic deafness with the highest morbidity in Chinese congenital deafness patient groups, and provides key support for researching a deafness causing mechanism of GJB2 gene deletion and researching and developing new technologies, new schemes or new equipment for treating and intervening GJB2 deletion deafness.

The present application relates to the application of RNF20 gene in the preparation of therapeutic drugs for azoospermia caused by RNF20 gene mutation, and the therapeutic drugs. The present application rescues the non-obstructive azoospermia of the young mouse with RNF20 gene deletion by introducing the RNF20 gene expression vector into the testicular tissue of the mouse through microinjection and electrotransformation. Further, the non-obstructive azoospermia of the adult mouse with RNF20 gene deletion is rescued by adding an autophagy inhibitor in the microinjection.

The invention discloses a shrimp and crab animal embryo microinjection method and a shrimp and crab animal gene editing method. According to the shrimp and crab animal embryo microinjection method, a fertilized egg embryo of a shrimp and crab animal in a cell stage is obtained firstly, then injection liquid is injected into the fertilized egg embryo through a microinjection needle, and the inner diameter of a needle point opening of the microinjection needle is not larger than 1 micrometer. According to the shrimp and crab animal gene editing method, after the shrimp and crab animal embryo microinjection method is used for completing microinjection, the survival rate is improved by optimizing culture conditions. According to the method, the reliability and the efficiency of performing a gene manipulation experiment on the macrobrachium rosenbergii embryo by utilizing a microinjection method are remarkably improved, and the method has important theoretical significance and practical significance in developing macrobrachium rosenbergii functional gene research, gene editing breeding and the like; and important reference is provided for microinjection operation of other shrimp and crab crustacean animal embryos.

The application discloses a needle breaking device for microinjection of Caenorhabditis elegans, and relates to the technical field of microinjection, which comprises a fixed needle breaking mechanism and an automatic needle breaking mechanism matched with the fixed needle breaking mechanism; the fixed needle breaking mechanism is provided with a fixed needle control lower disc and a rotating needle control cover body matched with the fixed needle control lower disc; the fixed needle control lower disc is provided with a plurality of radial needle control grooves with equal radian and used for mounting microinjection needles to be broken; and a corresponding end of the radial needle control grooves penetrates through one side of the outer periphery of the fixed needle control lower disc to allow the microinjection needles to be broken to pass out; the rotating needle control cover body is provided with a driving element used for driving the microinjection needles to be broken mounted in the radial needle control grooves to pass out from one side of the outer periphery of the fixed needle control lower disc at a length. The application has the effect of significantly improving the consistency of the breaking position and angle.