18 results about "Pronucleus" patented technology

The present disclosure generally relates to micromanipulation devices and methods for the manipulation of mammalian sub-cellular structures. Uses include intra cytoplasmic sperm injection procedures, embryo biopsy procedures, trophectoderm biopsy procedures, mitochondrial transfer procedures, pronuclear transfer procedures, maternal spindle transfer procedures, cumulus cell biopsy procedures and other sub-cellular manipulation procedures. Devices comprise a cellular manipulation dish further comprising multiple sub- cellular manipulation units having a microwell and a cell deposit well wherein the cell deposit opening is disposed adjacent to the microwell opening.

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

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

The present invention relates to the field of biotechnology. Specifically, the present invention relates to a method for rapidly extracting the nuclei of germinal vesicle (GV)-stage oocytes and pronuclei of zygotes. This method uses a self-made egg transfer needle to aspirate oocytes or fertilized eggs, and then uses a self-made glass needle to aspirate the nuclei of GV-stage oocytes and pronuclei of zygotes from the nuclear extraction droplet. A relatively large number of nuclei of GV-stage oocytes or pronuclei of fertilized eggs can be obtained in a short time, and expensive equipment and professional technicians are not required. The obtained nuclei of GV-stage oocytes or pronuclei of fertilized eggs have high integrity and low contamination rate, and have great application value.

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 invention discloses a method and system for embryo quality detection, relating to the field of embryo detection technology, including the following steps: S1: fertilized embryos are continuously cultured in an in vitro culture system containing specific metabolic marker detection components, and continuous dynamic imaging of the embryos is performed to obtain a complete dynamic developmental image sequence; S2: the dynamic developmental image sequence is segmented, and temporal developmental features are captured, identifying key time points such as pronucleus appearance and disappearance, cleavage, and blastocyst formation, and generating a segmentation mask corresponding to each frame of the dynamic developmental image sequence; this invention achieves full-process time-difference imaging to capture the temporal features of embryonic development, without trauma or interference, avoiding subjective bias in static observation; by segmenting the dynamic developmental image sequence and capturing temporal developmental features, the segmentation accuracy is high and the anti-interference ability is strong.

The present invention discloses a method for constructing a transgenic mouse with systemic overexpression of human α-Syn-NLS. First, the human α-Syn coding sequence and the SV40 nuclear translocation signal sequence are obtained by PCR. Then, an entry clone vector containing the target sequence is constructed through a BP reaction. After digestion with enzymes, the entry clone vector is constructed into a recombinant expression vector containing the target sequence through an LR reaction. After digestion with enzymes again, using the lentiviral LV overexpression vector pLV-EGFP:T2A:Puro-EF1A as the backbone vector, the recombinant expression vector pDown-hSNCA / SV40NLS containing the target sequence is constructed into the lentiviral vector, and virus packaging and purification are carried out to obtain the pLV virus overexpressing nuclear import α-Syn. The virus is injected by the method of pronuclear injection of fertilized eggs to obtain a transgenic mouse with systemic overexpression of nuclear import human α-Syn protein, which can stably overexpress α-Syn for a long time and solve the problem of nuclear import at the same time. This model has important value for studying the comprehensive functions and mechanisms of α-Syn nuclear import.

The invention discloses a method for constructing an anti-spinetoram neoseiulus californicus gene and an ovary targeting peptide of the anti-spinetoram neoseiulus californicus gene. According to the invention, a segment of yolk protein targeted peptide binding region is identified in Neoseiulus californicus, and the yolk protein targeted peptide binding region and a Cas9 sequence are fused and constructed on a PET28a carrier, and the NcvgP2C-Cas9 protein is obtained through prokaryotic expression. NcvgP2C-Cas9 protein, sgNcnAChR alpha 6 and an endosome escape reagent are mixed and injected into female adult mites, NcnAChR alpha 6 gene large fragment deleted neoseiulus californicus offspring is obtained and shows a phenotype with 23.2 times of resistance to spinetoram, and the resistance of a knockout strain to the spinetoram belongs to an autosome partial recessive genetic mode. The invention provides a new method for the genetic improvement of the drug resistance of Neoseiulus californicus, and enhances the compatibility between biological control and pesticide treatment strategies.

The invention discloses a construction method and application of an HRAS humanized mouse for carcinogenicity evaluation, and the construction method comprises the following steps: construction of a Tg-HRAS mouse: injecting a constructed vector into a mouse fertilized egg in a prokaryotic injection manner, randomly inserting a human HRAS gene segment containing an HRAS gene regulatory sequence and an expression sequence into a mouse genome, and constructing the Tg-HRAS mouse; the sequence fragment of the human HRAS gene is as shown in SEQ ID NO. 1; construction of a PMD18-T skeleton vector: taking the PMD18-T vector as a template, carrying out high-fidelity PCR (Polymerase Chain Reaction) amplification to obtain a 2.7 kb linearized PMD18-T vector, and carrying out gel recovery to serve as a skeleton of SLIC reaction; according to the construction method and application of the HRAS humanized mouse for carcinogenicity evaluation, by constructing the HRAS humanized mouse, carcinogenicity verification of the mouse is more efficient, the passing incidence rate is high, quick discovery can be achieved in the verification process, the purpose of shortening the verification period is achieved, the test cost is reduced, and the spontaneous tumor probability is high.

The invention relates to the technical field of hydrogel preparations, and discloses a hydrogel preparation, a preparation method and application in repairing sperm injury. The method comprises the following steps: step 1, constructing recombinant plasmids; step 2, plasmid transformation; step 3, protein purification; step 4, preparing the SKAP2 protein wrapped by the mEXOs; step 5, preparing an SKAP2 hydrogel preparation; according to the invention, the SKAP2 protein is expressed through prokaryotic escherichia coli, the milk exosome is extracted, then the SKAP2 protein is wrapped in the milk exosome, and then the exosome containing the SKAP2 protein is fused into the hydrogel preparation, so that the sperm motility of a mouse with asthenospermia can be remarkably improved in a skin smearing manner; comprising the effect of improving sperm motility decline of asthenospermia caused by heavy metal lead, organic endocrine disrupter phthalate and high temperature and aging, and the problem that no effective repairing preparation for asthenospermia exists in the prior art is solved.

The application relates to the technical field of hydrogel preparations, and discloses a hydrogel preparation, a preparation method and application in repairing sperm damage. The method comprises the following steps: step one, constructing a recombinant plasmid; step two, plasmid transformation; step three, protein purification; step four, preparing mEXOs wrapped SKAP2 proteins; and step five, preparing a SKAP2 hydrogel preparation. In the application, SKAP2 proteins are expressed by means of a prokaryote Escherichia coli, milk exosomes are extracted, the SKAP2 proteins are then wrapped into the milk exosomes, and the exosomes containing the SKAP2 proteins are then fused into a hydrogel preparation. The sperm activity of asthenospermia mice can be significantly improved through skin smearing, including improving the sperm activity reduction effect of asthenospermia caused by heavy metal lead, organic endocrine disruptors phthalate, high temperature and aging, and the problem that there is no effective repair preparation for asthenospermia in the prior art is solved.

The present invention discloses a method for detecting the pronuclear area of ​​an embryo, comprising: constructing a pronuclear detection model using a preprocessed embryo source file, wherein the preprocessed embryo source file includes multiple embryo images; the pronuclear detection model outputs an embryo pronuclear area detection result, wherein the embryo pronuclear area detection result includes one or more groups of pronuclear coordinates, target classification, and confidence; performing a comprehensive area calculation on the embryo pronuclear area detection result, and outputting a pronuclear area A; the comprehensive area calculation includes a balanced calculation method, a denoising calculation method, and an enhanced calculation method.

The application discloses a culture medium for improving in-vitro aging of oocytes after ovulation and application thereof. The culture medium is prepared by adding 0.1-30 muM of astragaloside IV into an oocyte culture solution. The culture medium is safe, non-toxic and free of side effects, can reduce the oocyte fragmentation rate, active oxygen level and abnormal spindle rate of the oocytes after 24 hours of in-vitro culture, can significantly improve the mitochondrial membrane potential, in-vitro sperm binding capacity, in-vitro fertilization pronucleus rate and cleavage rate of the mature oocytes after 24 hours of in-vitro culture, and can improve the quality of the late embryos. The application of the culture medium provides favorable support for assisted reproductive technology, and has a good application prospect.

The present application relates to a kind of anti-recombination canine interleukin-1β yolk antibody and its preparation method and application, the preparation method utilizes genetic engineering prokaryotic expression preparation antigen recombination canine interleukin-1β, antigen recombination canine interleukin-1β is emulsified after first immunization egg chicken with first immunoadjuvant, again the recombination canine interleukin-1β antigen is emulsified to egg chicken with second immunoadjuvant, and egg chicken is strengthened immunization, collect egg, separate egg yolk and extract the antibody in the yolk of egg, successfully obtain anti-recombination canine interleukin-1β yolk antibody, the titer of the yolk antibody is 1:409600, and higher purity, with better specificity and stability, can provide stable antigen and antibody material basis for canine IL-1β related detection, antibody preparation and subsequent application research, with wide application prospect.

Embodiments described herein use deep learning to automate measurement of key morphokinetic embryo features associated with viability and quality, in particular those relevant for clinical in-vitro fertilization (IVF). Systems and methods may, for example, acquire one or more digital images of one or more embryos; select one or more embryos in each digital image; and for each selected embryo, (i) computationally characterize the zona pellucida, detect the degree of fragmentation in the embryo, and for each embryo with a low fragmentation score, computationally classify the embryo's developmental stage based on whether cells constituting the embryo exceed a threshold number (e.g., nine). For an embryo consisting of a single cell, pronuclei may be detected and counted. Based on these measurements, a viability score may be assigned to the embryo.

The application discloses an embryo quality intelligent evaluation method, comprising the following steps: establishing a pronucleus stage instance segmentation model; establishing a pronucleus stage target tracking model based on DeepSort; establishing a cleavage stage cell detection model; establishing a cleavage stage cell fragmentation degree rating model; establishing a semantic segmentation model based on U-Net; establishing a quality rating model based on Inception V3; and based on the six models established above, establishing an embryo quality intelligent evaluation model based on a graph convolutional neural network, so as to evaluate the embryo quality. The application models different stages in the embryo development process respectively, fuses the embryo characteristics of different stages, trains the graph convolutional neural network, effectively combines the characteristic parameters with physical significance in different periods, establishes the embryo quality intelligent evaluation model, and by using the intelligent evaluation method instead of manual evaluation, the measuring efficiency is greatly improved while the high precision is maintained.

The invention discloses a construction method and application of an IER2-Flag gene knock-in mouse model, and the construction method comprises the following steps: determining a target site of an IER2 gene to be knocked in a mouse, and designing sgRNA with a sequence as shown in SEQ ID No: 2; the method comprises the following steps: carrying out recombination on double-stranded sgRNA formed by annealing and CRISPR-Cas9 plasmid PX459, and constructing a PX459-sgRNA recombinant plasmid of which the sequence is as shown in SEQ ID No: 3-4; the method comprises the following steps: by taking a recombinant plasmid as a template, carrying out PCR (Polymerase Chain Reaction) amplification on a double-stranded DNA (Deoxyribose Nucleic Acid) fragment of a T7 promoter sequence + sgRNA + tracr RNA, and carrying out in-vitro transcription to obtain single-stranded sgRNA + tracr RNA, a single-chain sgRNA + tracr RNA, a Donor sequence containing a 3 * Flag tag and Cas9 Protein are co-injected into a male pronucleus of a mouse fertilized egg, an F0-generation IER2-Flag gene knock-in mouse can be obtained until an F3-generation IER2-Flag gene knock-in mouse is obtained, and the IER2-Flag gene knock-in mouse model is obtained. According to the invention, the expression of the Flag tag is detected for the first time to further identify that the IER2 protein is continuously and strongly expressed in the enamel cell, and an important animal model is provided for the research on revealing a mechanism for regulating and controlling enamel mineralization by the IER2 gene.

Embodiments described herein use deep learning to automate measurement of key morphokinetic embryo features associated with viability and quality, in particular those relevant for clinical in-vitro fertilization (IVF). Systems and methods may, for example, acquire one or more digital images of one or more embryos; select one or more embryos in each digital image; and for each selected embryo, (i) computationally characterize the zona pellucida, detect the degree of fragmentation in the embryo, and for each embryo with a low fragmentation score, computationally classify the embryo's developmental stage based on whether cells constituting the embryo exceed a threshold number (e.g., nine). For an embryo consisting of a single cell, pronuclei may be detected and counted. Based on these measurements, a viability score may be assigned to the embryo.