36 results about "Plant Germ Cells" patented technology

The invention provides a method for overspeed preparation of gene edited goldfish. The method comprises the following steps: S1, obtaining gobiocypris rarus without primordial germ cells as receptor fish; s2, designing a target spot for the goldfish amh gene, introducing a Cas9 / gRNA complex into a goldfish fertilized egg, performing sequencing verification to obtain a mutant individual, and feeding the mutant individual as a donor fish; s3, taking gonads of donor fish, cutting into pieces, performing enzymolysis digestion, filtering, and performing density gradient centrifugation to enrich donor germline stem cells; s4, transplanting the germline stem cells of the donor fish into the receptor fish body, and feeding to obtain the gene edited goldfish sperms and ova. S5, hybridizing and feeding goldfish amh mutant sperms and ova produced by gobiocypris rarus to obtain gene edited goldfish individuals. According to the invention, gobiocypris gobiocypris is used as a receptor, the cross-subfamily gene edited goldfish is used as a donor, and an efficient, stable and easy-to-popularize method for preparing the gene edited goldfish is provided by utilizing a belly borrowing reproduction technology.

The invention discloses application of Kitl in promoting meiosis of germ cells cultured in vitro. By adding a small molecule Kit ligand (Kitl), germ cells (including primordial germ cells PGCs, primordial germ cell-like cells PGCLCs and the like) cultured in vitro are promoted to enter and complete the first meiosis earlier stage, meiosis key protein expression is improved, and homologous chromosome association and recombination efficiency is improved. The method has a wide transformation medical prospect in the fields of human assisted reproductive technology and reproductive medicine.

The application discloses a kind of chicken primordial germ cell transfection methods, including the following transfection steps: S1, the separation of chicken primordial germ cell is carried out, and grouping culture in culture medium, S2, according to the proportion of 3.5:1 of pPB-GFP transposon plasmid and transposase plasmid mPB dosage Two are mixed, S3, first dilute plasmid with opti-MEN, then dilute transfection reagent with opti-MEN, plasmid diluent and transfection reagent diluent are mixed to obtain compound, the transfection operation of chicken primordial germ cell is carried out by the culture medium of different serum adding amount and adding time in the application, the influence of different serum adding amount and different serum adding time on chicken primordial germ cell transfection efficiency can be understood, then the increase and decrease of the fluorescence brightness in chicken primordial germ cell after transfection are recorded, the growth of chicken primordial germ cell after transfection is understood, so that the most suitable culture medium can be selected, serum adding condition is optimized, and more efficient chicken primordial germ cell transfection can be realized.

The application belongs to the technical field of biology and particularly relates to a functional recombinant Cas9 protein targeted to an ovocyte of Procambarus clarkii and application thereof. The protein is NLs-VgSP-Cas9-NLs. The application directly delivers the recombinant Cas9 protein, thereby reducing the risk of integration of exogenous genes. By specifically targeting the ovocyte, off-target effects on somatic cells can be avoided, the efficiency of editing of germ cells is improved, and early development or genetic manipulation can be conveniently studied. The ovocyte of Procambarus clarkii is large, the recombinant Cas9 protein is efficiently delivered by receptor-mediated endocytosis on the surface of the ovocyte, and the problem that microinjection of zygotes cannot be implemented is solved.

The present disclosure provides bovine animals and methods to create recipient bovine animals for spermatogenic stem cell transplantation / complementation through modulation of the NANOS2 gene. In one embodiment genome editing was used to create bovine animals with insertions or deletions (indels) that inactivate or otherwise modulate NANOS2 gene activity so that resulting male bovines lack functional germ cells yet retain functional testicular somatic cells, and bovine females are fertile. These bovine males can then be transplanted or complemented with donor cells capable of giving rise to cells of the spermatogenic lineage, or spermatogenic stem cells and used for breeding.

The invention discloses a method for identifying germ cells of grass carps and carps by using dazl genes. The method comprises the following steps: cloning dazl cDNA sequences of the grass carps and the carps, and performing comparative analysis by using DNAMAN software; designing a universal non-specific primer, a grass carp specific primer and a carp specific primer according to sequence differences; extracting total RNA (Ribonucleic Acid) of testis of 12-month-old grass carp and carp, and reversely transcribing into cDNA Respectively carrying out PCR (Polymerase Chain Reaction) amplification by using three pairs of primers by taking the cDNA as a template; carrying out 1% agarose gel electrophoresis and imaging on the PCR product, and identifying and distinguishing germ cells according to the size of a strip; the method is simple in structure and reasonable in design, high-specificity primers are designed based on small difference of dazl gene sequences for the first time, precise amplification is achieved by combining with optimized annealing temperature (60 DEG C / 65 DEG C), special instruments are not needed, operation is easy and convenient, sensitivity is high, grass carp germ cells in carp gonads after transplantation can be rapidly identified, development of the'belly borrowing reproduction 'technology is supported, and the method is suitable for large-scale popularization and application. The grass carp breeding period is remarkably shortened, and the important industrialization prospect is achieved.

The present invention provides an endogenous germ cell-free fish, crustacean or mollusk having a chimeric gonad wherein the chimeric gonad comprises at least one transplanted germ cell, the transplanted germ cells have mutations that are present in the germ cell line and disrupt the development and / or function of somatic gonad cells that can be used as parent fish. The present invention also provides a method of preparing a parent fish, crustacean or mollusk for the production of sterilized fish, crustacean or mollusk that is sterilized or sex-determined, and the parent fish themselves.

The invention discloses a rapid breeding method for generating larimichthys crocea sperms based on spermatogonial stem cell transplantation. The rapid breeding method comprises the following steps: step S1, performing whole cryopreservation on larimichthys crocea testis; s2, separating the spermatogonial stem cells of the pseudosciaena crocea; s3, dyeing the spermatogonial stem cells of the pseudosciaena crocea; step S4, treatment of nibea albiflora receptors; and S5, transplanting the dyed donor larimichthys crocea spermatogonial stem cells into the nibea albiflora receptor testis. According to the method, when the nibea albiflora acceptor is treated, the traditional busulfan concentration of 40 mg / kg, twice injection and 35 DEG C high-temperature assistance is optimized into 60 mg / kg, single injection and 22 DEG C normal-temperature culture, optimal parameters induced by busulfan are screened out through experiments, the operation process is simplified under the action of ensuring that endogenous germ cells of the acceptor are reduced and immunological rejection is reduced, and the method is suitable for large-scale popularization and application. The harsh requirement of a high-temperature environment is avoided, and meanwhile the field planting efficiency of donor cells in receptors and the survival rate of the receptors are improved.

This invention discloses a method for the isolation, culture, and identification of chicken sexual gonadal cells, belonging to the field of biotechnology. The invention selects chicken embryos hatched to 18.5 days old, isolates the gonads after PCR sex identification, and uses 0.25% trypsin-EDTA digestion followed by filtration through a 70μm filter to obtain a single-cell suspension with a survival rate >90% and free from PGC contamination. A sex-specific culture system is established, allowing cells to be stably passaged and maintain a sex-specific phenotype. Through morphological, qRT-PCR, immunofluorescence, and Western blot multidimensional identification, accurate identification of male testicular Sertoli cells and female ovarian granulosa cells is achieved. This method is simple to operate, has good reproducibility, and produces high cell viability and purity. It can provide a stable gonadal microenvironment for the in vitro directed differentiation of chicken primordial germ cells, significantly improving gamete induction efficiency. It is suitable for research on avian reproductive and developmental mechanisms, transgenic breeding, and germplasm resource preservation.

The application belongs to the technical field of medicine, and discloses application of uridine-5' monophosphate and a compound thereof in preparation of a medicine for enhancing the proliferation ability of male reproductive cells. It is found for the first time that the uridine-5' monophosphate has the proliferation ability on the male reproductive cells, and the proliferation effect is best when the concentration of the uridine-5' monophosphate is 10 μM, the proliferation effect on the mouse TM3 cells is increased by 52.87% compared with a control, and the proliferation effect on the mouse TM4 cells is increased by 53.57%. In addition, the uridine-5' monophosphate has a synergistic ability when being compounded with xylonic acid lactone, and further improves the proliferation effect on the TM3 cells, thereby laying a foundation for treatment of male reproductive system diseases in the future.

An object of the present invention is to provide a method for simply and efficiently inducing differentiation of oogonia or prospermatogonia from primordial germ cell-like cells or primordial germ cells. The object is solved by a method for producing oogonia or prospermatogonia comprising a step of culturing primordial germ cell-like cells or primordial germ cells in a medium containing a BMP receptor type 2 signal activator to obtain oogonia or prospermatogonia.

This disclosure relates to methods, products, and compositions useful for inducing bovine induced blastoids. Methods enabling naive stem cell aggregation and induced blastoid formation are described. Aggregation and induced blastoid media suitable for promoting naive stem cell aggregation and preparing induced blastoids are also provided. Bovine induced blastoids may be used in selection and breeding programs, including the replication of pre-transplant embryos with desirable characteristics, as well as in vitro breeding, including cross-generation breeding cycles from parental germ cells and / or embryos to offspring embryos.

Disclosed are a chicken serum-free culture medium for promoting the proliferation of chicken primordial germ cells and use thereof. Ovotransferrin is used in the culture medium instead of chicken serum. The effect of using ovotransferrin, instead of chicken serum, in a culture system for PGCs is disclosed. Ovotransferrin can maintain the self-renewal of PGCs, thereby providing a basis for developing a stable culture system. The culture medium can avoid adverse effects caused by the undefined components of chicken serum, and the precise use of various growth factors can provide better culture conditions and reduce culture costs.

The invention provides a myxocyprinus asiaticus individual genetic resurgence method which comprises the following steps: S1, taking a myxocyprinus asiaticus gonad tissue, cleaning, cutting into pieces, adding an L-15 culture medium containing double antibodies and a DPBS buffer solution, and centrifuging to obtain the gonad tissue; s2, performing water bath on the gonad tissues to obtain resuscitated gonad tissues; s3, digesting, filtering and centrifugally purifying the resuscitated gonad tissues to obtain germline stem cells; s4, injecting morpholino to obtain gobiocypris rarus without primordial germ cells; and S5, transplanting the germline stem cells into gobiocypris rarus juvenile fish bodies, and culturing to obtain the myxocyprinus asiaticus gametes. S6, the generated myxocyprinus asiaticus gametes are fertilized, and myxocyprinus asiaticus individuals are obtained. According to the method, the sperms of the myxocyprinus asiaticus are successfully obtained by utilizing a gonad cryopreservation and abdomen borrowing reproduction technology, and a feasible way is provided for individual genetic resurgence of rare and endangered fishes.

The invention discloses an application of 24-Epi brassinolide in promoting the development of female germline stem cells. Belongs to the technical field of reproductive biology. Female germline stem cells (FGSCs) are important cell models for studying the development of germ cells, however, the regulation and control mechanism of natural products on the development of the FGSCs still needs to be deeply explored. The invention aims at disclosing the regulation and control effect of 24-Epi brassinolide (24-Epibrassinolide, EBR) on FGSCs development and a molecular mechanism of the 24-Epi brassinolide on the FGSCs development by utilizing an FGSCs in-vitro development system and combining LongRNA-seq and RIC-seq technologies, and a new strategy is provided for treatment of infertility and optimization of a clinical treatment scheme.

In some aspects, methods are provided for generating primordial follicles from human and other mammalian germ cells with high efficiency, high cell quality, and high reproducibility. In some aspects, methods and culture systems are provided for generating an ovarian organoid, such as a cell aggregate consisting of both somatic and germ cells from a source derived from primary and / or stem cells, the ovarian organoid is capable of progressing primordial germ cells into oocytes that initiate meiosis and are capable of follicular assembly. In some aspects, provided embodiments include the step of culturing an ovarian organoid in a fully defined serum-free medium and under conditions that generally employ a low defined serum protein component.

The invention discloses a bicolor fluorescence in-situ hybridization probe for identifying Eccyp307a1 and Vasa gene expression and application of the bicolor fluorescence in-situ hybridization probe, and belongs to the technical field of molecular biology. The invention provides a fluorescence in-situ hybridization probe for exopalaemon carinicauda, and the probe is designed for mRNA of Eccyp307a1 and Vasa in exopalaemon carinicauda and comprises a specific hypervariable region targeting the mRNA of Eccyp307a1 and Vasa in exopalaemon carinicauda. According to the invention, different substrate markers are adopted for Eccyp307a1 and Vasa, so that the expression of Eccyp307a1 and Vasa can be respectively marked by fluorescence with different colors, and the sensitivity is high. The fluorescence in-situ hybridization probe provided by the invention can accurately and intuitively locate the expression condition of Eccyp307a1 in germ cells, can be used for detecting and locating the expression of reproduction-related genes, and provides a powerful technical support for explaining the expression rule and potential interaction of Eccyp307a1 and Vasa in the development and reproduction process of organisms; the method is of great significance to the development research of crustacean animals, especially exopalaemon carinicauda.

Provided is use of PGAM2 in the breeding of Blcattle black cattle, which belongs to the technical field of molecular biological breeding, in which individuals having a high PGAM2 content are screened as high-quality breeding bulls. In the present disclosure, it is proved by means of experiments that PGAM2 may promote the proliferation and glycolytic metabolism of Sertoli cells to produce lactic acid, an energy substrate required for spermatogenic cell development. It regulates the formation of blood-testis barrier and facilitates the communication between Sertoli cells and germ cells, thereby promoting spermatogenesis. The present disclosure provides a new direction for the breeding of Blcattle black cattle for high quality beef production.

In some aspects, methods are provided for producing primordial follicles from human and other mammalian germ cells with high efficiency, high cell quality, and high reproducibility. In some aspects, methods and culture systems are provided for producing ovarian organoids, such as cell aggregates composed of both somatic and germ cells from primary and / or stem cell-derived sources, the ovarian organoids having the ability to advance primordial germ cells into oocytes that are competent to enter meiosis and form follicular aggregates. In some aspects, the embodiments provided include the step of culturing the ovarian organoids in fully normalized serum-free medium and under conditions using a generally low normalized serum protein component.

To develop novel therapeutic techniques that do not pose the risk of genotoxicity to help infertility patients. [Solution] A single-stranded negative-strand RNA viral vector containing a nucleic acid sequence encoding a protein associated with the formation or development of germ cells, or a pharmaceutical composition for preventing or treating infertility containing an RNA molecule encoding the protein, is provided.

The invention belongs to but is not limited to the technical field of gene engineering, and particularly relates to a preparation method and application of unlabeled sebastes schlegeli IGF2 recombinant protein, and by detecting the IGF2 expression level in different development stages of ovary, it is found that IGF2 has an important regulation effect on fertilized ovary matrix and embryo. The cDNA of sebastes schlegeli is used as a template for PCR amplification to obtain the sebastes schlegeli IGF2 mature peptide, and the amino acid sequence of the sebastes schlegeli IGF2 mature peptide is as shown in SEQ ID NO: 2. The recombinant protein comprises the IGF2 mature peptide, a His-Sumo tag connected with the N end of the IGF2 mature peptide, and a connecting peptide fragment between the His tag and the Sumo tag, and the amino acid sequence is shown as SEQ ID NO: 1. The invention also provides application of the sebastes schlegeli IGF2 mature peptide recombinant protein in promoting proliferation of primary ovarian cells and development of germ cells of sebastes schlegeli.

The application provides a method for genetic reactivation of individual Procyprinus palpebrosus, comprising the following steps: S1, taking the gonadal tissue of Procyprinus palpebrosus, washing, cutting, adding L-15 culture medium containing double antibodies and DPBS buffer solution to centrifuge the gonadal tissue; S2, water-bathing the gonadal tissue to obtain recovered gonadal tissue; S3, digesting the recovered gonadal tissue, filtering, centrifuging and purifying to obtain germ stem cells; S4, injecting morpholino to obtain rare gobi-ocypris rarus with deleted primordial germ cells; S5, transplanting the germ stem cells into the body of the rare gobi-ocypris rarus juvenile to culture, and obtaining the gametes of Procyprinus palpebrosus; and S6, fertilizing the generated gametes of Procyprinus palpebrosus to obtain the individual Procyprinus palpebrosus. The application successfully obtains the sperm of Procyprinus palpebrosus by using the cryopreserved gonadal tissue and the technology of parthenogenesis, and provides a feasible way for the individual genetic reactivation of rare and endangered fish species.

The invention relates to the technical field of animal models, in particular to construction and application of a Stard6 gene knockout mouse animal model, and the Stard6 gene knockout mouse animal model is obtained by mating a Stard6-flox mouse with a Star8-Cre mouse. A Stard6 gene knockout mouse model is constructed, and it is found that gene level expression and protein level expression of a specific Stard6 gene knockout mouse in male germ cells are remarkably reduced, so that testis development and spermatogenesis of a male mouse of the Stard6 gene knockout mouse are abnormal. Therefore, the problems that a common animal model cannot target a target gene, interference of other factors exists, an animal model with whole body knockout is prone to embryo death and the like are solved.

The invention provides a line establishment method of primordial germ cells from chick embryo blood. The line establishment method comprises the following steps: obtaining fertilized eggs incubated to a 14-17HH period; sucking chick embryo blood from the dorsal aorta by adopting a micro-injection needle with a specific size and specific suction pressure; carrying out centrifugal treatment, and resuspending the cells by adopting a PGCs complete culture solution added with an antifungal drug; carrying out subculture by adopting a PGCs complete culture solution until the PGCs monoclonal cell line is successfully established; according to the line establishment method, a traditional enzyme digestion separation mode is not adopted, dependence on feeder layer cells is avoided, and the undifferentiated state of the PGCs can be maintained directly through culture medium components of a specific formula by adopting the chick embryo blood obtained through specific operation; compared with the prior art, the operation is more efficient and simpler; more importantly, the technical scheme can be used for quickly and successfully establishing the line, is good in repeatability and high in line establishment success rate, and can be popularized and applied to in-vitro line establishment of the PGCs and subsequent biological design breeding and model animal production.

The invention provides a mononuclear transcriptomics technology-based analysis method for interaction of testicular sertoli cells and germ cells of plateau livestock, and belongs to the technical field of bioinformatics. The analysis method comprises the following steps: carrying out mononuclear transcriptome sequencing by taking testis tissues of plateau livestock at different development stages as materials, dividing a supporting cell subset and a germ cell subset according to marker genes, drawing a quasi-time differentiation track, screening differential expression genes and enriching functions, and constructing a supporting cell and germ cell interaction molecular network. According to the method, molecular characteristics, differentiation tracks and interaction networks of supporting cells and germ cells of the testis of the Tibetan sheep in different development stages are analyzed, and the problems that in the prior art, cell resolution is insufficient, development dynamics are deficient, variety specificity information is deficient and the like are solved; a brand new technical approach and theoretical basis are provided for clarification of cell molecular basis of low reproductive capacity of Tibetan sheep and mining of key regulatory factors.

Methods of preventing or restoring age-related loss of reproductive function in a mammal by contacting germ cells of the mammal with a composition comprising a flavonoid glycoside (e.g., rutin) and L-arginine are provided. Also provided are methods of preventing or restoring age-related reproductive function loss, preventing or restoring age-related fertility decline, and reducing the risk of age-related reproductive function loss by administering the composition to a mammal. Also disclosed is the use of the composition for preventing or restoring age-related fertility decline in a mammal.

This invention provides a method for isolating and identifying interstitial cells from Bactrian camels, belonging to the field of cell isolation technology. A mixture of 0.08–0.12% collagenase IV solution and 0.23–0.27% trypsin-EDTA at a volume ratio of 1:2 is used to digest Bactrian camel testicular tissue blocks. The mixture is then purified by hypotonic treatment to remove excess germ cells, differential adhesion purification to remove a large number of supporting cells, and Percoll density gradient centrifugation to remove a small number of supporting cells. The suspension, obtained from a 34%–60% Percoll gradient, is transferred to a new culture flask to obtain interstitial cells with a purity ≥95%. Identification is performed using morphological observation, immunofluorescence staining, and secretory function testing. The interstitial cells isolated from Bactrian camels using this invention can be used for research on the reproductive performance of Bactrian camels, laying the foundation for understanding the reproductive performance of Bactrian camels.