42 results about "Primordial germ cell" 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 a chicken sex identification method based on fluorescence labeling, and belongs to the technical field of gene engineering. The chicken sex identification method comprises the following steps: co-transfecting a CRISPR / Cas9 system and a WPG-EGFP donor plasmid into chicken primordial germ cells PGCs, screening to obtain EGFP positive PGCs, micro-injecting the EGFP positive PGCs into a receptor chicken embryo, hatching to obtain an EGFP reproductive chimera chicken, and identifying the sex through fluorescence labeling, the CRISPR / Cas9 system comprises sgRNA of a targeted chicken W chromosome WPG gene; the WPG-EGFP donor plasmid is constructed by inserting an EGFP gene into a WPG gene locus through a homologous recombination strategy. According to the method, the sex of the chicken can be visually and rapidly identified. The method solves the problems that a traditional sex identification method is complex in operation and depends on sample ages, has the advantages of high efficiency and accuracy, and is suitable for sex control and screening of poultry such as laying hens and broilers.

The invention discloses a method for generating haploid sperm-like cells through in-vitro differentiation of pluripotent stem cells and application of the haploid sperm-like cells. Human pluripotent stem cells are firstly differentiated into ectoderm-like cells, then the ectoderm-like cells are spontaneously gathered and converted into primordial germ cell-like cells under the induction of ectoderm induction factors, and then the primordial germ cell-like cells and testicular sertoli cells are subjected to mixed culture to form recombinant testicular tissues; the recombinant testis tissue enters meiosis under the induction of nutrition limitation and retinoic acid, then meiosis is completed under the stimulation of meiosis second-stage induction factors such as testosterone, follicle-stimulating hormone and pituitary extract, and interaction between primordial germ cell-like cells and testis supporting cells is enhanced by using small molecules SB431542, so that the meiosis of the testis is enhanced. And finally inducing to generate haploid sperm-like cells. According to the invention, a new tool can be provided for researching a molecular mechanism of male reproduction, and an important thought can be provided for research and transformation of asthenospermia, oligospermia, drug screening and the like.

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 present disclosure relates to deoxyribonucleic acid (DNA) editing agents, and their use in preparing genetically modified cells and birds. The present disclosure further relates to fertile genetically modified avians and genetically modified avian primordial germ cells (PGCs) for producing sterile genetically modified avians (birds) that can serve as surrogate hosts for donor PGCs. The present disclosure further relates to methods for producing fertile avian strains that can produce a population of embryos and offspring, in both sexes, all of which are sterile and viable, and further relates to their subsequent use as sterile surrogate hosts for donor PGCs.

The invention discloses a system for directionally differentiating chicken primordial germ cells into gametes in vitro and a regulation and control method, the system comprises a basic culture medium: based on DMEM / F12, adding 10% of fetal calf serum, 2% of chicken serum, 2 mmol / L of L-glutamine, 1 mmol / L of sodium pyruvate and 5.5 * 10 <-5 > mol / L of beta-mercaptoethanol; the spermatogonium induction culture medium further comprises BMP4, RA and GDNF; the oogonium induction culture medium also comprises BMP15, GDF9 and EGF (Epidermal Growth Factor). According to the system and the regulation and control method, the differentiation efficiency is improved, the culture period is shortened, and the number of cell clusters is remarkably increased.

The invention discloses a method for improving transfection efficiency of chicken primordial germ cells, and belongs to the technical field of cell transfection. The invention provides application of an EGFR (epidermal growth factor receptor) activator NSC 228155 in improvement of plasmid uptake efficiency and cationic liposome transfection efficiency of chicken PGCs, and a method for efficiently transfecting the chicken PGCs is constructed. Experimental results show that compared with a control group, the gene transfection efficiency of the chicken PGCs treated by the EGFR activator NSC 228155 under a DMRIE-C transfection reagent and the gene transfection efficiency of the chicken PGCs treated by the EGFR activator NSC 228155 under a LipofectamineTM 3000 transfection reagent are both remarkably improved, which indicates that the EGFR activator remarkably improves the transfection efficiency of the chicken PGCs; and the EGFR activator does not influence the cell characteristics of the chicken primordial germ cells. The method lays a foundation for establishing an efficient gene editing chicken technical system, and has a wide application prospect.

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 present application belongs to the technical field of genetic propagation, and particularly relates to application of targeted Rbm24a control of fish fertility. Specifically, the present application finds that Rbm24a is a new fish germ plasm component, which plays the function of a germ plasm organizer. Loss of function of the maternal protein will lead to the failure of primordial germ cells to form and exhibit a sterile phenotype, but does not affect the development of other tissues and organs, so that rbm24a is a new target gene for controlling fish fertility. Further, the present application also develops a strategy of inducing degradation of maternal Rbm24a protein by treatment with plant growth hormone, thereby achieving control of fish fertility, and also can produce sterile offspring by inducing degradation of Rbm24a through zGrad. The method is convenient, safe and thorough in controlling fish fertility, and therefore has good practical application value.

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.

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 invention relates to the field of bovine embryonic stem cell culture technology, and particularly to a long-term culture system for morphological bovine embryonic stem cells completely without a feeder layer and its applications. After trying various culture methods, this invention establishes a feeder-free bovine morphological embryonic stem cell line (FF-bESCs). Bovine morphological embryonic stem cells cultured under the feeder-free conditions provided by this invention can maintain stable proliferation capacity and pluripotency over a long period. The bovine morphological embryonic stem cells provided by this invention can undergo gene editing and can be effectively induced to differentiate into primordial germ cell-like cells (PGCLCs). This invention lays an important foundation for the standardized and large-scale culture of bovine embryonic stem cells under feeder-free conditions, and for their application in stem cell breeding and other fields.

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 present invention addresses the problem of providing a method for simply and efficiently inducing oocytes and prespermatogonium cells to differentiate from primordial germ cell-like cells or primordial germ cells. This problem is solved by a method for producing oogonium cells or prespermatogonium cells, which comprises a step for culturing primordial germ cell-like cells or primordial germ cells in a culture medium containing a BMP receptor type 2 signaling agonist to obtain oogonium cells or prespermatogonium cells.

The invention provides application of nicotinamide ribose in preparation of drugs for increasing the number of primordial germ cells and prolonging the growth cycle, and relates to the technical field of cell biology. Animal experiments prove that the supplement of nicotinamide ribose in the embryonic period can significantly increase the number of PGCs and prolong the post-birth growth cycle, and the action mechanism comprises: nicotinamide ribose promotes the proliferation of embryonic cells by up-regulating the NAD + level in PGCs, and enhances the expression of TFAP2C at the same time to maintain the undifferentiated state of cells and delay the meiosis initiation of female embryo PGCs and the differentiation initiation of male embryo PGCs; in post-birth effect evaluation, the number of follicles in ovaries of the female child rats in the NR group is remarkably increased, the fertility cycle is remarkably prolonged, the fertility of filial generations is not affected, and nicotinamide ribose is prompted to effectively maintain reproductive reserve and enhance the fertility by optimizing the development time sequence of PGCs. The invention provides a solid experimental foundation for the application of nicotinamide ribose in increasing the number of primordial germ cells and prolonging the growth cycle, and has a wide application prospect.

The application provides a screening method and system for markers in oocyte formation process, and relates to the technical field of gene regulation. The method comprises the following steps: determining cell-specific transcription factors and cell-specific genes based on human and mouse chromatin characteristic data and gene expression data; constructing a transcription factor-mediated gene regulation network; obtaining key oocyte-specific transcription factors by comparing human and mouse cell-specific transcription factors; obtaining key oocyte-specific genes by comparing human and mouse cell-specific genes; evaluating the expression levels of the key oocyte-specific transcription factors and the key oocyte-specific genes in embryonic stem cells, primordial germ cells and oocytes; and taking the key oocyte-specific transcription factors and the key oocyte-specific genes with expression levels higher than preset expression values as markers. The key transcription factors and genes are determined, and the efficiency of in-vitro embryonic stem cell differentiation into oocytes is improved.

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.

This invention discloses the application of the Src kinase inhibitor PP2 in improving the differentiation efficiency of pluripotent stem cells into germ cells, belonging to the field of bioengineering technology. The specific culture method is as follows: Human embryonic stem cells are prepared into a single-cell suspension and seeded into a cell culture plate; the cell culture plate is placed in a cell culture incubator for culture, and then the culture medium is replaced with the first-stage differentiation medium for inducing human embryonic stem cells to differentiate into epiblast cells for continued culture; the cell culture medium for epiblast cells is aspirated and replaced with the second-stage differentiation medium for inducing epiblast cells to differentiate into primordial germ cell-like cells, and the plate is placed in a cell culture incubator for culture. Then, the Src kinase inhibitor PP2 is added to the culture medium at a final concentration ranging from 2.5 to 10 μM, and culture continues. This invention significantly improves differentiation efficiency and provides more optimized experimental conditions for exploring the developmental mechanism of germ cells through a primordial germ cell-like cell induction system.

A method for inducing immature oocytes includes introducing four genes consisting of FIGLA, NOBOX, LHX8 and TBPL2, or transcripts or expressed proteins thereof, into at least one type of cell selected from the group consisting of pluripotent stem cells, epiblast-like cells and primordial germ cells. A method for producing mature oocytes includes introducing four genes consisting of FIGLA, NOBOX, LHX8 and TBPL2, or transcripts or expressed proteins thereof, into at least one type of cell selected from the group consisting of pluripotent stem cells, epiblast-like cells and primordial germ cells, and co-culturing the cell obtained after the introduction and ovarian somatic cells.

The invention discloses a bovine ectoderm stem cell line as well as an establishment method and application thereof. The bovine ectoderm stem cell line has the pluripotency of a bovine intermediate state stem cell line, expresses one or more pluripotency markers, can be stably passaged, still maintains pluripotency transcriptome characteristics and normal karyotype similar to those of an intermediate state (Formative) ectoderm cell after more than 112 generations, and has the potential of being differentiated into three-germ layers. The bovine intermediate-state stem cell line has the potential for myogenic differentiation, primordial germ cell-like cell differentiation and serving as somatic cell nuclear transplantation (SCNT) donor cells, which shows that the bovine intermediate-state stem cell line has wide application prospects in the aspects of promoting cell culture meat production and animal breeding.

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.

The invention provides a method for breeding high-yield green-shell laying hens by using a PGC gene editing technology, and belongs to the field of poultry breeding and gene engineering. According to EAV-HP insertion site information of a 5'flanking region of an SLCO1B3 gene, a group of sgRNA aiming at the gene region and a donor DNA template containing an EAV-HP insertion sequence and a regulatory element are designed and obtained. An EAV-HP sequence is directionally introduced into a chicken primordial germ cell genome by utilizing a CRISPR / Cas9 system, successfully edited cells are screened out and cultivated into gene edited chickens, and a new strain of laying hens capable of stably inheriting and laying green-shell eggs is established through a generation of breeding, so that the economic benefit of commercial laying hen breeding is remarkably improved.

This invention discloses the application of TRUB1 as a target for specific labeling and recognition of primordial, primary oocytes, or follicles. In microproteomic analysis of human oocytes at various developmental stages, this invention revealed that the expression of certain proteins in oocytes exhibits developmental stage specificity. Screening for stage-specific expressed proteins and comparing them with data from the Human Protein Atlas, GEO, PubMed databases, and published mRNA data, it was found that TRUB1 is highly expressed in primary oocytes. Histochemical experiments confirmed that TRUB1 is specifically expressed in primordial and primary follicle oocytes. Therefore, it is proposed that the TRUB1 molecule can be used as a target for specific labeling and recognition of primordial and primary oocytes, assisting researchers in identifying and determining the developmental stage of oocytes, and providing an effective molecular target for primordial, primary oocytes, or follicles to facilitate targeted intervention or related molecular biology research.

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.

Provided herein are methods and compositions for differentiating induced pluripotent stem cells into primordial germ cell-like cells by overexpressing transcription factors such as DLX5, HHEX, and / or FIGLA.

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 special culture medium for chicken primordial germ cells as well as a preparation method and application thereof, and belongs to the technical field of biology. The invention provides a special culture medium for chicken primordial germ cells, which takes KO-DMEM as a basic culture medium and further comprises the following components: SCF, bFGF, GDNF, Activin A, L-glutamine, non-essential amino acid and beta-mercaptoethanol. The GDNA and the bFGF are added to achieve a synergistic effect, so that the cell proliferation rate is remarkably increased, the line establishment period is shortened, a high-quality cell source is provided for gene editing, and the cost is also reduced.