26 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 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 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.

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.

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.

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 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 present application provides a method for directly transdifferentiating spermatogonial stem cells (SSCs) into neural stem cell-like cells (iNSCs). The iNSCs have proliferative activity, can be stably subcultured in vitro, and have the potential to differentiate into other neural cells such as neurons, astrocytes and oligodendrocytes. The method of the present application has high efficiency, can obtain more iNSCs in a relatively short period in vitro, and can obtain iNSCs with high purity, wherein the double positive rate of Nestin and Pax6 is more than 95%.

The invention discloses a system for promoting in-vitro differentiation of chicken oogonial stem cells and forming blastocysts, and belongs to the technical field of stem cell differentiation culture. According to the invention, retinoic acid (RA) and vitamin C (VC) are added on the basis of an oogonial stem cell culture medium to induce cells to start meiosis, and then follicle stimulating hormone (FSH), human chorionic gonadotropin (HCG), glial cell-derived neurotrophic factor (GDNF), progesterone (P4) and rosiglitazone are further added on the basis of the culture medium containing RA and VC to promote the recovery of meiosis, so that the effect of promoting meiosis is achieved. A complete system that the chicken oogonial stem cells are differentiated in vitro and form blastocysts is creatively established by inducing formation of secondary oocytes, direct evidence is provided for in-vitro reconstruction of poultry ovum generation, and a technical basis and application prospects are provided for preservation of female germ cells and large-scale production of the chicken oocytes.

The invention relates to the technical field of biology, in particular to a combined culture medium of chicken embryo primordial germ cells (Primordial Germ Cells, PGCs) and a cell culture method of the combined culture medium, calcium ion-free DMEM is used as a basis, various supplements, nucleoside, sodium pyruvate, amino acid, vitamins, additives, trace components, three antibodies, serum and specific cell factors are added, filtration sterilization is performed after osmotic pressure is adjusted, and the combined culture medium and the cell culture method of the combined culture medium are used for culturing the chicken embryo primordial germ cells (Primordial Germ Cells). The cell factors are added before use to adapt to the culture of the related PGCs. The method does not need a feeding layer, simplifies the operation, avoids exogenous pollution, improves the purity and survival state of the PGCs, promotes proliferation, has no sex preference, is adaptive to different sources of PGCs, is stable and reliable, and has a wide application prospect.

The invention discloses a serum-free additive E6 for a chicken primordial germ cell culture medium and application of the serum-free additive E6, and belongs to the technical field of animal husbandry and biology. The E6 serum-free additive is composed of six components necessary for in-vitro proliferation of chicken PGCs, and specifically comprises DL-alpha-tocopheryl acetate, BSA (fatty acid-free component V), human recombinant insulin, corticosterone, linolenic acid and triiodothyronine. After the E6 serum-free additive is added into a chicken PGCs culture medium, the basic form of cells can be effectively maintained, long-term in-vitro proliferation of the cells is supported, expression of specific marker genes and proteins such as reproduction and pluripotency is maintained, and meanwhile the long-term subculture capacity and gonad migration potential of the PGCs are guaranteed. The E6 serum-free additive disclosed by the invention has the advantages of simple and clear components, simplicity and convenience in preparation and relatively low cost, and can be widely applied to the fields of poultry germplasm resource preservation, gene editing, biological breeding and the like.

The invention discloses a method for efficiently differentiating monkey spermatogonial stem cells in vitro and application. The invention belongs to the technical field of biology, and particularly relates to a method for efficiently differentiating monkey spermatogonial stem cells in vitro and application. The active ingredients of the composition for promoting in-vitro differentiation of the monkey spermatogonial stem cells are retinoic acid, BMP4, testosterone and a basic differentiation medium. The concentration of the retinoic acid in the composition is 1 [mu] mol / L, the concentration of the BMP4 is 50 [mu] g / L, and the concentration of the testosterone is 0.1 mmol / L. According to the method, a culture system for in-vitro differentiation of the monkey spermatogonial stem cells (SSCs) into the functional haploid sperms is successfully established, the haploid differentiation efficiency can be obviously improved (the proportion exceeds 8%) after 96 hours of continuous treatment, and the blank of research on in-vitro differentiation systems and molecular mechanisms of the monkey SSCs is filled.

The invention discloses a method for breeding transgenic chickens with offspring stable genetic specific sites, and belongs to the technical field of animal husbandry and animal genetic engineering. The method comprises the following steps: separating and culturing chick embryo gonad primordial germ cells; a lentivirus-mediated exogenous gene is inserted into the PGCs, transgenic PGCs are screened, and the biological characteristics of the transgenic PGCs are verified; transplanting the transgenic PGCs to a receptor chick embryo to obtain a chimeric chicken; the method comprises the following steps: carrying out test cross to obtain an F1 generation, screening out a transgenic chicken with an exogenous gene integrated to 6528773-6528774 sites of a chicken 10 # chromosome through whole genome sequencing, and verifying genetic stability in F2-F3 generations. The result shows that the F1-F3 generation exogenous gene genetic mode accords with the Mendel segregation law, insertion sites are not lost or rearranged, and the growth performance and the reproductive performance are the same as those of wild chickens. According to the method, the problems of genetic instability and phenotype abnormality caused by random insertion are solved, and an efficient method is provided for stable breeding of transgenic chickens.

A chicken serum-free medium for promoting proliferation of chicken primordial germ cells (PGCs) and a use of the chicken serum-free medium are provided. In the chicken serum-free medium, ovotransferrin is used instead of chicken serum. The ovotransferrin is used instead of chicken serum in a PGC culture system, and ovotransferrin can keep the self-renewing of PGCs to provide a basis for exploring a stable culture system. The chicken serum-free medium can avoid the adverse effects caused by an unclear composition of chicken serum. In addition, the accurate use of various growth factors can provide excellent culture conditions and reduce the culture cost.