66 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 a method for separating, purifying and transplanting spermatogonial stem cells of lateolabrax japonicus, the spermatogonial stem cells of lateolabrax japonicus are separated and purified, marine fish larvae or freshwater fish larvae are used as receptor larvae, and then the spermatogonial stem cells of lateolabrax japonicus are transplanted to abdominal cavities of the receptor larvae. The spermatogonial stem cells obtained through separation are complete in number, structure and function and low in cell clustering rate. After the transplanted spermatogonial stem cells are subjected to fluorescence labeling, the migration and proliferation processes of donor germ cells can be observed in receptor fishes of marine fish fries or freshwater fish fries, so that the receptor fishes with the chimeric germline are obtained. According to the method, through xenotransplantation of the spermatogonial stem cells, a'belly borrowing reproduction 'breeding mode is provided for the marine fishes, a new way for gamete preservation and rapid propagation is provided for the marine fishes with the long reproductive cycle, and a new method and a new thought are provided for genetic improvement and germplasm resource preservation.

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 separating and transplanting spermatogonial stem cells of Chinese sturgeons, and belongs to the technical field of separation of germline stem cells of fishes. The separation method comprises the following steps: washing Chinese sturgeon testis tissues with an L-15 culture medium containing penicillin / streptomycin double antibodies, and carrying out physical shearing, digestive enzyme enzymolysis and screen filtration to obtain a Chinese sturgeon testis cell suspension; performing percoll density gradient centrifugation to obtain high-purity spermatogonial stem cells of the Chinese sturgeons; and dyeing, washing, resuspending and carrying out micro-injection transplantation to enter the gonad primordium reproductive ridge part of the juvenile acceptor fish. According to the physical characteristics of the spermatogonial stem cells of the Chinese sturgeons, the spermatogonial stem cells with high purity are obtained by adopting percoll density gradient centrifugation, and the purpose of abdomen borrowing reproduction is achieved by adopting a microinjection mode, so that the preservation of germ cells of endangered fishes and the reconstruction of artificial populations are realized; and a new technical approach is provided for protecting genetic resources of the Chinese sturgeons and breaking through artificial propagation bottlenecks.

The present disclosure relates to methods, products, and compositions useful for deriving bovine induced blastoids. Methods are described that allow for juvenile stem cell aggregation and induced blastocyst formation. The invention further provides an aggregation culture medium and an induced blastocyst-like culture medium which are suitable for promoting aggregation of the young stem cells and preparing the induced blastocyst-like. The bovine induced blastoids can be used in selection and breeding procedures, including the replication of pre-implant embryos with desired characteristics and in vitro breeding including a cross-generation breeding cycle from parent germ cells and / or embryos to progeny embryos.

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 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.

Provided is a method for obtaining the germ cell lineage of an oviparous vertebrate more efficiently than conventional techniques. A host oviparous vertebrate is prepared at a developmental stage after the development of black pigmentary cells in the retina and before the formation of multiple layers of germ cells in the genitals, and isolated undifferentiated germ cells from a donor oviparous vertebrate are transplanted into the host oviparous vertebrate.

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.

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.

The invention provides a method for promoting gonad maturation of plectropomus leopardus by applying a spectrum, which is characterized in that breeding systems under different illumination conditions, including full-spectrum, green-spectrum and blue-spectrum illumination conditions, are constructed, and are combined with a factory-like circulating water breeding system to breed the plectropomus leopardus for 60 days. Experimental results show that the blue light and the green light can significantly accelerate the development of male and female gonads, especially the blue light can promote the development of oocytes, and the green light can promote the development of male germ cells. The method effectively shortens the sexual maturity period of the plectropomus leopardus by accurately regulating and controlling the illumination condition, improves the culture efficiency, and provides a theoretical basis and technical support for regulating and controlling the gonad development environment in aquaculture.

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.

The present invention provides a primer set and method for analyzing the nanos1 gene of Macrobrachium rosenbergii, relating to the biotechnology field of reproductive-related genes in Macrobrachium rosenbergii. The Nanos1 gene obtained by the present invention has a cDNA sequence of 2811 base pairs (bp) long, encoding 243 amino acids. Nanos1 is specifically expressed in the ovary, and the expression level of nanso1 mRNA is highest in unfertilized eggs, significantly higher than after fertilization and at all stages of embryonic development. During embryonic development, expression is highest during fertilization, significantly higher than during the cleavage stage and extremely significantly higher than during the late embryonic development stage. The gene's expression level during the cleavage stage is significantly higher than from the blastocyst stage to the larvae stage; however, the expression level is lower and does not differ between the blastocyst stage and the larvae stage. Nanos1 mRNA is expressed in the cytoplasm of oogonia and primary oocytes (Oc1, Oc2, Oc3, and Oc4). Nanos1 is closely related to the development of female germ cells in Macrobrachium rosenbergii and plays an important role in the reproductive development of Macrobrachium rosenbergii, providing a theoretical basis and ideas for research on reproductive development and sex identification in Macrobrachium rosenbergii.

The present invention relates generally to the use of auxin-type phytohormones to induce diploid (2n) pollen formation in a plant. More particularly the invention relates to a method to modulate meiosis in male germ cells to ectopically induce meiotic restitution and associated formation of diploid pollen. This method provides a straightforward and human controlled way to create polyploid offspring (plant species with more than the usual two sets of chromosomes) with maximum integration of the genetic diversity of the parental line(s).

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 provides a separation method and an identification method of bactrian camel testis interstitial cells, and belongs to the technical field of cell separation. The method comprises the following steps: mixing 0.08-0.12% of collagenase IV solution and 0.23-0.27% of trypsin-EDTA (Ethylene Diamine Tetraacetic Acid) according to a volume ratio of 1: 2, digesting a bactrian camel testis tissue block, then carrying out hypotonic treatment purification to remove redundant germ cells, carrying out differential adhesion purification to remove a large amount of supporting cells, carrying out percoll density gradient centrifugation purification to remove a small amount of supporting cells, and carrying out freeze drying to obtain a finished product. And transferring the suspension with the percoll gradient of 34-60% into a new culture bottle to obtain the interstitial cells with the purity of more than or equal to 95%, and completing identification by combining morphological observation, immunofluorescence staining and secretion function detection. The separated bactrian camel testis interstitial cells can be used for double-hump reproductive performance research, and a foundation is laid for the reproductive performance of bactrian camels.

The invention aims to provide a phascolosoma esculenta sex or egg maturity nondestructive identification system and method, and the method comprises the following steps: step 1, to-be-detected phascolosoma esculenta is subjected to stress treatment with low-salt seawater, the salinity of the low-salt seawater is 7-9 PSU, and the stress time is 1-3 h; step 2, irradiating the to-be-detected phascolosoma esculenta body subjected to stress treatment by adopting a red light source to obtain a light transmission intensity value; and step 3, judging a result according to the light transmission intensity value. According to the system and the method, the time for identifying the sex of the phascolosoma esculenta and the maturity of the star eggs is 5-10 seconds, the identification accuracy rate can reach 92% or above, the operation is convenient and fast, and the body of the phascolosoma esculenta does not need to be damaged. Compared with a traditional mode of distinguishing the gender and judging the maturity of the star eggs by observing the form of the germ cells through a microscope, the method has the advantages that the identification time is shortened by more than 50%, and the asthenopia and the working intensity of operators are greatly reduced.