97 results about "Primordial germ cell" patented technology

The present invention provides a non-mouse, including human, pluripotential embryonic stem cell which can:(a) be maintained on feeder layers for at least 20 passages; and(b) give rise to embryoid bodies and multiple differentiated cell phenotypes in monolayer culture.The invention further provides a method of making a pluripotential embryonic stem cell comprising culturing germ cells and germ cell progenitors in a composition comprising a growth enhancing amount of basic fibroblast growth factor, leukemia inhibitory factor, membrane associated steel factor, and soluble steel factor to primordial germ cells under cell growth conditions, thereby making a pluripotential embryonic stem cell.Also provided are compositions useful to produce the pluripotent embryonic stem cells and methods of screening associated with the method of making the embryonic stem cell.

A culture system for maintaining avian PGCs for long periods in tissue culture is provided. This culture system uses LIF, bFGF, IGF and SCF. The resultant PGCs are useful for the production of transgenic and chimeric avians, in particular, chickens or turkeys.

Methods for modulating primordial germ cell (PGC) numbers and / or development in avians are provided. In one embodiment, the presently disclosed subject matter provides a method for modulating primordial germ cells numbers in an avian embryo comprising immunizing a female bird with an antigen associated with primordial germ cells, whereby an egg produced by the female bird comprises a sufficiently high concentration of antibodies specific for the antigen to modulate numbers of endogenous PGCs in an avian embryo present within in the egg. Also provided are methods for producing chimeric avians, methods for increasing the proportion of male birds in a plurality of eggs, methods of producing avian gametes, and methods for enhancing germ line transmission of nucleic acids in birds.

The invention discloses a method for remarkably improving fish genome editing efficiency. The method for remarkably improving fish genome editing efficiency comprises the following steps: designing a genome editing tool for specifically identifying and cutting a specified site sequence of the fish genome, designing a homologous donor corresponding to the specified site sequence and containing a knock-in exogenous gene fragment, introducing the genome editing tool, the homologous donor and mRNA for specifically and stably expressing fluorescent protein in primordial germ cells into fish animal embryos by using a codominant microinjection method, and selecting the embryos by detecting fluorescent protein expressed by the fluorescent protein mRNA and obtaining stable inheritable characters. The efficiency of the method for knocking the exogenous gene fragment into the specified site of the fish genome so as to obtain a first filial generation of fish with the knock-in exogenous gene fragment is remarkably higher than that of the prior art.

The invention provides a novel culture medium useful for the proliferation and maintenance of avian primordial germ cells (PGCs) and encompassing a medium base, leukemia inhibitory factor, basic fibroblast growth factor, stem cell factor and insulin-like growth factor, and an avian serum. The invention also provides a method for the proliferation and maintenance of avian primordial germ cells for extended periods encompassing the steps of isolating a population of PGCs from an avian and culturing the PGCs in a culture medium containing the growth factors and avian serum. The invention further provides methods of producing chimeric avians by isolating and culturing PGCs in a culture medium containing avian serum, transferring the PGCs into a recipient avian embryo, and allowing the recipient avian to develop into a chimeric bird. Another aspect of the invention provides a culture of avian PGCs grown and maintained in the culture medium containing avian serum.

The present invention is long-term cultures of avian PGCs and techniques to produce germline chimeric and transgenic birds derived from prolonged PGC cultures. In some embodiments, these PGCs can be transfected with genetic constructs to modify the DNA of the PGC, specifically to introduce a transgene encoding an exogenous protein. When combined with a host avian embryo by known procedures, those modified PGCs produce germline chimeric birds. These germline chimeric birds do not have PGC derived somatic cells or tissues. This invention includes compositions comprising long-term cultures of PGCs that can be genetically modified by gene targeting, that can accept large amounts of foreign DNA and that contribute to the germline of recipient embryos.

The invention discloses a vector for efficiently labeling zebra fish PGC, and a preparation method and a use of transgenic fish. A Gal4 / UAS transcription activation system is utilized and mRFP is used as a report gene to realize an inductive gene expression regulation technology in the primordial germ cells of zebra fish. The preparation method of the transgenic fish comprises the following steps: 1, respectively constructing activation transgenic line Tg (kop:KalTA4) and an effect transgenic line Tg (UAS:mRFP); and 2, hybridizing Tg (kop:KalTA4) male fish with Tg (UAS:mRFP) female fish to obtain the transgenic fish for efficiently labeling the primordial germ cells of zebra fish. The transgenic fish for efficiently labeling the primordial germ cells of zebra fish can be widely applied to the fish bioengineering.

The invention relates to a positioning and marking method for embryonic-period primordial germ cells (PGCs), in particular to a bastard halibut embryonic-period primordial germ cell tracking and positioning method. The bastard halibut embryonic-period primordial germ cell tracking and positioning method includes the steps of fixing collected various periods of embryo samples of the bastard halibuts by a 4% PFA solution; using a PBS (phosphate buffer saline) solution with 50% of deionized formamide to preserve the embryo samples at the temperature of -20 DEG C, and subjecting the fixed and preserved embryo samples to oolemma removing, gradient methanol dewatering and rewatering; after rewatering, washing the various periods of embryo samples with PBS buffering liquid without RNA ( ribonucleic acid) enzyme, pre-hybridizing at the temperature of 62-65 DEG C for 2-4 hours; after hybridization, adding a hybridization solution with bastard halibut RNA probes into the various periods of embryo samples subjected to pre-hybridization for hybridizing overnight at the temperature of 62-65 DEG C; after hybridization, subjecting the various periods of embryo samples to washing, antibody incubation and rewashing, keeping away from light, and developing colors to achieve marking for tracking and positioning of the embryonic-period primordial germ cells of the bastard halibuts. The bastard halibut embryonic-period primordial germ cell tracking and positioning method has the advantages that the problems that yolks and oolemma of the samples hybridized in situ conventionally are difficult to strip and a background color is too deep after color developing detection are solved, and operation steps are simplified.

The present invention is long-term cultures of avian PGCs and techniques to produce germline chimeric and transgenic birds derived from prolonged PGC cultures. In some embodiments, these PGCs can be transfected with genetic constructs to modify the DNA of the PGC, specifically to introduce a transgene encoding an exogenous protein. When combined with a host avian embryo by known procedures, those modified PGCs are transmitted through the germline to yield transgenic offspring. These germline chimeric birds do not have substantial contributions of PGC-derived phenotypes in somatic cells or tissues. This invention includes compositions comprising long-term cultures of PGCs that can be genetically modified by gene targeting, that can accept large amounts of foreign DNA and that contribute to the germline of recipient embryos.

Human pre-formed xenoantibodies play an important role in the hyperacute rejection response in human xenotransplantation. Disclosed are materials and methods for removing or neutralizing such antibodies. Also disclosed are materials and methods for reducing or eliminating the epitopes in the donor organs that are recognized by such antibodies. Such epitopes are formed as the result of activity by the enzyme α-1,3 galactosyltransferase. The porcine gene encoding α-1,3 galactosyltransferase is disclosed, as are materials and methods for inactivating (“knocking out”) the α-1,3 galactosyltransferase gene in mammalian cells and embryos. Included are nucleic acid constructs useful for inactivating the α-1,3 galactosyltransferase gene in a target cell. Also disclosed is a novel leukemia inhibitory factor (T-LIF) that is useful for maintenance of embryonic stem cells and primordial germ cells in culture.

A culture system for maintaining avian PGCs for long periods in tissue culture is provided. This culture system uses LIF, bFGF, IGF-I and SCF. The resultant PGCs are useful for the production of transgenic and chimeric avians, in particular, chickens or turkeys.

The present invention is a method for culturing avian PGCs comprising providing fibroblast cells, culturing fibroblast cells in a basal medium, collecting a conditioned medium, providing avain gonadal cells with PGCs from sex glands, and growing the PGCs in the conditioned medium supplemented with growth factors, wherein the conditioned medium provides the avian PGCs being culturing for a long period. Furthermore, the present invention is a method for preparing a medium for culturing avian PGCs comprising providing a basal medium, culturing fibroblast cells in the basal medium, collecting a conditioned medium; and adding growth factors into the conditioned medium, wherein the conditioned medium provides the avian PGCs being cultured for a long period. The present invention is the conditioned medium prepared by the above method.

The external cultivating method of primordial bird germ cell relates to transgene, animal cloning, rare animal preservation, tissue and cell engineering and other science fields. After it is inoculated to the CEF raising layer, the primordial bird germ cell is cultivated in liquid high-sugar DMEM cultivation medium with added calf serum in 10 úÑ, chicken serum in 2 úÑ, L-glutamine in 2mmol / L, sodium pyrovate in 1 mmol / L, beta ¿Cmercaptoethanol in 5.5í‡10-5 mol / L, optional amino acid 10 microliter / ml, hSCF in 15-20 ng / ml, mLIF in 10 U / ml, bFGF in 10-15 ng / ml, hIL-11 in 0.04 ng / ml, IGF in 10 ng / ml, and gentamycin sulfate in 100 U / ml. The present invention has simple operation and high repeatability, and lays the foundation of the relevant research.

The invention discloses a method for rapid separation and line establishment of chicken gonad primordial germ cells. The method comprises: separating and purifying primordial germ cells from the gonadof a 7-day-old chick embryo, and amplifying the separated primordial germ cells within 15 days by using a culture insert matched feeder layer method through an optimized culture system to obtain morethan one million cells so as to achieve rapid amplification and line establishment. According to the present invention, the method has characteristics of high success rate, short time, stability andhigh efficiency, can obtain more than 10<6> cells through the amplifying within in 15 d, and can establish the foundation for the poultry germplasm source conservation and preparation of transgenic chicken.

The present invention addresses the problem of providing a method for differentiating primordial germ cells into functionally mature GV stage oocytes by in vitro culture. The present invention pertains to a method for differentiating primordial germ cells into functional GV stage oocytes in vitro including (a) a step for forming secondary follicles by culturing primordial germ cells and feeder cells adjacent to the primordial germ cells under conditions that eliminate the effects of estrogen or factors having a similar function to estrogen, (b) a step for partially cleaving the bonds between the granulosa cell layer and the thecal cell layer among the oocyte, granulosa cell layer, and thecal cell layer that constitute the formed secondary follicles, and (c) a step for differentiating the oocytes into functional GV stage oocytes by culturing the oocytes and granulosa cell layer that constitute the secondary follicles and the thecal cell layer in medium including a polymer compound.

The vitrification freezing method of primordial chicken embryo germ cell is to vitrify and freeze primordial germ cell of chicken embryo sex gland in the 19th stage and 28th stage of growth. After it is added into vitrifying and freezing liquid, primordial chicken embryo germ cell is transferred into freezing tube, balanced at 4 + / -1 deg.c for 5 min, stilled on surface of liquid nitrogen for 0.5-1.5 min and set directly into liquid nitrogen. The present invention has simple and quick operation, and stable and reliable preservation effect, and may be used in preserving various traditional bird resources.

The invention discloses a method for directionally inducing and differentiating pig pluripotent stem cells into male germ cells and a special culture medium. The invention provides the special culture medium. The special culture medium comprises an EpiLC inducing medium, a PGCLC (primordial germ cell-like cell) inducing medium and an SSCLC (spermatogonial stem cell-like cell) inducing medium. The invention provides the method for directionally inducing and differentiating the pig pluripotent stem cells into PGCLC in vitro; experiments prove that the PGCLC can be further induced and differentiated to form SSCLC; in vivo, induced cells are injected into seminiferous tubules of mice of which endogenous sperms are removed, and the PGCLC and the SSCLC can survive and are further developed. Therefore, the invention provides a feasible and useful scheme for directional differentiation from the pig pluripotent stem cells to the germ cells, and lays a foundation for exploring a development mechanism and the like of the germ cells.

The invention relates to a technological method for promoting ectogenesis of premeiotic female germ cells by using activin A (ActA). The method comprises the following steps of: separating 12.5 dpc female fetal rat genital ridges by a mechanical method; cultivating the fetal rat genital ridges in vitro; collecting oocytes; differentiating and developing 12.5 dpc premeiotic germ cells into GV stage oocytes by an in vitro culture method and inducing maturation to obtain mature oocytes; and detecting the quality of the oocytes developed in vitro by an in vitro fertilization (IVF) technology to obtain IVF offspring developed in an early stage. The technological method for promoting the ectogenesis of the premeiotic female germ cells by using the ActA is established and is simple, convenient and practical, so a cellular and molecular mechanism of archecyte ectogenesis and follicle ectogenesis is explored theoretically, high-quality oocytes are provided for patients suffering from ovarian infertility, and huge social value and economic value are created.

The invention discloses a complete medium for cultivating cPGCs (chicken primordial germ cells) and a using method of the complete medium. The complete medium consists of KO-DMEM, B-27supplement, NAA, nucleosides, GlutaMAX II, [beta]-mercaptoethanol, ovalbumin, OVT, heparin sodium, Activin A, TGF-[beta]1, BMP4, FGF-2 and IGF-1. The using method comprises the following steps: cultivating collected 14HH chicken embryo blood in a culture system containing a cPGCs complete culture solution, and conducting primary culture for 10-15d so as to obtain purified cells; (2) adding the purified cPGCs in the cPGCs complete medium once again and conducting in-vitro culture and amplification; and (3) conducting counting and indirect immunofluorescence detection on the cPGCs obtained from the in-vitro culture and amplification, including SSEA-1 and DAZL surface markers . The medium provided by the invention, different from a conventional culture method which has serum and a feed layer, is concrete in ingredients, and the medium can facilitate researches on a molecular mechanism of cell differentiation.

The invention relates to a method of establishing a novel induction system to reprogram CEFs (chicken embryo fibroblasts) into PGCs (primordial germ cells) and belongs to the technical field of biology. In isolated primordial CEF, OCT4, SOX2, NANOG and LIN28 reprogramming factors are introduced to lentivirus exogenously; 21 days after continuous culture, iPSC (induced pluripotent stem cells) strain identification is performed with an SSEA-1 antibody; a positive cell line that is identified is replaced with a BMP4 / BMP8b / EGF induction system; iPGCs (induced primordial germ cells) are acquired byinduction. The problem is solved herein that the quantity of PGCs acquired in endangered species is low; germplasms resource protection and species restoration are achieved for the engendered chickenspecies.

This invention provides a method of producing an epiblast-like cell (EpiLC) from a pluripotent stem cell, which comprises culturing the pluripotent stem cell in the presence of activin A; a method of producing a primordial germ cell-like (PGC-like) cell a pluripotent stem cell, which comprises culturing the EpiLC obtained by the method above in the presence of BMP4 and LIF. Also provided are a cell population containing PGC-like cells as obtained by the method, and reagent kits for the EpiLC- and PGC-like cell-induction from a pluripotent stem cell.

The invention discloses a slide climbing method of PGCs (primordial germ cells) and belongs to the biotechnology field. The slide climbing method comprises the steps as follows: purifying the PGCs; washing the purified PGCs with a PBS (phosphate buffer solution) for three times, and centrifugally collecting the cells; selecting and putting 100-200 cells in PBS droplets on a glass slide under a microscope for washing for one time; selecting and putting the cells in a paraformaldehyde solution with the concentration of 4% on the glass slide for washing for one time; selecting and putting the cells on paraformaldehyde droplets with the concentration of 4% on the glass slide for fixation for 10-30 min; and removing the paraformaldehyde with the concentration of 4% through adsorption, and finishing slide climbing of the cells while fixing the cells. According to the slide climbing method, the problem that the purified PGCs are difficult to climb the glass slide is solved, and the time and the cost are saved.

The invention relates to a method for obtaining haploid germ cells through differentiation of human skin stem cells. The method comprises the following operation steps: after in-vitro separation, in-vitro culture is carried out on human skin derived stem cells: wherein separated human skin derived stem cells form a spherical cloning sample structure during in-vitro suspension culture, subculturing is carried out every four days and human skin derived stem cell cloning spheres are obtained after subculturing; human skin derived stem cells are induced to germ cell sample cells; human skin derived stem cells are induced and differentiated towards haploid germ cells, and after differentiation is carried out for about 18 days, DNA ploidy analysis can detect that 1.79% of haploid sample cells are formed. With the adoption of the method provided by the invention, primordial germ cells, of which the morphology and specific molecular marker expression are similar to those of normal primordial germ cells in the human body are obtained, the human haploid germ cells are obtained in a differentiation process of a proper condition culture medium and a good in-vitro model is provided for research on a generating mechanism of human germ cells.

The invention provides a method for searching for a target gene of a Wnt signal pathway in a process of differentiating embryonic stem cells into primordial germ cells and relates to the technical field of biology. According to the method, firstly, target gene prediction is carried out on a key transcription factor TCF7L2 of the Wnt signal pathway, GO function annotation is carried out on the predicted target gene, and a conservative target gene which is involved in germ cell development and stem cell differentiation is screened out as a to-be-selected target gene. In-vivo / in-vitro experiments are carried out to verify the regulation and control effect of a Wnt signal on the transcription level of the to-be-selected target gene, and when a transcription factor binding site in a promoter of the to-be-selected target gene is lost, whether or not the Wnt signal has a regulation effect on the transcription level of the to-be-selected target gene is detected; the binding effect of a beta-Catenin / transcription factor complex and the to-be-selected target gene is verified through ChIP-qPCR in order to determine whether or not the to-be-selected target gene has a targeting response effect on the Wnt signal. The method is simple and feasible.

The invention relates to a regulator sequence of flounder primordial germ cells, and specifically relates to a regulatory sequence of a flounder primordial germ cell Nanos3 gene and applications thereof. The 3'UTR sequence of the flounder Nanos3 gene is represented by the SEQ ID No.1. The nucleotide sequence represented by the SEQ ID No.1 regulates the target gene's specific expression in primordial germ cells. The gene sequence is a specific sequence of flounder, can be used to label PGCs of marine fishes such as flounder, zebra fish, and the like, and can specifically express target genes in PCGs.

The invention discloses the method used to directionally induce chick embryo primordial germ cell to differentiate into bone cell in vitro. It uses the inducer formed by 90% DMEM, 10% FBS, 5.5*10-5 beta-mercaptoethanol, 1*10-7mol / L dexamethasone, 0.01mol / L beta-sodium glycerophosphate, 0.05g / L vitamin C to process induction differentiating culturing in vitro for chick embryo primordial germ to differentiate into bone cell finally. It can build cell model for clinical medicine study, widely apply in tissue engineering field for providing reliable experience data, supply new source for cell substituting therapy and tissue therapy, even organ transplantation. The invention has the advantages of simple technology, convenient operation, strong repeatability, no need for special apparatus, only need to master universal cell separating and culturing operation.

Disclosed in this invention is a process for preparing an established avian embryonic germ cell line comprising the steps of: (a) culturing primordial germ cells (PGCs) isolated from an avian embryonic gonad in a medium supplemented with a cell growth factor and a differentiation inhibitory factor to obtain EG cell colonies; (b) culturing the EG cells in the same medium as in step (a) by employing a feeder layer until the EG cells are colonized; and (c) recovering and subculturing the EG cells in the same medium as in step (a) to establish the EG cell line.

A method for the production and collection of avian sperm comprises the steps of: providing primordial germ cells from a donor avian species; administering the primordial germ cells to a recipient avian species in ovo; incubating the recipient avian species to hatch; and then collecting sperm of the donor avian species from the recipient avian species. For example, the donor avian species may be a whooping crane, and the recipient avian species may be a sand hill crane. In another example, the donor avian species may be a turkey, and the recipient avian species may be a chicken.