190 results about "Blastocytes" patented technology

Methods are disclosed for generating HLA homozygous parthenogenetic human stem cell (hpSC-Hhom) lines from both HLA homozygous and HLA heterozygous donors. These hpSC-Hhom lines demonstrate typical human embryonic stem cell morphology, expressing appropriate stem cell markers and possessing high levels of alkaline phosphatase and telomerase activity. Additionally, injection of these cell lines into immunodeficient animals leads to teratoma formation. Furthermore, in the case of HLA heterozygous donors, the hpSC-Hhom lines inherit the haplotype from only one of the donor's parents. SNP data analysis suggests that hpSC-Hhom lines derived from HLA heterozygous oocyte donors are homozygous throughout the genome as assessed by single-nucleotide polymorphism (SNP) analysis. The protocol as disclosed minimizes the use of animal-derived components, which makes the stem cells more practical for clinical application.

A method for isolating an inner cell mass comprising the steps of immobilizing a blastocyst stage embryo having a zona pellucida, trophectoderm, and inner cell mass, creating an aperture in the blastocyst stage embryo by laser ablation, and removing the inner cell mass from the blastocyst stage embryo through the aperture. The aperture is through the zona pellucida and the trophectoderm. The laser ablation is acheived using a non-contact diode laser. The inner cell mass removed from the blastocyst stage embryo is used to establish human Embryonic Stem Cell lines.

Instead of immersing human reproductive cells in a single culture medium throughout the various procedures used in IVF, a process is provided by which the reproductive cells may be moved through a sequence of distinct culture media as the various IVF procedures are carried out. In one implementation, the culture media specifically formulated to provide a physical environment similar to that found within the female reproductive tract and conducive to growth and development of human reproductive cells during the various stages of the IVF process. In this regard, specifically formulated culture media can be applied to support the reproductive cells in one or more of the following procedures: oocyte retrieval and handling; oocyte maturation; ordinary fertilization; oocyte, zygote and embryo examination and biopsy; embryonic development to the eight-cell stage; embryonic development to the blastocyst stage; embryo transfer; and cryopreservation.

The invention discloses a split-range embryo culture solution and a preparation method thereof, belonging to the technical field of cytology and biology. The split-range culture solution added with three embryotrophic factors comprises a cleavage solution and a blastula solution, wherein the cleavage solution and the blastula are respectively used in a fertilized egg before and after 8-cell cycle. The embryotrophic factors added into the embryo culture solution disclosed by the invention have synergistic effect, so that the defect that the split-range culture solution loses the embryotrophic factors secreted by an embryo is avoided, the fertilized egg has higher embryo stem cell pluripotency during the growth in the culture solution disclosed by the invention, the development situation of the fertilized egg is equivalent to the in-vivo embryo growth, the in-vitro growth and development of the fertilized egg is improved, and the quality of the embryo is improved.

The invention discloses an in-vitro maturating method for sheep oocyte, a pretreatment solution and a kit. The related pretreatment solution applied to in-vitro maturating of sheep oocyte is characterized by comprising sheep small follicle fluid and routine culture-solution compositions for in-vitro maturating of sheep oocyte. The disclosed in-vitro maturating method for sheep oocyte is characterized in that a pretreatment step is increased on the basis of a conventional in-vitro maturating method. Also, experiments in the invention verify that oosperm obtained after an oocyte which is cultured by employing the maturating method is subjected to in-vitro fertilization has the cleavage rate and the blastocyst rate both substantially higher than those of an oocyte cultured by employing a conventional maturating method. The provided pretreatment solution and the in-vitro maturating culturing method are capable of effectively promoting in-vitro maturation of sheep oocyte, and have the characteristics of no hazard to occyte, few limits, good effect and the like.

It is revealed that an organ such as pancreas can be regenerated by utilizing a fact that the deficiency of an organ is complemented by injecting an induced pluripotent stem cell (iPS cell) into a developed blastocyst in a blastocyst complementation method. Thus, the present invention has solved the above-described object. This provides a method for producing a target organ, using an iPS cell, in a living body of a non-human mammal having an abnormality associated with a lack of development of the target organ in a development stage, the target organ produced being derived from a different individual mammal that is an individual different from the non-human mammal.

A novel population of multipotent cardiac precursor (MCP) cells derived from human blastocysts derived stem cells is disclosed, methods for the preparation thereof and use of the cells for in vitro testing. Basement cells derived from hBS cells are also disclosed and method for the preparation of MCP cells from basement cells. The MCP cells have the following characteristicsi) at least 1% of the cells exhibit no antigen expression of one or more markers for undifferentiated cell, the marker being selected from the group consisting of SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and Oct-4,ii) at least 1% of the cells exhibit no protein expression of one or more of a neural marker including nestin or GFAPiii) at least 1% of the cells exhibit protein and / or gene expression of one or more of a mesodermal marker including brachyury, vimentin or desminiv) at least 1% of the cells exhibit protein and / or gene expression of Flk-1 (KDR).Furthermore, the MCP cells have a characteristic morphology. They grow as clusters of small, round and phase-bright cells; individual cells are 5-20 μm in diameter and each cluster is composed of 2-500 cells. They form clusters of round or elongated shape, that appear as loosely adherent cell clumps that as illustrated in FIG. 2 panel a, b and c. Furthermore, they have a relatively high nucleus-to-cytoplasma ratio, e.g. 1:2-1:64 of the total volume of the cell and / or appear as balloons on a string, as illustrated in FIG. 18, schematic sketch. Moreover, the MCP cells are non-contracting.

The invention discloses a blastocyst nutrient solution and a preparation method thereof. The blastocyst nutrient solution comprises a composition for treating and culturing a zygote, a bactericide, a selectively added indicator and a selectively added human serum albumin. Each index of the blastocyst nutrient solution reaches the standard of an in-vitro nutrient solution, the use is safe, and whether the nutrient solution is deteriorated or not can be easily determined, and therefore a powerful guarantee is provided to the assisted reproductive technology.

The invention discloses a swine in-vitro embryo culture solution. The swine in-vitro embryo culture solution disclosed by the invention is obtained through adding ligustrazine, with the final concentration of 0.05-5.00 microgram / milliliter, into an embryo culture solution NCSU-23. A traditional Chinese medicine monomer component, namely ligustrazine, is added into the swine in-vitro embryo culture solution disclosed by the invention; shown by experiments, through carrying out in-vitro culture on the swine parthenogenetic activated embryos and the swine in-vitro fertilized embryos by using the culture solution, the development efficiency of the embryos and the number of the cell masses and total cells in blastulae can be effectively increased. The swine in-vitro embryo culture solution has the advantages that a foundation is laid for the development of other biotechnologies, medical research and animal husbandry, and meanwhile, a certain theoretical basis for clarifying the development mechanism of early swine embryos is provided.

The invention provides a method for improving the blastocyst rate of in-vitro embryos of animals. According to the method, the developmental capacity of a parthenogenetic embryo and an in-vitro fertilization embryo can be remarkably improved by adding type I interferon into an embryonic development culture solution for the in-vitro culture of the embryos; compared with the blastocyst rate obtained under the culture of the conventional culture solution, the blastocyst rate is improved by 7 to 13 percent; and the blastocyst hatching rate is also remarkably higher than the blastocyst hatching rate under the culture of the conventional culture solution. By the method, the early development capacity of in-vitro embryos of cattle and sheep is improved, so that the in-vitro production efficiency of embryos of the cattle and sheep is improved, and high-quality embryos are provided for the scientific research and production field in which the embryos of the cattle and sheep are taken as materials.

The invention discloses a method for non-invasive preimplantation hereditary detection of embryos, and belongs to the technical field of biological detection. The method comprises the following steps:carrying out whole genome amplification on a blastocyst culture solution sample by using a kit, carrying out short tandem repeat sequence analysis on an amplification product and DNA samples of parents to detect maternal pollution, carrying out library preparation and next-generation sequencing detection on the amplification product to determine whether the number of chromosomes is abnormal or not; and optimizing a pre-amplification mixed solution and an amplification mixed solution by the provided kit. According to the method provided by the invention, the blastocyst culture solution can besubjected to parent source pollution detection, so that whether the chromosome aneuploidy detection result of the culture solution is accurate and reliable or not is judged. The invention provides a detection method for judging whether granular cells are completely removed or not, and the inhibition effect of components in the culture solution on amplification is effectively avoided through optimization of the kit, so that amplification uniformity is good, and the single cell amplification yield is high. The detection method is simple, the result is accurate, and data quality is improved.

The invention discloses multifunctional vulvae care solution and a preparation method thereof. The care solution is prepared by mixing the following Chinese traditional medicines by weight portion to obtain the medicine solution by decoction and then adding with the following western medicines by weight portion; the Chinese traditional medicines are as follows: 3 potions to 7 portions of ebony, 4 portions to 8 potions of cuttlebone, 1 portions to 3 portions of white vitriol, 15 portions to 25 portions of herba houttuyniae, 12 portions to 18 portions of radix isatidis, 12 portions to 18 portions of Chinese pulsaiila root, 7 portions to 13 portions of glabrous greenbrier rhizome, 7 portions to 13 portions of radix sophorae tonkinensis, 7 portions to 13 portions of belamcanda bhinensis DC., 15 portions to 25 portions of dayflower, 15 portions to 25 portions of groundsel, 12 portions to 18 portions of agrimony, 7 portions to 13 portions of raspberry and 3 portions to 7 portions of Chinese tumion seed; the added western medicines are vitamin B2 metronidazole, jasmine top note flavors, benzalkonium bromide solution and jie-er-yin liquid; stilbestrol and / or philter and / or saxol. The pH value of the care solution is about 6.5, and the care solution can inhibit the reproductions of bacteria and blastocyte, adopts a plurality of heat-clearing and detoxifying drug, and has the functions of sterilization, desinsection, relieving itching and anticancer as well as detoxification of cervical erosion.

The invention provides a kind of transgenic inhuman mammal by producing klone gene without mouse model. Roll outside self murder gene into the mammal. The expression box contains promoter, self murder gene relation with promoter and termination codon. The natural masculine gene will express in testes organize after transgenic animal builds system. The transgenic animal can provide blastosphere for eliminating gene technic to improve the efficiency of the technic and to solve the problem that mouse genetic background is impure.

The present invention relates to a genetically modified pig comprising at least one site for integration of at least one transgene. The invention also pertains to a porcine embryo, blastocyst, foetus, donor cell and / or cell nucleus, derived from said genetically modified pig. In another aspect, the invention relates to any genetically modified porcine blastocyst, wherein the genetically modified genome comprises at least one site for integration of at least one transgene.

The invention relates to the use of cells from cell lines, which are derived from early embryonic stages, for the donor-specific increase in transplantation tolerance and for repairing damaged tissue. Areas of application of the invention include the field of medicine and the pharmaceutical industry. The aim of the invention is to produce a donor-specific immunotolerance in order to prevent a rejection of the transplanted tissue due to an immune response and thus to be able to limit the administration of immune suppressive agents. In order to produce a donor-specific immunotolerance, embryonic stem cell-like cell lines (ECL) are obtained from blastocysts and are transfected with genetic material of the donor, which codes for the MHC haplotypes. The cells produced in such a manner are administered to the recipient before the transplantation for producing an immunotolerance against the tissue to be transplanted or for regenerating already damaged tissue.

The invention provides a method for enhancing the ectogenesis of sheep oocyte. The method adopts an adenylate cyclase activator Forskolin (FSK), phosphodiesterase inhibitor cilostamide (CIL) and 3-isobutyl-1-methylxanthine (IBMX), treats the sheep oocyte in a combining manner and improves the capability of the ectogenesis of the sheep oocyte. The method comprises the following detailed steps of: picking the ovary of the killed sheep, putting the ovary of the killed sheep into normal saline for cleaning for 3-4 times; pumping the 2-8mm follicles on the surface of the ovary by using a 10ml syringe which absorbs 1ml ovum-pumping liquid containing 100mu mol / L FSK and 500mumol / L IBMX and is provided with a number-9 needle head, and injecting the follicular fluid recovered in the syringe into a35-60mm vessel; picking the oocyte under a microscope, putting the oocyte into the pumped follicular fluid, cleaning the oocyte for three times with in-vitro mature fluid containing 20mu mol / L CIL, and putting the oocyte in a CO2 incubator for culture; then cleaning the oocyte for three times with in-vitro mature fluid which does not contain CIL, putting the oocyte in the CO2 incubator for culture and adding into in-vitro fertilization fluid drop; unfreezing the seminal fluid with water bathing, and commonly incubating with the oocyte; and calculating the cleavage rate after 24 hours and calculating the blastocyst rate after 168 hours.

The invention discloses a kit for screening high-quality blastulas. According to the kit, birth safety is forecasted before a human assisted reproduction blastula is implanted, and the kit comprises products of expression quantities of 398 genes as shown in a table 2 in wall trophoblast cells and polar trophoblast cells. The human assisted reproduction blastula is normally developed, and a pregnant woman normally delivers if the expression quantity of each gene in the 398 genes of the wall trophoblast cells of the human assisted reproduction blastula (a blastula implanted before fifth days of in-vitro fertilization) is twice or more or half or less that of the same gene in the 398 genes of the polar trophoblast cells. The kit has important application values for scientific judgment of transplantation strategies and decisions and possibility reduction of perinatal period complications of pregnant women and sick children in assisted reproduction.

The invention discloses a kit for predicting birth safety before people-assisted reproduction blastosphere implantation. The kit comprises products, wherein the products are used for detecting the expression quantity of 97 genes shown in the table 5 and from a cell of a trophectoderm of a people-assisted reproduction blastosphere. If the expression quantity of the 97 genes from the cell of the trophectoderm of a people-assisted reproduction blastosphere meets the evaluation standard, the people-assisted reproduction blastosphere can develop normally and a pregnant woman can have eutocia; otherwise, the people-assisted reproduction blastosphere cannot develop normally, the possibility of affecting baby health and occurring perinatal complication is increased along with the increase of the number of the genes not meeting the evaluation standard, and the transplantation strategy and decision-making are further affected. With adoption of the kit, the possibility of reducing the perinatal complication and the number of babies suffering from illnesses is reduced.

The invention discloses an improved method and a kit for detecting quality of an assisted reproductive technology by a mouse embryo array, and particularly relates to a method for detecting the quality of the assisted reproductive technology (ART) by utilizing cell number at each stage of mouse embryo development and UTF1 positive cell number expressed by blastocyst in the mouse embryo assay (MEA), belonging to the technical field of cytology and biology. At present, based on the internationally common standard MEA, the following two standards are increased: (1) total number of cells contained in embryo at each 12-hour embryonic development period; (2) number of UTF1 positive cells expressed by the blastocyst and number of CDX2 cells expressed by trophoblast cell as well as a proportion of the UTF1 positive cells and the CDX2 cells to total cells of the blastocyst. By comparing the two standards for in-vitro cultivated and developed embryo with the in-vivo embryo at the corresponding period, relatively sensitive and effective means are provided for quality control, including optimizing in-vitro culture conditions and optimizing embryo culture liquor, of the detected assisted reproductive technology, and reference standards are provided for improving the assisted reproductive technology.

The invention discloses a method for preparing an expression vector of adenoviruses. The method comprises the following steps: using a genome of wild type adenoviruses AdC6 as a base, deleting an E1 coding region and an E3 coding region according to a genome direct cloning method, and inserting I-Ceu I and PI-Sce I digestion sites in the E1 deleted region. Simultaneously, OFR6 of human-derived adenoviruses Ad5 is used for replacing ORF6 of AdC6, the success rate of packaging recombinant viruses and the titer of the packaged recombinant viruses in human-derived kidney blastocyte (293A) are improved, and the replication-deficient expression vector of the recombinant adenovirus is obtained. According to the direct molecular cloning method, long-fragment plasmids are constructed, the method issimple and easy to operate, and the method can be used for construction of any long-fragment vectors. The construction of the expression vector of the adenoviruses can be completed through several times of cloning connection, and the steps are simple; the E1 and E3 regions are deleted, the Ad5 ORF6 is replaced, and the deletion and replacing can be completed in the PCR amplification process of DNA fragments; the method is easier and quicker in horizontal bacteria screening and positive cloning, and the construction time of the adenovirus vector is shortened.

The invention discloses a special culture medium and method for culturing porcine trophoderm stem cells. The special culture medium is composed of bFGF, activin A, Y27632, Knockout SR and beta-mercaptoethanol. The culture method comprises the following steps: previously coating a stem cell culture dish with an extracellular matrix; digesting porcine blastula with proteinase to remove the zona pellucida, transferring into the stem cell culture dish, and adding the special culture medium; and culturing until a trophoderm stem cell cloning group with the total cell count of 1000-2000 is formed, digesting into unicells, transferring into a new extracellular-matrix-coated culture dish, and carrying out cell subculture. The special culture medium has the advantages of definite components and high use safety, and avoids the pollution of heterogenous cells. The culture method is simple and efficient, and has the advantages of high cloning formation rate, low apoptosis rate, high proliferation and subculture capacity, high safety and high stability when being used for porcine trophoderm stem cell culture.

The present invention provides methods and compositions for integrating a nucleotide sequence of interest into a genome. Specifically, the method of the present invention comprises: contacting the lentiviral vector with oocytes, early embryos or blastocysts under conditions that allow entry of the viral vector and subsequent integration of the nucleotide sequence of interest into the genome. The method also includes culturing the oocyte or embryo contacted with the lentiviral vector under conditions that allow the formation of a pre-implantation embryo. The pre-implantation embryo can be conceived to be transferred into a recipient vertebrate, allowing it to develop into at least one genetically modified animal. The methods and compositions of the invention thus enable the production of genetically modified animals, in particular vertebrates, especially mammals.

The invention discloses primer pairs and probes for detecting thalassemia genes. The primer pairs and probes include primer pairs and probes of beta-thalassemia CD41 / 42 and alpha-thalassemia-SEA; theprimer pair of the beta-thalassemia CD41 / 42 is a sequence 1 and a sequence 2, and the probes are a sequence 3 and a sequence 4; and the primer pair of thealpha-thalassemia-SEA is a sequence 5 and a sequence 6 or a sequence 5 and a sequence 7, and the probes are a sequence 8 and a sequence 9. The invention further provides a use method of the primer pairs and probes for detecting the thalassemia genes. The use method comprises the following steps of S1, collecting a sample and extracting DNA; S2, carrying out an amplification reaction by utilizing one kind of primer pair and probes; S3, detecting the fluorescence intensity and determining the genotype; and S4, repeating the process by utilizing the other kind of primer pair and probes. The method has the beneficial effects that the fluorescence detection accuracy is enhanced through the designed primer pairs and probes and the two kinds of primer pairs of the same type; and rapid, accurate and harmless detection is realized by extracting free DNA of plasma and blastocyst culture media of pregnant women.

The invention provides a constructing method of 1.3 copy C gene type HBV transgenic mice. Through blastocyst injection of ES cells, the method prepares targeted integration 1.3 copy C gene type HBV (Hepatitis B virus) transgenic mice. First, targeted integration1.3 copy C type HBV expression vectors are constructed, and then an electrical transduction transformation method is employed to transform recombinant plasmids into ES cells; after the and cultivation, targeted integration recombinant ES cells are obtained; then the targeted integration recombinant ES cells are digested into single cells and injected into blastocysts; and then the blastocysts are transplanted to uteruses of pseudopregnant female mice. The method sets up the 1.3 copy C type HBV transgenic mice for the first time home and abroad, is specifically applied to the field of hepatitis B related researches, and provides a more ideal animal model for the researches on prevention and treatment of hepatitis B in China.

The invention provides a composition for oocyte and skin adult stem cell culture and applications thereof. The composition can improve the in-vitro maturation rate of oocyte, cleavage rate of ovum, and rate that embryo grows into mulberry and blastocyst; and thus the growth from in-vitro fertilized ovum to embryo is greatly promoted. At the same time, the differentiation from skin adult stem cells to fibroblast is effectively prohibited by the composition, or the self renewing and regeneration of skin adult stem cells are maintained. The composition comprises cysteamine, leukemia inhibitory factor, and Rho kinase inhibitor Y27632.

The invention relates to the technical field of medical artificial intelligence, in particular to an embryo development potential prediction method and system, equipment and a storage medium. The method comprises the steps: inputting an embryo initial image into a blastocyst prediction model, and obtaining an embryo feature vector; inputting the embryo feature vector into a bidirectional long-short-term memory network to obtain embryo development features; based on a cross-modal feature fusion mechanism, obtaining fusion features according to clinical data and the embryonic development features; and inputting the fusion features into a first multi-layer sensor, and predicting to obtain the embryo pregnancy rate. According to the invention, multi-focal-segment embryo videos shot in the early stage are analyzed, and the fusion features with the space-time characteristic is obtained by utilizing a multi-focal-segment selection model and a time transfer model, so that the pregnancy rate of the embryo cultured in vitro is predicted in real time, and the prediction accuracy is improved; meanwhile, by predicting the blastocyst forming probability and the euploid probability, doctors are assisted in early embryo screening, and therefore the labor cost is reduced.

The invention discloses a breeding method for transgenic pigs expressing sIFITM3 (swine interferon induced transmembrane 3) genes. The breeding method comprises the steps as follows: firstly, the construction of swine fibroblast cell lines stably expressing the sIFITM3 genes comprises the following steps: the step a refers to the construction and the linearization of pcDNACAsIITM3 vectors, wherein, primers are designed, lymph node tissue RNA (Ribose Nucleic Acid) of pigs is extracted, the sIFITM3 genes are obtained through RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification, ethanol precipitation is carried out, and sterile water is used for dissolving; the step b refers to liposome transfection, wherein, swine fibroblasts expressing sIFITM3 are constructed; the step c refers to screening and proliferation of nuclear donor cells transfected with the sIFITM3 genes; secondly, recombination blastula acquisition based on a manual nucleus transplantation method comprises the following steps: the step a refers to the preparation of recipient cells; the step b refers to enucleation and injection of the nuclear donor cells; and thirdly, the embryo transplantation and the acquisition of the transgenic pigs comprise the following steps: the step a refers to embryo transplantation, wherein, bred blastulas are transferred to a fallopian tube of a surrogate sow, and then detection is carried out; and the step b refers to transgenic individual identification. The breeding method is feasible, and is simple and convenient to operate; in addition, the pigs are the transgenic pigs expressing sIFITM3, and have the potential capability to resist animal virus such as foot-and-mouth disease virus, Japanese encephalitis virus, swine influenza virus and the like.

The invention discloses a method for culturing a single porcine embryo in vitro, and belongs to the field of agriculture-animal husbandry and veterinary medicine. The method particularly involves thatfeeder layer cell micro-droplets are adopted to be co-cultured with a single embryo so as to obtain a better development result; the development process of single embryo culture is consistent with the development process of traditionally used multi-embryo culture, and the single embryo attached with a transparent belt or removing a transparent belt can obtain a porcine parthenogenetic developmentblastosphere in 7 days. The method for culturing the single porcine embryo in vitro has the advantages that the provided feeder layer cell micro-droplets preparation is simple, a reliable original material for studying the development of a single embryo can be provided according to the provided method, and the needs of tracking, observing and further researching the development process of the single porcine embryo can be fully met; the provided single embryo culture method can be widely applied to single embryo culture of various species.