58 results about "Totipotent" patented technology

The present invention provides methods of producing transgenic livestock animals. The methods generally involve first introducing a nucleoprotein made up of nucleic acid and a recombinase into a totipotent or pluripotent cell to produce a recombinant totipotent or pluripotent cell and then growing the recombinant totipotent or pluripotent cell to produce the transgenic livestock animal. The invention further provides kits for use in generating transgenic non-human animals of the invention.

Human non-embryonic adult totipotent and pluripotent stem cells are isolated in a simplified serum-free and feeder cell-free process. Most remarkably, certain stem cells, and especially BLSCs, are extremely small, fail to exclude trypan blue, but are nevertheless able to proliferate from even high dilutions. Therefore, so obtained stem cells can be used to prepare true monoclonal stem cell populations, which are useful in numerous uses, including therapeutic, prophylactic, diagnostic, and research uses.

The invention provides a use method for a de-host extraction test kit of a metagenome sample, and relates to the field of biological detection. The method comprises the following steps: pathogen enrichment: a step of adding a sample into a first centrifuge tube with a volume of 1.5 mL, carrying out centrifuging for 5-10 min at a room temperature, and removing a supernatant; host cell lysis: a stepof adding 200-1000 [mu]L of a lysis buffer solution, namely Buffer G1 into the first centrifuge tube, and carrying out incubating at 20-40 DEG C for 10-30 min; host DNA removal: a step of centrifuging the first centrifuge tube at a room temperature for 2-5 min so as to remove a supernatant, adding 200-500 [mu]L of the lysis buffer solution, namely the Buffer G1 and 1-3 [mu]L of totipotent nuclease into the first centrifuge tube again, carrying out incubating at 30-40 DEG C for 10-30 min, carrying out centrifuging so as to remove the supernatant, adding 500-1000 [mu]L of the Buffer G1, and washing a precipitate twice; wall breaking of microbial thalli: a step of adding 100-300 [mu]L of lysozyme to resuspend the precipitate, transferring a resuspension to a lysis tube filled with glass beads, placing the lysis tube at 30-40 DEG C, carrying out incubating for 10-30 min, and carrying out vortex uniform mixing treatment for 5-10 min; and microbial lysis and nucleic acid extraction. The usemethod provided by the invention is wide in application range, good in compatibility, good in extraction effect, high in efficiency and low in cost.

The invention relates to a method for extracting sub-totipotent stem cells from a chorion of a fetal surface of a placenta. The method comprises the steps of removing an amnion and stagnated blood from the placenta, and then repetitively washing the surface of the placenta to perform sterilization; shearing the chorion of the fetal surface in a glass utensil, removing placenta lobule tissues remained on the surface as much as possible, and shearing the chorion into small blocks; washing small tissue blocks by using a screen and a great amount of normal saline, and removing residual blood cells; performing tissue digestion: performing oscillatory digestion in a constant temperature shaker by using mixed enzymes; adding a proper amount of FBS for termination after digestion, performing filtration through a filter screen, and adding a great amount of normal saline to wash filter residues to obtain cells as many as possible; performing centrifugation, abandoning supernatant, adding saline water for washing and performing centrifugation to obtain mononuclear cells; performing magnetic bead sorting (OCT-4 positive, Nanog positive and STRO-1 negative) to obtain target cells. The invention further relates to the sub-totipotent stem cells obtained by adopting the method and pharmaceutical use thereof. The sub-totipotent stem cells have excellent characteristics.

Stem cells from human sources can have a variety of useful applications in disease treatment and biotechnology. More particularly the umbilical cord matrix cell cultures of the invention have a variety of totipotent, pluripotent, or multipotent cells for a variety of end uses from a non-controversial, universally available, species-specific source. The technology can have application to any amniotic animal, including agricultural and laboratory animals and humans. The invention relates to isolating the stem cells, culturing the stem cells, maintaining the stem cells, transforming the stem cells into useful cell types using genetic or other transformation technologies, stem cell and tissue banking and using untransformed or transformed cells in disease treatment.

The subject invention provides totipotent, chlorophyllous, cell cultures of maize. In addition, the methods of producing such cultures are applicable to other related species, including cereals such as rice, oats, barley, and heat. The subject cultures are valuable for herbicide studies, studies for enhancing photosynthesis, and genetic manipulation, such as plastid transformation. The methods of the subject invention are capable of providing high percentages of totipotent cells. These cells are capable of sustained cell division and are competent for regeneration over long periods; they provide high-quality target tissue for nuclear and organelle transformation. The invention also describes methods for the introduction of heterologous DNA into the chloroplast genome. The present invention also provides methods, vectors, and gene constructs for enhancing expression of a recombinant nucleic acid sequence in transgenic plants and plant tissues.

The invention discloses a preparation method of sub-totipotent stem cells. The method comprises the following steps: acquisition of tissues, disinfection, isolation, washing and shearing of the tissue, collagenase digestion, and acquisition of the sub-totipotent stem cells. The invention discloses a method for isolating sub-totipotent stem cells from umbilical cord, placenta amnion, chorion or decidua basalis tissues, and the obtained sub-totipotent stem cells have the advantages of uniformity, stability and short growth cycle.

The present invention provides a method for producing mesenchymal cells for production of mesenchymal cells for formation of a tooth, the method comprising: culturing totipotent stem cells in the presence of a differentiation inducer to produce a cell population after differentiation induction treatment, the cell population containing CD44-positive and CD29-positive cells or CD44-positive and CD 106-positive cells; and selecting, from the cell population after the differentiation induction treatment, the CD44-positive and CD29-positive cells or CD44-positive and CD 106-positive cells as the mesenchymal cells for the formation of the tooth. The present invention also provides a method for producing a tooth comprising: positioning, in a support carrier capable of retaining cells in a state of contacting therewith, a first cell mass substantially consisting of only either one of mesenchymal cells and epithelial cells and a second cell mass substantially consisting of only the other one of the mesenchymal cells and epithelial cells, the first and second cell masses being not mixed with each other but made to closely contact with each other; and culturing the first and second cell masses; wherein the mesenchymal cells comprise the mesenchymal cells for the formation of the tooth.

The invention discloses a human adipose sub-totipotent stem cell isolated-culture method and a stem cell bank establishing method. The human adipose sub-totipotent stem cell isolated-culture method includes the following steps of human adipose tissue collection, sub-totipotent stem cell separation and obtaining, sub-totipotent stem cell culture and proliferation, sub-totipotent stem cell cryopreservation, product quality control, stem cell bank establishing and stem cell revivifying and annual inspection. Mixed collagenase used by the method can effectively separate sub-totipotent stem cells from adipose tissues, is homogenous in tissue digestion, no obvious tissue residual is produced, the cell activity is good, wall adherence is quick, and the homogeneity is good. Cryopreservation liquid adopted by the method can effectively maintain the cell activity, a gradient cooling box is adopted to cryopreserve cells, the cell activity is not affected, and the human adipose sub-totipotent stem cell isolated-culture method is simple in operation, convenient and high in efficiency.

The invention relates to a method for making an at least double layered cell aggregate and/or an artificial blastocyst, and/or a further-developed blastoid termed blastoid, by forming a double layered cell aggregate from at least one trophoblast cell and at least one pluripotent and/or totipotent cell, and culturing said aggregate to obtain an artificial blastocyst. This artificial blastocyst has a trophectoderm-like tissue that surrounds a blastocoel and an inner cell mass-like tissue. The cell aggregate can be formed from toti- or pluripotent stem cell types, or induced pluripotent stem cell types, in combination with trophoblast stem cells. Formation of a blastoid can be achieved by culturing the cell aggregate in a medium preferably comprising one or more of a Rho/ROCK inhibitor, a Wnt pathway modulator, a PKA pathway modulator, a PKC pathway modulator, a MAPK pathway modulator, a STAT pathway modulator, an Akt pathway modulator, a Tgf pathway modulator and a Hippo pathway modulator. The invention further relates to a method for growing an at least double layered cell aggregate into an artificial blastocyst, and into a further-developed blastoid, a fetus or a live animal. The invention further pertains to an in vitro cell culture comprising the mentioned compounds and/or cell aggregates.

The invention provides a special culture medium and a special culture method for sub-totipotent stem cells. Enough stem cells can be obtained through one passage, and are stored. The culture medium contains no serum, and can be conveniently used. The special culture medium for the sub-totipotent stem cells includes 95% of DMEM/F12, 5% of a serum substitute, 5mg/ml of human albumin, 5.5[mu]g/ml of linoleic acid, 10ng/ml of a basic fibroblast growth factor, 2*10<-8>mol/ml of dexamethasone, 10ng/ml of a human platelet-derived growth factor, 10ng/ml of an epidermal growth factor, 50[mu]mol/L of beta-mercaptoethanol and 2mmol/L of L-glutamine. The culture medium contains no serum, has a good culture effect, and allows a required number of frozen sub-totipotent stem stems to be reached only through culturing one generation.

The invention belongs to the field of cells, and discloses a method for inducing fetal bovine fibroblast to reprogram. According to the invention, the fetal bovine fibroblast is subject to permeabilized treatment by a digitonin solution, and is subject to co-incubation with xenopus oocytes extract; whole medium containing 2mMcaCl2 is adopted to block reversible pores in the cell membrane, extract treated medium is adopted to cultivate and induce the fetal bovine fibroblast to reprogram to obtain fetal bovine fibroblast pluripotent stem cells. The method discloses by the invention is efficient, stable, and safe; the alkaline phosphatase staining of the fetal bovine fibroblast pluripotent stem cells disclosed by the invention shows a positive result; the acetylation degree of histone H3K9 is lowered; Oct-4 protein and pluripotent marker gene Oct-4, Nanog are expressed; therefore the fetal bovine fibroblast undergoes reprogramming, the gene expression mode of cells is changed to show characteristics of stem cell, partial growing totipotent is restored, and the fetal bovine fibroblast can be used as nuclear donor cells to prepare clone embryo for body cell clone.

The invention discloses an expression and purification method of a totipotent nuclease. The method comprises the following steps: (1) expression steps: E. coli containing a target gene growing in a basic culture medium, adding an inducer isopropyl-P-D-thiogalactose to induce expression of a target protein, the target gene containing a totipotent nuclease sequence, the front end of the totipotent nuclease sequence containing a tag for coding 6 x His, and the target protein being an inclusion body; (2) inclusion body renaturation: diluting and renaturing the inclusion body obtained in the step (1) to obtain the soluble and active target protein; and (3) purification steps: conducting affinity chromatography for purification, enzyme digestion and inversion of the target protein to obtain thetotipotent nuclease with high purity. According to the present invention, the production cost of the totipotent endonuclease can be effectively reduced, the purification time can be shortened, and thetotipotent endonuclease with high activity can be obtained by the provided method, and the method is conducive to the application of the totipotent endonuclease in scientific research and industrialfields.

The invention discloses a method for induced differentiation of human placenta sub-totipotent stem cells to obtain cardiac muscle cells. The method comprises the following steps: inoculating human placenta sub-totipotent stem cells into a 24-pore plate containing a stem cell culture medium; after cell attachment, removing the stem cell culture medium; and adding a cardiac muscle cell culture medium for culture for 28 days, and changing the culture medium every 3 days. The stem cell culture medium comprises a DMEM culture medium, hydroxyethyl starch, human serum albumin, potassium bicarbonate, ethylenediamine tetraacetic acid sodium salt, sodium selenite, transferrin, hyaluronic acid, taurine, placental growth factors, fibroblast growth factors, glutathione, lipoic acid, astragalus polysaccharide, codonopsis pilosula polysaccharide and jujube polysaccharide. The cardiac muscle cell culture medium comprises an RPMI-1640 culture medium, trehalose, taurine, fibroblast growth factors, endothelial cell growth factors, potassium bicarbonate, amino acid chelated selenium, glutamine, progesterone, coenzyme I, lipoic acid, salvianolic acid B, polygonatum polysaccharide and ginsenoside.

The invention discloses an inducing and culturing system-establishing method for totipotent stem cell quteASCs of mice or human beings. The materials are early embryo of mice growing for 3-8 days (obtained through in-vitro fertilization or collected from the uterus of a parent body), and the early embryo of the human beings growing for 3-14 days (obtained through in-vitro fertilization or collected from the uterus of a parent body). The inducing method comprises the following two key technical processes: 1, carrying out inducing to establish excess type stem cells ASCs, wherein the key inducing factor is AF; and 2, carrying out further inducing to complete the system establishing technological process of the novel pleuripotent stem cells quteASCs, wherein the key inducing factor is ABCL. According to the invention, through the specific inducing method, the novel stem cells quteASCs of the mice or the human beings are successfully obtained through the specific inducing method, and the totipotency of the novel stem cells quteASCs of the mice or the human beings for developing to the individual tissue of animal or the placenta of the animal is verified through the experimental means.

The invention discloses a method for separating and purifying human amniotic fluid stem cells. The method is to use an embryo antigen-4 at a special stage as a screening marker to separate and purify the human amniotic fluid stem cells which have homogeneous characteristics and are positively expressed by the embryo antigen-4 at the special stage from the human amniotic fluid stem cells which are adherently cultured. Experimental results show that the method can obtain the human amniotic fluid stem cells which have homogeneous characteristics, good capacities of proliferation and inductive differentiation, and cell ratio of G0 stage to G1 stage cells of more than 90 percent, and can express specificity markers of human totipotent stem cells such as the embryo antigen-4, Nanog and Oct-4 at the special stage. The invention provides a new way to obtain the human amniotic fluid stem cells, and lays a foundation for the further application of the human amniotic fluid stem cells.

The invention of the present patent application provides, a histamine-hyperproductive animal, which is a non-human animal or progeny thereof obtained by ontogenesis of totipotent cell transfected with a polynucleotide encoding histidine decarboxylase and has the above polynucleotide in the cellular chromosome to produce histamine at a high level in the somatic cells. Thus, analysis of the pathogenesis and pathological consequence in various disorders associated with histamine in human as well as development of therapeutic techniques and remedies for these disorders will be developed.

The invention provides a totipotent stem cell culture method. A polypeptide which is artificially synthesized is taken as an extracellular matrix and spread on the surface of a cell culture dish, totipotent stem cells are inoculated to the surface of the culture dish and injected into a culture medium without a feeder layer, and the totipotent stem cells after inoculation are used for culture and passage. According to the method provided by the invention, the amplification of the totipotent stem cells can be supported, and the cell content of Oct4+ cells can be always kept above 80%. Through at least 40 days of culture (six passages and about 40 times of cell doubling), the cells can still realize high expression of various markers Oct4 of the totipotent stem cells, and after the amplification culture, the karyotype can still be kept consistent.

Methods and compositions are provided for generating F0 fertile XY female animals. The methods and compositions involve making XY pluripotent or totipotent animal cells, in vitro cell cultures, or embryos that are capable of producing a fertile female XY animal in an F0 generation. Such cells, embryos, and animals can be made by silencing a region of the Y chromosome. Optionally, the cells can also be cultured in feminizing medium such as a low-osmolality medium and/or can be modified to decrease the level and/or activity of a Sry protein. Methods and compositions are also provided for silencing a region of the Y chromosome in an XY pluripotent or totipotent animal cell, or in vitro cell cultures, embryos, or animals derived therefrom, by maintaining an XY pluripotent or totipotent animalcell in a feminizing medium. Methods and compositions are also provided for maintaining a population of XY pluripotent or totipotent animal cells in a feminizing medium and selecting cells or clones having increased capabilities for producing a fertile female XY animal in an F0 generation. Methods and compositions are also provided for screening for compounds with feminizing activity or for optimizing concentrations of components in feminizing media.

Provided are a sub-totipotent stem cell product and apparent hereditary modifying label thereof, a method for inducing the generation of the sub-totipotent stem cell product and identification for the apparent hereditary modifying label of the differentiated potential of stem cells. Also provided is a use of a histone modifying state of a sub-totipotent gene and/or differentiated related gene to predict the apparent hereditary modifying label of the differentiated potential of stem cells.