115 results about "Zona pellucida" patented technology

This invention provides a method for deriving precursors to pluripotent non-embryonic stem (P-PNES) and pluripotent non-embryonic stem (PNES) cell lines. The present invention involves nuclear transfer of genetic material from a somatic cell into an enucleated, zona pellucida free human ooplastoid having a reduced amount of total cytoplasm. The present invention provides a new source for obtaining human and other animal pluripotent stem cells. The source utilizes as starting materials an oocyte and a somatic cell as the starting materials but does not require the use, creation and/or destruction of embryos or fetal tissue and does not in any way involve creating a cloned being. The oocyte never becomes fertilized and never develops into an embryo. Rather, portions of the oocyte cytoplasm are extracted and combined with the nuclear material of individual mature somatic cells in a manner that precludes embryo formation. Murine, bovine, and human examples of the procedure are demonstrated. Subsequently, the newly constructed P-PNES cells are cultured in vitro and give rise to PNES cells and cell colonies. Methods are described for culturing the P-PNES cells to yield purified PNES cells which have the ability to differentiate into cells derived from mesoderm, endoderm, and ectoderm germ layers. Methods are described for maintaining and proliferating PNES cells in culture in an undifferentiated state. Methods and results are described for analysis and validation of pluripotency of PNES cells including cell morphology, cell surface markers, pluripotent tumor development in SCID mouse, karyotyping, immortality in in vitro culture.

The present invention relates to nuclear methods and embryos developed therefrom. In particular, the present invention relates to a method of nuclear comprising the step of transferring a somatic cell nuclei into a zona pellucida-free, enucleated oocyte.

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.

The invention belongs to the technical field of biomedicine and biological detection, in particular to two linear epitope minimal motif peptides capable of being identified by human zona pellucida protein-4 (ZP4) monoclonal antibodies MA-1662 and MA-1671 on human ZP4 and extended eight peptides and application thereof. The invention provides two candidate epitopes which can be used for developing human immune birth control recombinant multi-epitope vaccines, and the amino acid sequences of the candidate epitopes are shown as SEQ ID No. 1 and marked as huZP4147-151 or shown as SEQ ID No.2 and marked as huZP4256-260; and the invention also provides candidate epitopes which can be used for developing novel high-specificity high-sensitivity recombinant multi-epitope detection antigens for diagnosing serum ZP antibodies of ZP autoimmune infertile women, and the amino acid sequences of the candidate epitopes are shown as SEQ ID No.3-SEQ ID No.6.

The invention discloses an electroporation method for transfection of mammal embryo with siRNA (small interfering Ribose Nucleic Acid). The electroporation method comprises the following steps: (1) weakening an embryo zona pellucid; (2) preparing electroporation solution containing siRNA; and (3) conveying the embryo into the prepared electroporation solution containing siRNA, standing at the room temperature, and then conveying the embryo and the electroporation solution into an electric turn trough for electroporation. The electroporation method comprises the steps of weakening the embryo zona pellucid and complexing the siRNA, and then placing the embryo under the action of a pulsed electric field to increase the permeability and the membrane conductance of the embryo instantaneously, and thus the siRNA which is difficult to penetrate through a cell membrane under the normal physiological condition enters the cell. Statistic observation and analysis prove that the stable positive transfection efficiency of survival embryos exceeds 95%, and the practical effective utilization rate of the collected total embryo exceeds 70%. The electroporation method is a safe, simple and efficient transfection technology of mammal embryo with siRNA.

The invention relates to an integrated detection reaction orifice plate and protein chip reagent box for detecting six sterility indexes. And the reaction orifice plate comprises a substrate and reaction orifices on the substrate and the reaction orifices comprise 3-384 sample orifices, 1-300 negative contrast orifices, and 1-300 positive quality control orifices, where at the bottom of each reaction hole is solid carrier, coated with micro lattice of Sperm antigen (SAg), Zona pellucida antigen (ZPAg), Endometrium antigen (EMAg), Ovary antigen (OAg), Trophoblast antigen (TAg), and Human Chorionic Gonadotropin antigen (HCGAg) antigens or more. And the reaction orifice plate and reagent can simply and conveniently, rapidly and accurately implement simultaneously detection of six sterility indexes for many persons.

Microfluidic embryo scaled channels (14) for handling and positioning embryos provide the opportunity to evaluate and treat embryos in improved manners. Fluid flow is used to move and position embryos within microfluidic channels and channel geometrics may be used to place embryos at specific locations. Surface properties and compliance (deformation) properties of embryos are evaluated as a predictor of viability. The microfluidic channels provide the opportunity for fine controls of pressure to conduct various evalutions at forces slightly below which damage to embryos is known to occur. Measurement of the distance and/or which embryos roll in a same pressure gradient microfluidic channel provides information, with healthy embryos traveling slower or a shorter distance as they demonstrate more stiction to channel walls. Positioned at a constriction (14a, 14b, 24, 26), health embryos also appear to deform less than unhealthy embryos that are more readily pulled into a constriction. In addition, healthy embryos appear to resume their shape better. Fluid from microfluidic channels is easily collected downstream without altering the embryo environment, providing a better opportunity for chemical analysis of fluid chemical analysis than convention manual handling and sampling techniques. Zona pellucida removal of mammalian embryos is achieved as embryos are moved through flow to a precise location where lysing agent can be washed over the embryo to achieve zona removal. Cumulus removal is realized with a series of constrictions to cut cumulus followed by fluid flows to remove cut cumulus from the embryo.

A vibration type microinjection device capable of ensuring smooth piercing of membranes having different properties such as a zona pellucida, a cell membrane and a nuclear membrane included in a fertilized egg with high accuracy and efficiency is provided.

A vibration type microinjection device comprises a vibrator (28) which is connected in series with a micropipette (8) and which has a piezoelectric actuator (29) installed in a housing, and a signal control device (21) for controlling an electric signal applied to the piezoelectric actuator (29), wherein vibration is applied in the longitudinal direction of the micropipette (8) via the vibrator (28) by inputting an electric signal to the piezoelectric actuator (29). By such configuration, smooth piercing of membranes having different properties such as a zona pellucida, a cell membrane and nuclear membrane included in a fertilized egg is realized with high accuracy and efficiency.

The invention provides a serum-free separating and culturing method for a sheep embryo stem cell, comprising the following steps of: removing a zona pellucida of sheep blastula cultured in vitro with a Tyrode's Solution and then inoculating the sheep blastula to a serum-free culture solution of the sheep embryo stem cell and fixing by using a needle head; placing under the conditions that the temperature is 38.6DEG C and the saturated humidity is 5 percent CO2 for culturing; during the culturing, replacing the culture solution in a half quantity every 48 hours with the pH value of 6.8-7.2 and primarily culturing for 7-9 days; and carrying out the transfer culture once according to the ratio of 1:2-1:4 by adopting a conventional mechanical method, that is to say, transferring the sheep embryo stem cell cultured in vitro to the 16th generation. The serum-free culture solution of the sheep embryo stem cell is prepared from D-MEM/F-12+80ng/mL bFGF+3muMCHIR 99021+10mu L/mL N2+20mu L/mL B27+10mu L/mL NEAA+2mM L-Glutamine+0.2mM beta-Mercaptoethanol. Bared embryos are fixed at the bottom of a culture dish by using a No.32 needle head so as to avoid the damage to the cells in the blastula, and a baked embryo trophocyte is stripped off. The preferable inoculating amount of the baked embryos is 45-60/groups. The method can ensure that the formation rate of primary colony of sheep embryo stem cells is improved to 33 percent (14/42).

The invention provides an animal model with male reproductive disorders of a non-human mammalian animal, as well as a preparation method and application of the animal model, and further identifies the functions of a Prss37 gene and a protein. The Prss37 gene of the animal model is inactivated, thereby affecting the appearance of ADAM3 in mature sperms produced by the animal model, indirectly affecting the combination of the sperms-zona pellucida and the migration of the sperms from the uterus to the oviduct and resulting in the serious male reproductive disorders. The model, the Prss37 gene and the protein thereof can be used for screening ADAM3 ripening accelerators, as well as sterility infertility treatment agents or contraceptive medicaments.

The present inventors discovered that genes could be introduced specifically into trophectodermal cells with high efficiency, by infecting blastocysts with viral vectors carrying an arbitrary polynucleotide, or by using a nucleic acid transfection reagent in blastocysts, from which zona pellucida (extracellular matrix covering preimplantation early embryos to protect them from infection of viruses and the like) is removed. This method has no risk of infecting cells of the inner cell mass, which develops into a fetus in the future, with the introduced polynucleotide because the trophectoderm serves as a barrier. The present invention provides methods for introducing foreign genes into only placenta but not fetus, which enables rescue of genetically mutant animals from embryonic lethality due to placental abnormality and allows their birth. Furthermore, it is possible to analyze expression and effect of genes that regulate placental formation or placental function by using these methods.

he invention discloses a method for producing somatic cell cloned bovine blastocysts. According to the method, to bovine ear limbal fibroblasts are used as nuclear transfer donorcells, and mature bovine oocytes cultivated in vitro are used as nuclear transfer receptor cells; the oocytes are denucleated by extrusion and removed with the zona pellucida; the oocytes and the donor cells are adhered in an arrangement way of oocytes-donor cells-oocytes and treated with electro-fusion to obtain reconstructed embryos; and the reconstructed embryos are activated and cultured in vitro to obtain the cloned blastocysts. The method can improve the production efficiency of the cloned bovine blastocysts.

The invention provides a method for detecting chromosome abnormality of embryos by the aid of blastocyst cultivation solution without zona pellucida. The method which is particularly an in-vitro non-therapeutic method for detecting the chromosome abnormality of the embryos by the aid of the blastocyst cultivation solution includes steps of (a), providing the cultivation solution from blastocyst cultivation systems; (b), carrying out gene detection on the cultivation solution so as to authenticate whether chromosomes of the embryos are abnormal or not. The zona pellucida of the embryos in the blastocyst cultivation systems is removed before the embryos are cultivated, the embryos are cultivated in the blastocyst systems for 3-6 days, and preferably, the cultivation solution is separated from the cultivation systems after 4 days. The method has the advantages that contamination and interference risks due to redundant sperms and parent-source granular cells in IVF (in-vitro fertilization) and ICSI (intracytoplasmic sperm injection) technical procedures can be eliminated, and whether the chromosomes of the embryos are abnormal or not can be accurately authenticated.

A method of enhancing in vitro development of a mammalian embryo is disclosed which comprises supplementing the culture medium with a prostaglandin, or a prostaglandin analog, in an amount effective to promote complete hatching of the embryo (i.e., freeing of the embryo from the zona pellucida). The quality of human blastocysts is enhanced in vitro by culturing with a prostacyclin agonist, Iloprost. The in vivo implantation potential and live birth potential of an in vitro fertilization embryo is thereby enhanced and establishment of a viable pregnancy is facilitated.

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 invention discloses a method for removing mouse oocyte nuclei by adopting a zona pellucida solution cavity method. The method comprises the following steps of: performing central slight dripping and peripheral slight dripping in a plastic utensil; making an acid Tyrode's solution react with a zona pellucida under a microscope; forming a pore on the zona pellucida 1-6 seconds later by dissolving till the zona pellucida becomes thin and soft and cytoplasm protrudes outwards; absorbing a first polar body and surrounding cytoplasm out; releasing an oocyte; and denucleating and observing integrality by adopting trypan blue dyeing, wherein the denucleation success rate is over 99.1 percent. In the preparation method, used equipment is simple and the denucleation operation can be completed under a micromanipulator; a reagent has the advantages of simple components, easiness of preparation, low cost, low difficulty of operation, easiness of comprehension, soft denucleation operation, small mechanical damage, quick denucleation and high efficiency; the average denucleation time of each oocyte is 1-6 seconds, the survival rate of each denucleated oocyte is over 97 percent and the success rate of denucleation is over 99 percent; and the method is suitable for researching mouse nucleus transplantation in a Piezo-free laboratory and early events relevant to embryonic development.

The invention discloses a piezoelectric ultrasonic cutting system and method for oocyte zona pellucida. The piezoelectric ultrasonic cutting system comprises an oocyte posture adjusting chip, an oocyte holding device and an oocyte ultrasonic cutting scalpel, wherein the oocyte posture adjusting chip is used for performing posture adjustment and bottom fixation and holding on oocytes; the oocyte holding device comprises a bent holding micro-needle and a micro-needle clamp used for clamping the bent holding micro-needle; the end of the bent holding micro-needle is provided with a single-side inwards recessed arc holding needle head; a bayonet is formed inside the bent holding micro-needle, and a notch is formed in the end of the bent holding micro-needle; the oocyte ultrasonic cutting scalpel is used for cutting the oocyte zona pellucida from the notch position. According to the piezoelectric ultrasonic cutting system disclosed by the invention, the defect that the oocytes are damaged due to longitudinal conduction of ultrasonic energy can be effectively overcome, the zona pellucida is cut from a tangential direction of the oocyte surface layer by the oocyte ultrasonic cutting scalpel, and damage of the oocytes can be reduced.

The invention provides a sperm-oocyte interaction vitro detection method, comprising: a. extracting sperms by a swim-up method or density gradient method; b. mixing the sperms and oocyte extracted from the step a according a certain proportion in the culture medium and setting a blank control of the oocyte in the culture medium; c. removing the sperms which are bonded with the surface of the oocyte in the step b and detecting the number of sperms bonded with the transparent area of the oocyte under an inverted phase contrast microscope; d. separating the oocyte from the sperms bonded with the transparent area of the oocyte and respectively collecting the separated sperms and the oocyte; e. coating the sperms in the blank control of the oocyte and sperms separated by the step b on the glass slide, drying and fixing the sperms using the fixation fluid, cleaning the fixed sperms and adding fluorescein-agglutinin label, re-cleaning the sperms after reaction, detecting the sperm acrosome reaction rate under a fluorescence microscope; f. detecting the number of sperms penetrating into the oocyte separated by the step d under the inverted phase contrast microscope. The detection method reduces the consumption of the label resource and the detection cost and time and increases the detection efficiency.

The present invention relates to method of digesting the zona pellucida of oocyte. The method is to set oocyte inside glutathione solution for digesting. The method of digesting the zona pellucida of oocyte in glutathione solution has simple operation, short digesting period, less damage to cell and low cost.