The way enucleation of oocytes and embryos of mammals
The invention relates to biotechnology cloning mammals. The method involves cutting a transparent shell, removal of nuclear material the same enucleation the introduction of directed tangentially to the cage with pull-transparent shell to contact with the outer surface of the suction pipette and the formation of folds, which cut the translational movement of the beveled portion of the tip enucleation micropipette. The method is distinguished by the simplicity and allows you to speed up the process of micromanipulation, ensures high safety of the operation cells, almost not traumatic, allows enucleation and reconstruction of the recipient cells with high efficiency. 1 C.p. f-crystals, 2 tab. The invention relates to the field of biology, in particular to biotechnology cloning mammals, and can be used to improve efficiency in the refurbishment of embryos for the transplantation of karyoplasts in the centrosome.The importance of developing effective ways enucleation of mammalian embryos is determined by the fact that enucleation is one of the most time-consuming stages in reconsolidating fragment - Cytoplast and karyoplast. The main condition of successful enucleation of cells is the integrity of the cytoplasmic membrane environment as Cytoplast and karyoplast. Only since the development of nontraumatic way enucleation of oocytes and zygotes (MC Grath, Solter, 1983) has made possible the development work on the cloning of mammals.Known method of enucleation and reconstruction of embryos (McGrath, Solter - Nuclear transplantation in the mouse embryo is made by and cell fusion // Science, 1983, 220 V., 4603, R. 1300-1302). The essence of this method lies in the fact that the zygote remove pronuclei without perforation of the plasma membrane, slow absorption in micropipette pronuclei part of cytoplasm surrounded by a plasma membrane. Removal of nuclear material from recipient cells is performed using a micropipette with a beveled sharp tip having an inner diameter of 15-20 μm (enucleation the introduction). With this method enucleation of oocytes requires that the tip enucleation micropipette was prohibet transparent membrane very deep and could easily come under the shell not only the tip, but its wide beveled part. However, very often the piercing transparent shell is only about is this at all the shell. Transparent sheath when bending to a considerable depth, the tip of the micro instruments reaches the opposite side of the membrane inside the cell, resulting in a puncture occurs in the cytoplasmic membrane sharp tip enucleation micropipette, which in turn leads to rapid cell death. So soon after the development of this method were attempts aimed at its improvement.Closest to the proposed invention to the technical essence and the achieved effect (prototype) is a method proposed by Tsunoda Y., Yasui T., Nakamura K., Uchida T., Sugie T. - Effect of cutting the zona pellucida on the pronuclear transplantation in the mouse // J. of Experimental Zoology, 1986, 240, 119-125. The essence of this method lies in the fact that the transparent shell of the egg is pre-cut by 10-20% sharp glass needle with a diameter of 5-7 microns. The egg keep using the suction pipette and tangentially in perivitelline space (from the polar cells) injected glass needle, while making two punctures in a transparent envelope at the entrance and exit perivitelline space egg. Then the needle together with fixed thereto egg move close to the side wall formed by the cut and the needle is released. When enucleation of recipient oocytes initially grasp the suction pipette on the side opposite the cut in the transparent shell, then through this incision enter enucleation micropipette and suck nuclear material.Transplantation of karyoplasts in nuclearvacuum egg (Cytoplast) carry out the same pipette through an incision in a transparent shell.The main disadvantage of this method enucleation of oocytes (Tsunoda et al., 1986) is the use of two tools, the first glass needle to obtain cut in the transparent shell of the egg, and then enucleation micropipette for enucleation of oocytes and transplantation karyoplasts, with the need to replace one instrument to another during the experiment that creates a very large inconvenience in carrying out such fine works as enucleation and reconstruction of cells. In addition, the cut in the transparent shell of the eggs cannot be done directly in one step, you must perform additional operations, such as the introduction of glass needles in perivitelline space, and the movement of the needle attached to it the egg close to the wall of the suction pipette and implementation of several motions of the needle the BA should also include the use of two chambers, one of which is required to obtain cuts in a transparent shell eggs, the second for enucleation of oocytes and transplantation karyoplasts. Moving eggs with cuts from one camera to another, with an intermediate treatment of oocytes with cytochalasin B in a manipulative environment, increases the total duration of manipulation and makes it difficult to find the location of the incision in the transparent shell of the eggs. All of the above disadvantages of the known method is complicate and slow the process micromanipulator.The aim of the present invention is to develop an efficient method for the enucleation of oocytes, which will simplify and accelerate the process of enucleation and reconstruction of cells and provide a high integrity of the cytoplasmic membrane operated cells.Summary of the invention the method involves the use of one enucleation micropipette as to obtain cut in the transparent shell of the eggs, and for enucleation and reconstruction of cells. Cut in a transparent egg shell is made in one step, while enucleation the introduction is not embedded in perivitelline space cells, and affects only the surface ESET formed a crease in a transparent shell.In the proposed method, the enucleation and reconstruction of eggs (in particular, oocytes) is carried out in the following sequence: using enucleation micropipette with an incision in a transparent shell of the egg (from the polar cells). To do this, the egg is fixed by using the suction pipette, and enucleation micropipette have in relation to the cell so that its beveled cutting portion in contact with the transparent egg shell and the outer surface of the suction pipette at the same time. Then a light pressure enucleations the introduction of transparent membrane tighten it until it touches the outer surface of the suction pipette with the formation of folds (loops), and cut out one or two translational motions beveled part enucleation micropipette (Annex 1).After that, using the suction pipette, the egg is fixed on the side opposite the cut in the transparent layer, and then using the same enucleation microinjectors are enucleation of the oocyte or zygote and transplanted karyoplast in perivitelline space enableireland.ie cells through an incision in Ionoi micropipette and forms of production. In each case dimensions enucleation micropipette can be varied depending on the magnitude of karyoplasts (more than karyoplast, the more dimensions enucleation micropipette).In our conditions for microsurgical manipulation karyoplasts derived from blastomeres of 2-cell embryos, used a beveled micropipette with an external diameter of 25-30 microns, made on the installation drawing micropipettor and sharpened on the grinding device at an angle 40o.The advantages of the proposed method (on prototype) are to use one enucleation micropipette as for cutting the transparent sheath, and enucleation and transplantation karyoplasts that allows you to avoid depressurization enucleation system and the new configuration. All this greatly simplifies the process of micromanipulation and shortens its duration. This eliminates the need for: - preparation of supplementary tool - glass needle and the additional working chamber for cutting the transparent shell of the egg; in the replacement of glass needles in the holder of the micromanipulator on enucleation micropipette, in the course of the experiment; in perennializing environment.Use enucleation micropipette for cutting the transparent sheath is preferable than glass needle, because the cutting process is faster, at one time, while enucleation the introduction does not penetrate into perivitelline space cells, and affects only the surface of the transparent shell, which makes the procedure very safe for the cells. Use one of the working chamber for making cuts in a transparent shell eggs and to micromanipulation makes it easy to find the sections operated in the cells.Although the presence of incision and facilitates penetration of any enucleation micropipette under a transparent shell with the aim of enucleation or transplantation karyoplasts, it is preferable to use a beveled enucleation micropipette than enucleation micropipette with smooth edge.The proposed method allows to simplify and accelerate the process of micromanipulation and ensures high safety of the operation of the eggs.The method is illustrated by the following example.The test of the proposed method enucleation and reconstruction performed on oocytes of mice using as karyoplasts - core entih cells were used oocytes at metaphase II.As cell donors karyoplasts were used early blastomeres of 2-cell embryos (after 0.5-1 hour after the first division of the crushing stage G1). To get an individual blastomeres donor nuclei - in 2-cell embryos was previously removed transparent shell with a 0.5% solution of pronase (prepared in phosphate-buffered saline Dulbecco without CA++and Mg++(FSB) for 2-3 minutes. Available from transparent shell embryos were transferred into a solution of the FSB (without CA++and Mg++and pietroboni to get an individual blastomeres. To manipulate used the medium M2.Before microsurgery egg together with blastomeres were incubated in manipulation medium containing cytoskeletal inhibitor cytochalasin B at a concentration of 5 µg/ml All microsurgical manipulations were carried out in the cell type Fombrun. For microsurgery (proposed method) used the following instruments: 1. The suction pipette (outer diameter of the tip of 100-120 microns, an inner diameter of 25-30 microns).2. One enucleation the introduction, is angled at 40o(outer diameter of 25-30 microns), were used for cutting the transparent sheath, and anyluckyday a set of manipulators and inverted microscope, equipped with optics of Namestovo.First, with the help enucleation micropipette was made the cut in a transparent shell eggs (from the location of the polar bodies), then enucleation was performed, removing the polar body and the adjacent region of cytoplasm containing the metaphase plate. When reverse engineering karyoplast 2-cell embryos were injected in perivitelline space of enucleated oocytes through an incision in a transparent shell. As a result of the manipulation of the reconstructed cell consisted of two parts: Cytoplast - enableireland.ie egg and karyoplast of blastomere of 2-cell embryo separated cytoplasmic membranes.Electroline of karyoplast with cytoplasm spent in the chamber with parallel electrodes at a voltage pulse of 1.5 to 2.5 kv/cm-1 and the duration of its action 10 µs in a solution of 0.5 M mannitol, 0.1 mm MgS4, 0.05 mm lactate Sa and SA - 0.5 mg/ml of electrical Stimulation consisted of two phases: using AC (500 KHz, 7 sec) oriented reconstructed cells in an electric field, followed by a rectangular pulse (50 V, 10 µs). Fusion was performed using three successive electric pulses is All fused reconstructed cells were cultured in microcaps environment M l6 under mineral oil in a humidified atmosphere with 5% CO3within 96 hours. Every 24 hours was observed the development of reconstructed cells.In the result of micromanipulation - enucleation of recipient cells and transplant them karyoplasts 2-cell embryos in 9 experiments of 113 eggs (with polar body) nuclearman 111 (98.2 per cent) and reconstructed 108 (97,2%) (table 1). The effectiveness of the merger karyoplast with cytoplasm was 76,8% (83/108) In the cell culture, reconstructed using karyoplasts derived from blastomeres of 2-cell embryos, noted the following development effectiveness: up to 2-cells 98,7% (82/83), up to 4-cells to 90.3% (75/83), up to 8 cells 81,9% (68/83), up to the morula to 78.3% (65/83), to blastocyst 71,0% (59/83) (table 2).Thus, the proposed method has high efficiency for enucleation and reconstruction recipient cells, and does not adversely affect the subsequent stages of the cloning technology is merging karyoplast with cytoplasm and development of reconstructed cells. The proposed method is easy to perform and allows you to accelerate the processes of micromanipulation, ensures high safety of the operation cells, almost not traumatic.Edenichnyh cases the destruction of the eggs with enucleation (2/113) and reverse engineering (3/111).
Claims1. The way enucleation of oocytes and embryos of mammals, including the cutting of the transparent membrane and the removal of nuclear material from the egg, characterized in that the incision in a transparent egg shell produce enucleations the introduction, directed along the tangent to the cage with pull-transparent shell to contact with the outer surface of the suction pipette and the formation of folds, which cut the translational movement of the beveled portion of the tip enucleation micropipette.2. The method according to p. 1, characterized in that the cut in the transparent shell and removing nuclear material from the egg to produce the same enucleation by using the pipettor.
SUBSTANCE: method involves using product containing spermatozoa treated by drying with freezing to humidity level of 1% and having injured membrane or spermatozoon head so that spermatozoon nucleus retains its genetic validity enough for fertilization. The spermatozoon heads fertilize an isolated oocyte after rehydration and microinjection being done. The retained genetic integrity is enough for fertilizing an oocyte and producing living descendants. Method involves collecting living mature spermatozoa, making spermatozoa suspension in special purpose physiological medium, freezing the spermatozoa suspension for producing frozen spermatozoa, drying the frozen spermatozoa or spermatozoa heads in vacuum to humidity level of 1%, making rehydration of spermatozoa or spermatozoa heads with injured membrane and selecting those retaining nucleus of genetic integrity. The selected spermatozoa or spermatozoa heads are used for fertilizing isolated oocytes with living descendants being produced.
EFFECT: enhanced effectiveness in producing living descendants.
39 cl, 5 dwg, 1 tbl
SUBSTANCE: the present innovation deals with individual matching donor sheep to recipient sheep at similar antigenic composition of blood types being correspondent to the value of antigenic similarity index being ra=0.51-1.00, where ra - antigenic similarity index. Moreover, the mentioned antigenic similarity index should be calculated by the following formula: where S - the number of similar antigens in a donor sheep and in a recipient sheep, n1 - the number of detected antigens in a donor sheep, n2 - the number of detected antigens in a recipient sheep. The present innovation enables to increase the level of adaptability of transferred ovine embryos by 25%.
EFFECT: higher efficiency of embryo transfer.
3 ex, 4 tbl
SUBSTANCE: method includes ovary removal after animal slaughtering, their transportation, ovocytes extraction from ovary, their cultivation to metaphase II stage, preparation of boar sperm, combined cultivation of ovocytes and spermatozoids and cultivation of ovulums. Besides, ovary is separated from animals no later than 30 minutes after the moment of animal immobilisation. Ovary is transplanted in free of antibiotics salt solution at 35-37°C during 1 hour. Ovocytes are taken from ovary by dissectioning of visible antrum-containing follicles. Received ovocytes are split into groups by quantity of surrounding cumuluce cell layers, namely: ovocytes surrounded by 4-5 layers of cumuluce cells, 2-3 layers and one layer of cumuluce layer. Each group of ovocytes is cultivated in medium containing 10 IU of human chorionic gonadotropin. To fertilise ovocytes in vitro, ejaculated sperm of pigs is used, which is dissolved with glucose-chelate-citrate-sulphate diluter and cleaned from semen plasma dissolved with mTBM (mouse thymic virus) medium and incubated during 25 minutes under incubator conditions at 5% CO2 and 38.5°C. Produced zygotes are cultivated in NCSU-23 medium free of phenol red and enriched with 0.1% of amino acids during 6-7 days; in addition, 0.17 mM Na-pyruvate and 2.75 mM Na-lactate are introduced into NCSU-23 medium during the first three days of cultivation.
EFFECT: increase of mature ovocytes yield and their fertilisation ratio in vitro.
1 tbl, 1 ex
FIELD: package industry.
SUBSTANCE: container 10 includes vessel (21) with biological medium, germinal cells and/or one or more embryos. Vessel (21) has CO2 permeable wall, CO2 permeable gasket (28) and closure medium (30) for selective access to internal vessel cavity. Buffer chamber (60) for air saturated with CO2 surrounds vessel at least partially and is formed by shell (61). Buffer chamber is connected to CO2 permeable wall. Such container construction is intended specifically for intravaginal cultivation, and permeable gasket (28) prevents vaginal secretion from entering buffer chamber. Buffer chamber (60) normalises water pH in vessel after the vessel leaves environment saturated with CO2.
EFFECT: prevented negative effect for long-term embryo(s) cultivation.
53 cl, 19 dwg
SUBSTANCE: method includes oophorectomy in animals after slaughter, transportation of oophorons, extraction of oocyte-cumulus complexes by aspiration of follicles content, irrigation of the complexes and their cultivation in a medium not covered with oil, in the presence of serum gonadotropin of pregnant mares, human chorionic gonadotropin and 5% estrous serum of bovine cattle. At that, follicles of diametre from 2 to 8 mm are used to obtain oocytes. After extraction of the oocyte-cumulus complexes are immediately placed in a washing medium containing 0.5 mM of isobutylmethylxanthine to synchronise reinitiation of meiosis. Before the cultivation the selection of oocytes based on their morphological evaluation is carried out. All the procedures of isolation, irrigation and selection are carried out in a medium containing 0.5 mM of isobutylmethylxanthine. Oocyte-cumulus complexes were cultured in a medium modified by insertion of 50 ng/ml of bovine prolactin hormone.
EFFECT: increase of proportion of mature oocytes in vitro, and their ability for further development.
1 ex, 1 tbl
SUBSTANCE: method comprises extracting the oocytes-cumulus complexes from ovaries, cultivating them to the stage of metaphase II, co-cultivating of oocytes and sperm cells, cultivating the impregnated ootids. And additionally the follicle-stimulating and luteinising hormone is administered in the form of the combined preparation Menopur, estradiol, heparin, caffeine and antibiotic. The cultivating is carried out in the base medium. Extraction of the oocytes-cumulus complexes from ovaries is carried out from live sheep under anaesthesia by puncturing ovarian follicles during laparotomy. Superovulation stimulation is preliminary carried out. In the maturation medium the preparation Menopur is added, the concentration of both components that are part of the preparation, is 0.075 IU/ml, and estradiol is added to the maturation medium in the concentration of 10 mcg/ml. For impregnation the newly obtained sperm of tups is used. For tup sperm capacitation and to the maturation medium the combination of heparin is added in an amount of 5 units/ml and caffeine in an amount of 0.2 mg/ml. The cultivation and impregnation is carried out in four well Petri dishes in 500 mcl nutrient medium under 200 mcl paraffin oil.
EFFECT: use of the method enables to increase the proportion of output of oocytes.
3 cl, 11 dwg, 3 ex