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).

Claims

1. 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.

 

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