A spindle nucleocytoplasmic replacement method for reducing residual mitochondria

Abstract

The present invention discloses a spindle nucleoplasm replacement method for reducing residual mitochondria. The method removes the spindle-containing cell body and performs a decytoplasm operation again, so that the cytoplasm around the spindle is almost detached, and the remaining cell body has almost no cytoplasm, showing a phenomenon that the cell membrane almost tightly wraps the spindle, thereby greatly reducing the residual mitochondria. In the spindle nucleoplasm replacement, the method of the present invention can be used to press the spindle-containing cell body after the decytoplasm is soaked in an inactivated Sendai virus solution and then pressed on the membrane of the recipient egg, so as to promote the efficient fusion of the spindle cell body into the recipient egg.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a spindle nucleocytoplasmic replacement method for reducing residual mitochondria. Background Art

[0002] At present, the known spindle nucleoplasm replacement, in the micromanipulation steps, only has the process of taking out the spindle cell body, and the operation of implanting the spindle cell body into the enucleated egg, and there is no operation of removing the spindle cell body from the cytoplasm. However, the current direct implantation of the spindle cell body into the enucleated egg after taking out the spindle cell body has the problem of more residual cytoplasm (including mitochondria). This makes the risk of mutant mitochondria in the residual mitochondria amplifying beyond the threshold ratio and causing disease greater, so the technical safety is still low. Therefore, there is an urgent need for a spindle nucleoplasm replacement method that reduces residual mitochondria. Summary of the invention

[0003] In our research on nucleocytoplasmic replacement, we found that if the cytoplasm around the nucleus is removed as much as possible, it will help reduce the amount of residual mitochondria that the nucleus carries with it when it is replaced in another enucleated egg, thereby reducing the problem caused by the increase in the proportion of residual mitochondria during subsequent development. Therefore, we proposed a spindle nucleocytoplasmic replacement method that can greatly reduce residual mitochondria.

[0004] The present invention provides a spindle nucleocytoplasmic replacement method for reducing residual mitochondria, the method comprising the process of removing the spindle cell body in an in vitro micromanipulation and implanting the spindle cell body into an enucleated egg, characterized in that after the removal operation and before the implantation operation, a cytoplasmic mechanical detachment operation of the spindle cell body is added, and during the implantation operation, a fusion-promoting compression operation is added.

[0005] Preferably, the mechanical detachment of the cytoplasm from the spindle cell body uses a culture medium that can soften the cell membrane, and through the action of mechanical swinging or squeezing force, the cytoplasm is prompted to smoothly detach from the cell body with the cell membrane without disintegration, thereby significantly reducing the initial mitochondrial residues of nucleocytoplasmic replacement.

[0006] Further preferably, the fusion-promoting pressure operation during the implantation operation is that after the spindle cell body is decytoplasmicated and immersed in an inactivated Sendai virus solution, a part of the cell body is sucked in with a microtube, and the other part is exposed outside the tube and pressed on the egg membrane to be fused, and maintained for a certain period of time to promote cell membrane fusion.

[0007] The present invention provides a spindle nucleocytoplasmic replacement method for reducing residual mitochondria, the method comprising:

[0008] (1) Using micromanipulation to aspirate the spindle-containing cell body from the donor egg to obtain the spindle cell body;

[0009] (2) Using micromanipulation to remove the cell body containing the spindle from the donor oocyte and discard it, the remaining oocyte is the enucleated recipient oocyte;

[0010] (3) placing the spindle cell body obtained in step (1) into a culture medium for mechanical detachment of cytoplasm, sucking one side of the cell body with a microscopic glass tube to expose the other side, and making at least most of the spindle body inside the tube mouth (none or only a small part is exposed outside the tube), shaking the glass tube relative to the bottom of the dish or squeezing the exposed part close to the edge of the droplet toward the interface with the mineral oil to detach the cytoplasm on one side, then directly spitting out most of the spindle body outside the tube, leaving the remaining cytoplasm inside the tube, repeating the above mechanical detachment operation, so that the remaining cytoplasm in the tube is detached; or all of the spindle cell body can be spit out of the tube, sucking from the other side of the cell body to expose the other side that has not been detached, and making at least most of the spindle body inside the tube mouth (none or only a small part is exposed outside the tube), repeating the above detachment operation, and obtaining a spindle cell body with detached cytoplasm on both sides;

[0011] (4) using a micropipette to bring the spindle cell body obtained in step (3) into the inactivated Sendai virus droplet, expel a portion and soak it for tens of seconds;

[0012] (5) Through the opening of the zona pellucida of the recipient egg, the spindle cell body soaked in the inactivated Sendai virus droplets is expelled from the tube and pressed against the membrane of the recipient egg for tens of seconds to several minutes. When the cell membranes are firmly bonded, the cell body is gently expelled and the pipette is withdrawn.

[0013] Preferably, before sucking out the cell body containing the spindle in step (1) and step (2), a hole is made in the zona pellucida at the first polar body of the donor egg and the recipient egg, and a microscopic glass tube (including a microscopic insemination needle) is used to enter through the opening of the zona pellucida to suck out the first polar body and discard it.

[0014] Further preferably, in step (1) and step (2), a microscope glass tube is used to enter through the opening of the zona pellucida to suck out the cell body containing the spindle.

[0015] Further preferably, in step (3), shaking the glass tube relative to the bottom of the dish is shaking the glass tube or moving the bottom of the dish.

[0016] Further preferably, the soaking time in step (4) is 20-90 seconds.

[0017] Further preferably, it is characterized in that in step (5), the spindle cell body is partially protruded out of the tube and pressed on the recipient egg membrane for 20-600 seconds.

[0018] Further preferably, in step (5), the opening of the zona pellucida of the recipient egg is adjusted to the opposite side of the oviduct holding it (for example, if it is held at the 9:00 position, the opening is adjusted to the 3:00 position; conversely, if it is held at the 3:00 position, the opening is adjusted to the 9:00 position), and the microscope tube enters from the zona pellucida opening, and the spindle cell body portion soaked in the inactivated Sendai virus droplets is expelled from the tube and pressed against the recipient egg membrane.

[0019] More preferably, the egg is a mammalian egg such as a mouse egg or a human egg.

[0020] Further preferably, the culture medium capable of softening cell membranes comprises basic components of in vitro culture medium and added components, wherein the added components comprise substances with appropriate concentrations for softening cell membranes during mechanical detachment of the cytoplasm, and the added components are selected from one or more of polyvinyl pyrrolidone (PVP), protein components, and cytochalasins.

[0021] Further preferably, the concentration of polyvinyl pyrrolidone (PVP) is less than 7%, and / or the concentration of the protein component is greater than 20%, and / or the concentration of cytochalasin is less than 5 μg / ml.

[0022] Further preferably, the basic components of the culture medium include one or more of sodium chloride, potassium chloride, magnesium carbonate, magnesium sulfate, calcium chloride, sodium pyruvate, sodium lactate, gentamicin, phenol red, sodium bicarbonate, and 4-hydroxyethylpiperazineethanesulfonic acid.

[0023] The principle of the above technology is:

[0024] Ingredients that make the cell membrane soft and sticky are added to the decytoplasmic culture medium. A part of the cell body is sucked in with a pipette to expose the part to be detached. Repeated shaking or squeezing near the edge of the droplet can cause the cytoplasm and cell membrane between the part exposed outside the tube and the part not exposed outside the tube to gradually stretch, and the stretched part gradually becomes thinner, and finally separates from each other and the cell membranes are closed separately to remain intact without rupture; if the part exposed outside the tube contains a rigid substance with a certain relative hardness, and part of the substance is inside the tube, such as the spindle is relatively rigid, when the cell body sucked in by the pipette contains most of the spindle and only a small part of the spindle is exposed outside the tube, then when mechanical repeated swinging or squeezing by the edge of the droplet is performed, the spindle exposed outside the tube will not break off, but only the cytoplasm around it will be pulled apart by the mechanical force, and the cell membrane around it will be stretched and close to the spindle, and finally wrapped and closed along the contour of the spindle to separate from the detached part. Then, most of the spindle can be directly expelled from the tube, and the residual cytoplasm can be left in the tube, and the above mechanical decytoplasm operation can be repeated to remove the residual cytoplasm in the tube; or all the cell bodies in the tube can be expelled, and the cell bodies can be reversed by 180 degrees to expose the residual cytoplasm to be removed while at least most of the spindle is inside the tube mouth (no or only a small part of the spindle is exposed outside the tube), and the above decytoplasm operation can be repeated. In this way, the residual cytoplasm around the spindle, especially at its two ends, can be further removed.

[0025] After the above-mentioned decytoplasm, the volume of the spindle cell body becomes smaller. If the fusion operation is performed according to the conventional method, the cell membrane contact surface and contact tension are very small, and the fusion efficiency is extremely low whether it is the electric fusion method or the virus fusion method. Increasing the pressure to promote fusion operation can solve this problem. Specifically, the spindle cell body after decytoplasm is soaked in the inactivated Sendai virus solution (the cell body can be sucked with a straw and only the exposed part of the cell body is soaked), and then sucked and transferred to the recipient egg with a microtube. If the egg is fixed on the left side under micromanipulation (that is, the 9:00 position of the egg), the opening of the zona pellucida of the egg is adjusted to the 3:00 position, and the microtube enters from 3:00 (conversely, if the right side is sucked at 3:00, the opening of the zona pellucida is adjusted to the 9:00 position for entering the tube), and the cell body part is expelled from the tube and pressed on the membrane of the recipient egg to be fused (forming a certain depression) for a certain time. This can increase the cell membrane contact surface and contact tension and promote cell membrane fusion.

[0026] The beneficial effect of the present invention is that: in the spindle nucleoplasm replacement, the present technology can perform a decytoplasm operation on the cell body containing the spindle, so that the cytoplasm around the spindle is almost separated, and the remaining cell body has almost no cytoplasm, showing a phenomenon that the cell membrane almost tightly wraps the spindle, thereby greatly reducing the mitochondrial residue. Through mitochondrial staining and quantitative fluorescence measurement, it can be found that: in human eggs, the mitochondrial content of the spindle cell body after the technology is treated is reduced to 0.1%-20% before the treatment. In the spindle nucleoplasm replacement, the method of the present invention can be used to compress and fuse the spindle-containing cell body after the decytoplasm, so as to promote the efficient (about 90% or more within 30 minutes) fusion of the spindle cell body into the recipient egg, while about 90% of the cell bodies that have not been subjected to the compression and fusion treatment will never fuse into the egg. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a schematic diagram of steps A to G of the spindle nucleocytoplasmic replacement method;

[0028] Figure 2 This is an observation diagram of the mouse egg spindle nucleocytoplasmic replacement operation. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific embodiments to provide a better understanding of the present invention.

[0030] A spindle nucleocytoplasmic replacement method for reducing residual mitochondria, the specific process is as follows Figure 1 As shown,

[0031] In the figure, A to E, the left side is the operation of the donor egg to obtain the spindle body, and the right side is the operation of the donor egg to obtain the enucleated egg (recipient egg). F to G are the fusion operation of the spindle body obtained above and the recipient egg. The steps are:

[0032] A. For an egg (large inner oval) containing a spindle (diamond in the inner part), a hole is made in the zona pellucida (large outer circle) at the first polar body (small oval), and the microscope glass tube (parallel black lines) will enter from the opening of the zona pellucida.

[0033] B. Enter the microscopic glass tube through the zona pellucida opening to aspirate and discard the first polar body.

[0034] C. Use a microscope glass tube to enter the opening of the zona pellucida and suck out the cell body containing the spindle. Discard the spindle cell body of the right egg if it is not needed, and continue the operation on the spindle cell body of the left egg.

[0035] D. Place the spindle cell body in the culture medium for mechanical detachment of the cytoplasm, use a microscopic glass tube to suck one side of the cell body, expose the other side, and make at least most of the spindle body inside the tube mouth (none or only a small part is exposed outside the tube), shake the glass tube relative to the bottom of the dish (just shake the glass tube or just move the bottom of the dish repeatedly), or squeeze the exposed part close to the edge of the droplet toward the interface with the mineral oil (not shown). Because the culture medium for mechanical detachment of the cytoplasm makes the cell membrane of the cell body soft and sticky, it is easy to be gradually stretched and thinned by mechanical force, and close again by itself after breaking, without causing the cell membrane to rupture and disintegrate. After the cytoplasm on one side is detached, most of the spindle can be directly spit out of the tube, leaving the residual cytoplasm in the tube, and repeating the above mechanical decytoplasm operation to detach the residual cytoplasm in the tube; or the entire cell body can be spit out of the tube, sucked in from the other side of the cell body to expose the other side that has not yet been decytoplasmed, and at least most of the spindle is inside the tube mouth (none or only a small part is exposed outside the tube), and then repeating the above decytoplasm operation to detach the residual cytoplasm outside the tube.

[0036] E. After the above operation D, the spindle cell body with both sides free of cytoplasm was obtained. The cell body was brought into the droplet of inactivated Sendai virus using a micropipette, and a part of it was expelled and soaked for tens of seconds. The right side is the recipient egg that was enucleated after the above operation C.

[0037] F. After the above immersion, the cell body is brought to the droplet with the recipient egg. Through the opening of the zona pellucida of the recipient egg, the spindle cell body part soaked in the inactivated Sendai virus droplet is expelled from the tube and pressed on the egg membrane for tens of seconds to several minutes. After the cell membranes are firmly bonded, the cell body is gently expelled and the pipette is withdrawn.

[0038] G. After several minutes or tens of minutes, the egg and the cell body completely fuse, and the spindle enters the recipient egg.

[0039] Example Mouse egg spindle nucleocytoplasmic replacement

[0040] Preparation of inactivated Sendai virus (HVJ-E) solution:

[0041] HVJ Envelope Cell Fusion Kit (Ishihara Sangyo Kaisha Ltd, cat. no. GenomONE-CF EX) was used. During the preparation process, the solution was placed on ice. A bottle of inactivated Sendai virus envelope freeze-dried powder was added to 260 μl of buffer provided in the kit for suspension. Then, in order to avoid the HVJ-E stock solution from losing activity due to multiple freezing and thawing, the conventional method is: aliquoting into 5 μl / vial, storing at -80°C for 3 months, and for the fusion operation of nucleocytoplasmic replacement, a small tube of HVJ-E solution was thawed before the operation and used directly without dilution. The method of the present invention is: aliquoting into 2 μl / vial, storing at -80°C for 3 months, and for the fusion operation of nucleocytoplasmic replacement, a small tube of HVJ-E solution was thawed before the operation and further diluted in multiples (such as 1:2 to 1:50, etc.) before use.

[0042] Source of experimental materials

[0043] Two strains of female mice (such as BALB / c mice and C57BL / 6 mice) were used in the experiment to induce ovulation. About 15-18 hours after HCG injection, the mice were killed by cervical dislocation, and the cumulus mass was obtained by incision of the ampulla of the fallopian tube. The mouse eggs were digested with hyaluronidase to obtain mouse eggs, and mature mouse eggs were selected for nuclear cytoplasmic replacement between the two strains.

[0044] Specific steps for preparing mouse oocyte decytoplasmic spindle cell bodies

[0045] When performing the spindle nucleoplasm replacement operation, the position and morphology of the spindle are observed with the aid of a spindle observation instrument on a microscope; the operation of removing the spindle cell body is performed conventionally, and the culture medium used is the following conventional culture medium (the basic components and concentrations in the aqueous solution are as follows):

[0046] Sodium chloride (97.6mM), potassium chloride (4.7mM), magnesium carbonate (or magnesium sulfate) (0.2mM), calcium chloride (2.04mM); sodium pyruvate (0.33mM), sodium lactate (21.4mM); gentamicin (50μg / ml), phenol red 0.001%wt / vol); sodium bicarbonate (25.0mM), 4-hydroxyethylpiperazineethanesulfonic acid (25mM), and then routinely added: protein component is 10%, and cytochalasin B is 5μg / ml.

[0047] However, in order to reduce the residual mitochondria caused by nucleocytoplasmic replacement, the present invention uses a new culture medium (culture medium for mechanical detachment of the cytoplasm) to remove the spindle cell body. In addition to the basic components and concentrations mentioned above, the added components and their concentrations of the culture medium are different from the conventional ones, namely: polyvinyl pyrrolidone (PVP) is 5%, the protein component is 30% and / or cytochalasin B is 1.25 micrograms / ml.

[0048] With the culture medium, one side of the spindle cell body is sucked into a microscope glass tube to expose the other side, and at least most of the spindle is inside the tube mouth (none or only a small part is exposed outside the tube). Because the part of the spindle exposed outside the tube is a rigid substance with a certain relative hardness, and most of the substance is inside the tube, the spindle exposed outside the tube will not break off during mechanical repeated swinging or droplet edge squeezing, but only the cytoplasm around it is pulled apart by the mechanical force, and the cell membrane around it is stretched and tightly attached to the spindle, and finally wrapped along the outline of the spindle, causing the cytoplasm and cell membrane between the part exposed outside the tube and the part not exposed outside the tube to gradually stretch, the stretched part gradually becomes thinner, and finally separate from each other, and the cell membranes are closed separately to remain intact without membrane rupture. Then, most of the spindle can be directly expelled from the tube, leaving the residual cytoplasm in the tube, and the above-mentioned mechanical decytoplasm operation can be repeated to remove the residual cytoplasm in the tube; or the entire cell body can be expelled from the tube, and the cell body can be sucked in again 180 degrees to expose the residual cytoplasm to be removed while at least most of the spindle is inside the tube mouth (none or only a small part of the spindle is exposed outside the tube), and the above-mentioned mechanical decytoplasm operation can be repeated to remove the residual cytoplasm outside the tube.

[0049] After obtaining the mouse decytoplasmic spindle cell body by the above operation, the mouse egg spindle is replaced and fused. The specific process diagram is shown in Figure 1 As shown (see the above implementation description), the process diagram is as follows Figure 2 As shown, the upper left picture shows the swinging state of the spindle cell body when the cytoplasm is removed (the spindle appears bright white in the glass tube, while the cell body inside and outside the tube is darker, with only the outline visible), the middle left picture is a bright field picture of the spindle cell body after the cytoplasm is removed, and the lower left picture is an observation picture of the spindle in the middle left picture (the spindle appears spindle-shaped white light), which shows that the cell body is almost occupied by the spindle, indicating that there is little residual cytoplasm; the upper middle picture shows that the spindle cell body after the cytoplasm is removed is sucked by a pipette, exposing about half of it immersed in a droplet of inactivated Sendai virus ( The concentration is 1:10); the lower middle picture shows that after the virus is soaked, it is transferred to one side of the egg, and a microscopic pipette is inserted from the opening of the zona pellucida. About half of the cell body is expelled to press the cell membrane on one side of the egg (to form a depression), and it is maintained for about 10 seconds to 120 seconds. After the cell membranes are firmly bonded, the cell body is gently expelled and the pipette is withdrawn; the picture on the right shows the state of the cell body after compression and fusion. The upper right part is a bright field picture, and the lower right part is a spindle observation picture (the spindle appears in a spindle-shaped white light), which shows that the spindle has been integrated into the recipient egg.

[0050] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A method for spindle nucleocytoplasmic replacement to reduce residual mitochondria, comprising the process of removing the spindle cell body in an in vitro micromanipulation and implanting the spindle cell body into an enucleated egg, characterized in that: After the removal operation and before the implantation operation, a cytoplasmic mechanical detachment operation of the spindle cell body is added, and a fusion-promoting compression operation is added during the implantation operation; The mechanical detachment of the cytoplasm from the spindle cell body uses a culture medium that can soften the cell membrane, and through the action of mechanical swinging or squeezing force, the cytoplasm is facilitated to be smoothly detached from the cell body with the cell membrane without disintegration, thereby significantly reducing the mitochondrial residues at the beginning of the nuclear cytoplasm replacement; The fusion-promoting compression operation during the implantation operation is to soak the spindle cell body after the cytoplasm is removed in an inactivated Sendai virus solution, and then a part of the cell body is sucked into the microtube, and the other part is exposed outside the tube and pressed on the egg membrane to be fused, and maintained for a certain period of time to promote cell membrane fusion; Specifically, the method includes: (1) Using micromanipulation to aspirate the spindle-containing cell body from the donor egg to obtain the spindle cell body; (2) Using micromanipulation, the cell bodies containing the spindles in the donor oocytes are removed and discarded, and the remaining oocytes are enucleated recipient oocytes; (3) placing the spindle cell body obtained in step (1) into a culture medium for mechanical cytoplasm removal, and using a microscopic glass tube to suck one side of the cell body to expose the other side, so that at least most of the spindle is within the tube mouth, with no or only a small part exposed outside the tube, shaking the glass tube relative to the bottom of the dish or squeezing the exposed part close to the edge of the droplet toward the interface with the mineral oil to release the cytoplasm on one side, and then directly spitting out most of the spindle out of the tube, leaving the remaining cytoplasm in the tube, repeating the above mechanical cytoplasm removal operation, so that the remaining cytoplasm in the tube is removed; or the spindle cell body can be spit out of the tube, sucked from the other side of the cell body, exposing the other side that has not yet been cytoplasm removed, and so that at least most of the spindle is within the tube mouth, with no or only a small part exposed outside the tube, repeating the above cytoplasm removal operation, to obtain a spindle cell body with cytoplasm removed from both sides; (4) Using a micropipette, place the cell bodies obtained in step (3) into a droplet of inactivated Sendai virus, expel a portion of the cell body, and soak it for a certain period of time; (5) Through the opening of the zona pellucida of the recipient egg, the spindle cell body soaked in the inactivated Sendai virus droplets is expelled from the tube and pressed against the membrane of the recipient egg for a certain period of time. When the cell membranes are firmly bonded, the cell body is gently expelled and the pipette is withdrawn; The culture medium for mechanical detachment of the cytoplasm includes the following components and concentrations: 97.6mM sodium chloride, 4.7mM potassium chloride, 0.2mM magnesium carbonate or magnesium sulfate, 2.04mM calcium chloride; 0.33mM sodium pyruvate, 21.4mM sodium lactate; 50μg / ml gentamicin, 0.001% wt / vol phenol red; 25.0mM sodium bicarbonate, 25mM 4-hydroxyethylpiperazineethanesulfonic acid, 5% polyvinylpyrrolidone, 30% protein component and / or 1.25μg / ml cytochalasin B.

2. A spindle nucleocytoplasmic replacement method for reducing residual mitochondria according to claim 1, characterized in that: Before aspirating the spindle-containing cell body in step (1) and step (2), a hole is made in the zona pellucida at the first polar body of the donor egg and the recipient egg, and a microscopic glass tube is used to enter through the opening of the zona pellucida to aspirate and discard the first polar body.

3. A spindle nucleocytoplasmic replacement method for reducing residual mitochondria according to claim 1, characterized in that: In step (3), shaking the glass tube relative to the bottom of the dish means shaking the glass tube or moving the bottom of the dish.

4. The method for spindle nucleocytoplasmic replacement for reducing residual mitochondria according to claim 1, characterized in that: The soaking time in step (4) is 20-90 seconds.

5. The method for spindle nucleocytoplasmic replacement for reducing residual mitochondria according to claim 1, characterized in that: It is characterized in that In step (5), the spindle cell body partially emerges from the tube and presses on the recipient egg membrane for 20-600 seconds.

6. A spindle nucleocytoplasmic replacement method for reducing residual mitochondria according to claim 1, characterized in that: In step (5), the opening of the zona pellucida of the recipient egg is adjusted to the opposite side of the oviduct holding the egg. If the egg is held at the 9:00 position, the opening is adjusted to the 3:00 position. Conversely, if the egg is held at the 3:00 position, the opening is adjusted to the 9:00 position. The microscopic tube enters from the opening of the zona pellucida, and the spindle cell body part soaked in the inactivated Sendai virus droplets is ejected from the tube and pressed against the membrane of the recipient egg.

7. A method for spindle nucleocytoplasmic replacement to reduce residual mitochondria according to any one of claims 1 to 6, characterized in that: The egg is a mammalian egg or a human egg.

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