Method for rapidly extracting oocyte nucleus in GV stage and pronucleus in zygotic stage

Abstract

The invention relates to the technical field of biology, in particular to a method for rapidly extracting an oocyte nucleus in a GV (germinal vesicles) stage and a pronucleus in a zygotic stage. According to the method, a self-made egg transfer needle is adopted to suck oocytes or fertilized eggs, then oocyte nucleuses and pronucleus in the zygote stage are sucked from nucleus extraction liquid drops through a self-made glass needle, a large number of GV-stage oocyte nucleuses or pronucleus samples in the fertilized egg stage can be obtained within a short time, expensive equipment and professional technicians are not needed, and the method is simple and convenient to operate. The obtained GV-stage oocyte nucleus or pronucleus sample in the fertilized egg stage is high in integrity and low in pollution rate, and has great application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for rapidly extracting oocyte nuclei at the GV (germinalvesicle) stage and pronuclei at the zygote stage. Background Art

[0002] Oocyte nuclei and fertilized egg binuclei (2PN) are extremely important in reproductive biology and medical research. However, due to the rarity and preciousness of oocyte and fertilized egg samples, especially human samples, which are even rarer and more precious, transcriptomic and proteomic research requires a certain amount of RNA and protein for detection. Currently, the research on the nuclei of oocyte and embryo samples is lagging behind and vacant.

[0003] In the past, methods for extracting nuclei from other tissues or cell lines mainly separated nuclei by grinding / digestion and density gradient centrifugation. In the process of separating nuclei using the above methods, because nuclei are sticky and will stick to the gun tip / tube wall and cause loss, at least a certain number of cells (at least tens of thousands or millions) are required to start the extraction of nuclei. For rare samples such as oocytes and embryos, even the minimum of 10,000 cells is difficult to obtain (requiring hundreds of mice and high cost). Therefore, the previous nuclear extraction methods for tissues and cell lines are not suitable for rare samples such as oocytes and embryos.

[0004] In the embryo field, the existing cell nucleus transplantation technology requires the use of a micromanipulator to extract the cell nucleus, but the technical requirements are high, and non-professional technicians need to practice for a long time (3-6 months), and it is difficult to quickly get started and master it. Using a micromanipulator to extract the cell nucleus requires operating on each oocyte one by one, and it is impossible to operate on dozens or even hundreds of oocytes at the same time in batches, which takes a long time. In addition, the micromanipulator is a high-end and precise instrument platform (tens to millions), which has high requirements for the laboratory platform. Not all laboratories have high-end micromanipulation systems.

[0005] Therefore, up to now, there is no method to obtain a large number of GV stage oocyte nuclei or fertilized egg pronuclear samples in a short period of time. Summary of the invention

[0006] In order to overcome the defects of the prior art, the present invention provides a method for rapidly extracting the GV stage oocyte nucleus and / or the zygote stage pronucleus.

[0007] Specifically, the method comprises using a homemade egg transfer needle to absorb the GV stage oocyte nucleus and / or fertilized egg from the zona pellucida digestive fluid, and then using a homemade glass needle to absorb the oocyte nucleus and / or the zygote stage pronucleus, wherein the preparation method of the homemade egg transfer needle is: placing the middle part of a glass capillary with an outer diameter of 1.00 mm, an inner diameter of 0.8 mm, and a length of about 10 cm in the outer flame of an alcohol lamp, heating while rotating, and when the glass tube becomes soft, rapidly stretching it horizontally, cutting it with a grinding wheel, and truncating it, observing under a stereomicroscope to confirm that the cross section of the needle tube is flush and smooth, the inner diameter of the drawn egg transfer needle is slightly larger than the diameter of the GV oocyte zona pellucida, and it is connected to the oral suction silicone tube for use; the preparation method of the homemade glass needle is: placing an outer diameter of 1.00 mm, an inner diameter of 0.8 mm, and a length of about 10 cm in the outer flame of an alcohol lamp, heating it while rotating, and when the glass tube becomes soft, rapidly stretching it horizontally, cutting it with a grinding wheel, and truncating it, and observing under a stereomicroscope to confirm that the cross section of the needle tube is flat and smooth, the inner diameter of the drawn egg transfer needle is slightly larger than the diameter of the GV oocyte zona pellucida, and it is used after being connected to the oral suction silicone tube; Fix the upper end of the 300mm glass capillary on the clamp on the high-precision capillary vertical puller (PC-100), align it with the heating wire and tighten it, lift the sliding clamp below and fix it on the lower end of the capillary, adjust the temperature of the platinum wire solenoid of the needle puller to 69.2℃, then press the start button, wait for the glass capillary to be pulled into two required capillary needles, use a grinding wheel to cut or a needle forging instrument to obtain a glass needle with a diameter of 30~40μM.

[0008] In one embodiment, the method further comprises digesting the zona pellucida of the oocyte and / or digesting the zona pellucida of the fertilized egg.

[0009] In one embodiment, the method for extracting the nucleus of the GV stage oocyte is to use an egg transfer needle to blow 20-25 oocytes with the zona pellucida removed into a nuclear extraction solution, let it stand for 5-10 seconds, change to a glass needle with a caliber of about 30~40μM, suck the oocyte into the glass needle, and then slowly blow it into a drop of sample cleaning solution, quickly use a glass needle with a caliber of about 30~40μM to pick up the cell nuclei scattered in the cleaning drop in turn, and blow them into a 200μL adsorption tube.

[0010] In one embodiment, the method for extracting the pronucleus of a fertilized egg is to blow the fertilized egg with the zona pellucida removed into a droplet of nuclear extraction liquid, let it stand for 5-10 seconds, replace it with a glass needle with a caliber of about 30-40 μM, suck the fertilized egg into the glass needle, and then slowly blow it into a droplet of sample cleaning liquid, and quickly pick up the pronuclei scattered in the cleaning droplet with a glass needle with a caliber of about 30-40 μM.

[0011] In one embodiment, the method for digesting the zona pellucida of oocytes is as follows: 20-25 prepared GV stage oocytes are sucked with an egg transfer needle, and blown into an acidic zona pellucida digestion solution for about 3-5 seconds, during which the zona pellucida is gradually digested. The eggs with the zona pellucida removed are immediately transferred to a cleaning solution droplet, and the solution is repeatedly blown and washed several times; the eggs are then transferred to a new cleaning solution droplet, and the solution is repeatedly blown and washed several times to fully remove the zona pellucida digestion acid component.

[0012] In one embodiment, the method for digesting the zona pellucida of fertilized eggs is: use an egg transfer needle to suck up to 10 fertilized egg embryos, blow them into acidic zona pellucida digestion solution for about 3-5 seconds, and then immediately transfer the fertilized eggs with the zona pellucida removed to a cleaning solution droplet, and blow and wash them repeatedly several times; then transfer them to a new cleaning droplet, and blow and wash them repeatedly several times.

[0013] In one embodiment, the method for preparing the nuclear extract is: purchase a commercial 10% Triton X-100 solution (Millipore, 648463), and dilute the 10% Triton X-100 solution 10,000 times with a sample cleaning solution. The specific operation is: in a 1.5mL EP tube, add 990μL of sample cleaning solution and 10μL of 10% Triton X-100 solution, mix thoroughly, and prepare a 0.1% Triton X-100 solution; then take 10μL of 0.1% Triton X-100 solution and add 990μL of sample cleaning solution, mix thoroughly, and prepare a nuclear extract containing 0.001% Triton X-100 and 5‰ HSA.

[0014] In one embodiment, the preparation method of acidic zona pellucida digestion solution is: in a biological safety cabinet, add 250 μL of concentrated HCL to 50 mL of sample cleaning solution, invert it upside down dozens of times to mix it thoroughly, and prepare a 5‰ HCL solution, which is the acidic zona pellucida digestion solution.

[0015] Compared with the prior art, the present invention has the following significant improvements: ① In terms of instrument and equipment requirements, the present invention only requires an ordinary stereoscope (usually priced at tens of thousands of yuan) to be implemented, and the requirements for the laboratory platform are relatively low. Other reagents and consumables involved are relatively common and low in price.

[0016] ② In the present invention, a capillary glass needle with an inner diameter of 30~40μM is used. In the early test process, capillary glass needles with various inner diameters (90μM, 60μM, 50μM, 40μM, 30μM, 20μM) were tried. After testing, it was found that the glass needle with an inner diameter of 30~40μM is most suitable for the separation of cell nuclei. On the one hand, it is reflected in that the glass needle of this caliber can just break the cell membrane by friction with the oocyte membrane without damaging the nuclear membrane (18~24μM); on the other hand, it is convenient for the subsequent cell nucleus cleaning process, and the cytoplasm is cleaned more cleanly to avoid contamination of the cell nucleus sample. The present invention only requires the experimenter to master the most conventional and basic mouse ovulation promotion and egg retrieval experimental operations. The cell nucleus can be separated using a capillary glass needle with a suitable caliber. Compared with the need to use a micromanipulator, the present invention does not require high requirements for experimental technicians and can be quickly started.

[0017] ③ In the process of cell nucleus separation, this patent has been repeatedly explored and verified (the effects of different concentrations of 0.05%, 0.01%, 0.005%, and 0.001% were tested respectively), and it was confirmed that a low concentration of 0.001% Triton X-100 should be added. During the separation process, if the concentration of Triton X-100 is too high, the yolk membrane will be easily broken, and it may rupture before the sample is collected, causing the loss of embryonic cytoplasm, organelles or pronuclei; but if the concentration is too low, the membrane permeability is insufficient, and the pronucleus cannot be released from the entire embryo. 0.001% Triton X-100 can increase the permeability of the cell membrane to a certain extent, making it easier to absorb the cell membrane and release the cell nucleus.

[0018] ④ Nuclear separation is performed on multiple oocytes (20-30) at the same time. On the one hand, multiple cells are blown and sucked at the same time using a capillary glass tube. The squeezing between the oocytes also helps to rupture the cell membrane and release the nucleus; on the other hand, the speed of nuclear separation is increased and the time is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 Extraction of GV stage oocyte nucleus (left) and fertilized egg pronucleus (right); Figure 2 This is the immunofluorescence staining of GV stage oocyte nuclei; Figure 3 Western Blot was used to detect cytoplasmic contamination in GV stage oocyte nuclear samples. DETAILED DESCRIPTION

[0020] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0021] Example 1 Method for extracting GV stage oocyte nuclei 1. Prepare the glass capillary needle for GV stage oocyte nuclear extraction

[0022] 1. Prepare the egg transfer glass needle: Hold the two ends of a glass capillary tube (outer diameter: 1.00 mm, inner diameter: 0.8 mm) of about 10 cm horizontally with both hands, place the middle of the glass tube in the outer flame of the alcohol lamp, and heat it while rotating. When the glass tube becomes soft, quickly stretch it horizontally, cut it with a grinding wheel, and cut it off. Observe whether the cross section of the needle tube is flat and smooth under a stereomicroscope. The inner diameter of the egg transfer needle drawn by the above method is between 90 and 100 μM, which is slightly larger than the diameter of the zona pellucida of the mouse GV oocyte, and use it after connecting it to the oral suction silicone tube.

[0023] 2. Prepare the glass needle for taking the GV stage oocyte nucleus and fertilized egg pronucleus: 1) Needle pulling: Fix the upper end of the glass capillary (outer diameter: 1.00mm, inner diameter: 0.8mm) on the clip on the high-precision capillary vertical puller (PC-100), align it with the heating wire and tighten it. Lift the sliding clamp below and fix it on the lower end of the capillary. Adjust the temperature of the platinum wire solenoid of the needle puller to 69.2℃. Then press the start button and wait for the glass capillary to be pulled into two required capillary needles.

[0024] 2) Broken needles: Use a grinding wheel to cut or a needle forging instrument to obtain glass needles with a diameter of about 30~40μM and place them in a needle box for use.

[0025] 2. Prepare the reagents required for nuclear extraction 1. Prepare sample cleaning solution: To prevent the sample from adhering to the bottom of the operating dish, add a certain concentration of human serum albumin (HSA) to the PBS solution. The specific operation is to add 250μL HSA to 50mL PBS solution in a clean bench, turn it upside down dozens of times to mix it thoroughly, and prepare a sample cleaning solution containing 5‰ HSA. Filter the prepared solution with a 0.22 μM filter and dispense it into 1.5mL EP tubes. Store it in a refrigerator at 4℃. The shelf life is 1 month.

[0026] 2. Prepare acidic zona pellucida digestion solution: In a biosafety cabinet, add 250 μL of concentrated HCL to 50 mL of sample washing solution, invert it several dozen times to mix thoroughly, and prepare a 5‰ HCL solution. This is used to digest and remove the outer zona pellucida of the oocyte.

[0027] 3. Prepare nuclear extract: Purchase commercial 10% Triton X-100 solution (Millipore, 648463). Use sample cleaning solution to dilute the 10% Triton X-100 solution 10,000 times. The specific operation is as follows: In a 1.5mL EP tube, add 990μL sample cleaning solution and 10μL 10% Triton X-100 solution, mix well, and prepare a 0.1% Triton X-100 solution; then take 10μL 0.1% Triton X-100 solution and add 990μL sample cleaning solution, mix well, and prepare a nuclear extract containing 0.001% Triton X-100 and 5‰ HSA.

[0028] 3. Superovulation of female mice 3-week-old ICR female mice were purchased from the company and kept in an SPF-level barrier environment (free access to food and water, temperature, humidity, dark: light time = 12:12 hours). The mice were allowed to stabilize in the barrier environment for at least one week before being used in the experiment. At 1-2 pm, 5 IU of pregnant mare serum gonadotropin (PMSG) was injected intraperitoneally into ~4-week-old ICR female mice to promote the development of oocytes into fully grown GV-stage oocytes.

[0029] IV. Obtaining GV-stage Oocytes 1. Mouse killing and sampling: Female mice were killed 44-48 hours after PMSG injection to obtain complete and clean ovaries (this process avoids sticking to adipose tissue and lipid droplets). The ovaries obtained by dissection were placed in M2 medium (Sigma, MR-015-D) preheated at 37°C and containing 2.5μM milrinone (MCE, HY-14252) for the time being, waiting for egg retrieval.

[0030] 2. Release the cumulus-oocyte complexes: Use M2 culture medium containing 2.5 μM milrinone to make a 200 μL droplet, place about 6 ovaries in the culture droplet, fix the ovaries with ophthalmic forceps in the left hand, and use a 1 mL syringe needle in the right hand to scratch the follicles on the surface of the ovary under a stereoscope to release the cumulus-oocyte complexes (COCs) into the culture medium liquid.

[0031] 3. Remove the granulosa cells around the GV oocytes: Use a glass needle with a diameter of about 80μM to repeatedly blow and suck the COCs clumps to remove the granulosa cells around the GV oocytes by mechanical force. Use 200μL of M2 medium containing 2.5μM milrinone to make about 6-7 culture drops in a 3.5cm culture dish, each culture drop has about 30μL of liquid, and add ~2mL of mineral oil to the culture dish until the surface of the culture drop is completely covered. Collect the GV oocytes with peripheral granulosa cells removed into the prepared culture dish, and place 20~25 GV oocytes in each drop.

[0032] The method for obtaining fertilized eggs is similar to the above method.

[0033] 5. Nuclear Extraction 1. Digestion of zona pellucida of oocytes: Use an egg transfer needle to suck up 20-25 prepared GV stage oocytes, blow them into the acid zona pellucida digestion solution for about 3-5 seconds, and the zona pellucida is gradually digested. Immediately transfer the zona pellucida-free oocytes to a drop of cleaning solution, and blow and wash them repeatedly several times; then transfer them to a new drop of cleaning solution, and blow and wash them repeatedly several times to fully remove the zona pellucida digestion acid components.

[0034] 2. Extraction of GV stage oocyte nuclei: Use an egg transfer needle to blow 20-25 oocytes with the zona pellucida removed into the nuclear extraction solution, let it stand for 5-10 seconds, change to a glass needle with a caliber of about 30~40μM, suck the oocytes into the glass needle, and then slowly blow them into the sample cleaning solution droplet. It can be seen that the oocyte membrane ruptures, the cytoplasm flows out, and the GV oocyte nucleus remains intact. Use a glass needle with a caliber of about 30~40μM to quickly pick up the cell nuclei scattered in the cleaning droplet one by one, and blow them into a 200μL adsorption tube. It takes about 2~3 minutes to perform one round of operation, and 20~25 GV oocyte nuclei can be obtained. If you need to collect more GV stage oocyte nuclei, repeat the above steps until a sufficient number of cell nucleus samples are collected. The bright field image of the extracted oocyte nucleus can be seen Figure 1 (Left).

[0035] 3. Zona pellucida digestion of fertilized eggs: Use an egg transfer needle to suck up to 10 fertilized egg embryos, blow them into the acidic zona pellucida digestion solution for about 3-5 seconds, and then immediately transfer the fertilized eggs with the zona pellucida removed to the cleaning solution droplets, and blow them repeatedly for several times; then transfer them to new cleaning drops and blow them repeatedly for several times.

[0036] 4. Extraction of fertilized egg pronuclei: Blow the fertilized egg with the zona pellucida removed into the nuclear extraction droplet, let it stand for 5-10 seconds, change to a glass needle with a diameter of about 30~40μM, suck the fertilized egg into the glass needle, and then slowly blow it into the sample cleaning droplet. It can be seen that the cell membrane of the fertilized egg ruptures, the cytoplasm flows out, and the pronucleus remains intact. Quickly use a glass needle with a diameter of about 30~40μM to pick up the pronuclei scattered in the cleaning droplet. The bright field image of the extracted pronuclei of some fertilized eggs can be seen Figure 1 (right).

[0037] Example 2 Immunofluorescence detection of GV stage oocyte nuclear sample integrity 2.1 Oocyte zona pellucida digestion: Use an egg transfer needle to suck up 20-25 GV stage mouse oocytes, blow them into the acidic zona pellucida digestion solution for about 3-5 seconds, and then immediately transfer the eggs without zona pellucida to a drop of cleaning solution and blow them repeatedly several times; then transfer them to a new drop of cleaning solution and blow them repeatedly several times.

[0038] 2.2 Extraction of GV oocyte nuclei: Use an oocyte transfer needle to blow 20-25 oocytes with the zona pellucida removed into the nuclear extraction droplet, let it stand for 5-10 seconds, change to a glass needle with a caliber of about 30~40μM, suck the oocyte into the glass needle, and then slowly blow it into the sample cleaning droplet. It can be seen that the oocyte membrane is broken, the cytoplasm flows out, and the GV oocyte nucleus remains intact. Quickly use a glass needle with a caliber of about 30~40μM to pick up the cell nuclei scattered in the cleaning droplet.

[0039] 2.3 Fixation of GV-stage oocyte nuclei: Make a 10 μL drop of 4% paraformaldehyde fixative solution on the bottom of a 35 mm glass-bottomed confocal culture dish, and cover it with mineral oil to prevent the drop from evaporating. Blow the GV-stage oocyte nucleus picked up by the glass needle into the 4% paraformaldehyde fixative drop, and the nucleus will gradually settle and adhere to the bottom of the confocal culture dish, and fix it at room temperature for ~30 minutes.

[0040] 2.4 Wheat germ agglutinin (WGA) and Hoechst33342 staining and fluorescence microscopy observation, the specific steps are as follows: 1) Prepare dye working solution: Use PBS solution to dilute WGA dye (Thermo Fisher, W32466) and Hoechst33342 (Invitrogen, 62249) dye according to the working concentrations recommended in the instructions to obtain a working solution Mix containing the two dyes.

[0041] 2) Staining: Use an egg transfer needle to absorb the 4% paraformaldehyde fixative droplet and blow in the dye working solution Mix liquid, repeat several times until the droplet containing the GV oocyte nucleus is completely replaced with the working solution Mix containing WGA and Hoechst33342. Incubate at room temperature for 15-20 minutes in the dark.

[0042] 3) Washing: After staining, use an egg transfer needle to absorb the working solution Mix liquid of the dye and blow in PBS liquid. Repeat the operation several times until the droplet containing the cell nucleus is completely replaced by PBS liquid.

[0043] 4) Confocal microscope photography: Place the confocal culture dish under a confocal fluorescence microscope for observation and data collection. Figure 2 As shown, the cell nuclear membrane is intact and undamaged, and the morphology of the DNA chromatin region is normal, which proves that this method of extracting the cell nucleus will not cause damage to the cell nucleus.

[0044] Example 3 Western Blot detection of cytoplasmic contamination in GV stage oocyte nuclear samples 3.1 Oocyte zona pellucida digestion: Use an egg transfer needle to suck up 20-25 GV-stage mouse oocytes, blow them into the acidic zona pellucida digestion solution for about 3-5 seconds, and then immediately transfer the eggs without zona pellucida to a drop of cleaning solution and blow them repeatedly several times; then transfer them to a new drop of cleaning solution and blow them repeatedly several times.

[0045] 3.2 Extraction of GV oocyte nuclei: Use an egg transfer needle to blow 20-25 oocytes with the zona pellucida removed into the nuclear extraction droplet, let it stand for 5-10 seconds, change to a glass needle with a caliber of about 30-40 μM, suck the oocyte into the glass needle, and then slowly blow it into the sample cleaning droplet. It can be seen that the oocyte membrane is broken, the cytoplasm flows out, and the GV oocyte nucleus remains intact. Quickly use a glass needle with a caliber of about 30-40 μM to pick up the cell nuclei scattered in the cleaning droplet, blow them to the bottom of a 200 μL low-adsorption EP tube, and temporarily place them on ice. One operation takes about 2-3 minutes, and about 20-25 GV oocyte nuclei can be obtained. According to previous studies, at least 100 cells are required for Western blot detection of oocyte samples. Therefore, repeat the above operation several times to obtain more cell nucleus samples. At the same time, GV oocyte samples after digestion of the zona pellucida were directly collected as controls.

[0046] GV stage oocyte samples were collected as follows: Sample 1: GV stage oocyte nuclei, N=160, Sample 2: GV stage oocytes, N=100, Sample 3: GV stage oocytes, N=160 3.3 GV oocyte nuclear protein denaturation, the specific steps are as follows: 1) After the sample is collected, add 2×SDS-PAGE protein loading buffer (Biyuntian, P0015B) corresponding to the volume of the sample in the EP tube to the 200μL EP tube, and gently vortex for 5-10 seconds; 2) Place the EP tube in a 4°C precooled benchtop centrifuge and centrifuge at 10,000 rpm for 5 minutes; 3) Then, place the EP tube in a PCR instrument and heat it at 98°C for 5 minutes to fully denature the proteins in the cell nucleus.

[0047] 3.4 Western Blot detection of GV oocyte nucleus, the specific steps are as follows: 1) Sample loading and gel electrophoresis: Add the prepared samples 1, 2, and 3 to the wells of the polyacrylamide gel, add protein markers to the lanes on both sides of the samples, and then separate the proteins according to their molecular weight by electrophoresis; 2) Transfer: transfer the separated proteins from the gel to the PVDF membrane; 3) Blocking: Incubate the PVDF membrane with QuickBlock™ Western Blocking Solution (Biyuntian, P0252-100ml) at room temperature for about 1 hour to prevent nonspecific antibody binding; 4) Primary antibody incubation: Use Anti-GAPDH antibody (Abcam, ab8245) as a cytoplasmic internal reference and Anti-LaminA+C antibody (Abcam, ab238303) as a nuclear internal reference, and dilute accordingly according to the concentration recommended in the instructions. Cut the PVDF membrane according to the location of GAPDH and LaminA+C proteins, add the corresponding antibodies and incubate overnight at 4°C; 5) Washing: Wash the PVDF membrane several times with 1× washing buffer TBST to remove unbound primary antibody; 6) Secondary antibody incubation: Incubate the membrane with the host-derived secondary antibody (HRP-labeled) corresponding to the primary antibody, and incubate on a shaker at room temperature for 1 hour; 7) Washing: Wash the PVDF membrane several times with 1× washing buffer (TBST) to remove unbound secondary antibody; 8) Chemiluminescent color development: Add developer to the membrane, perform chemiluminescent color development, expose, and observe the target band. For Western Blot results, see Figure 3 No GAPDH protein band was detected in the oocyte nuclear sample, indicating that there was no cytoplasmic contamination in the oocyte nuclear sample, confirming that the purity of the cell nucleus extracted by this method was high and there was no cytoplasmic contamination.

[0048] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for rapidly extracting GV stage oocyte nuclei and / or zygote stage pronuclei, characterized in that: The method comprises the steps of using a homemade egg transfer needle to absorb the nucleus of a GV stage oocyte and / or a fertilized egg from a zona pellucida digestive fluid, and then using a homemade glass needle to absorb the nucleus of an oocyte and / or a zygote stage pronucleus, wherein the preparation method of the homemade egg transfer needle is as follows: placing the middle of a glass capillary tube with an outer diameter of 1.00 mm, an inner diameter of 0.8 mm, and a length of about 10 cm in an outer flame of an alcohol lamp, heating while rotating, and when the glass tube becomes soft, rapidly stretching it horizontally, cutting it with a grinding wheel, and truncating it, observing under a stereomicroscope to confirm that the cross section of the needle tube is flush and smooth, the inner diameter of the drawn egg transfer needle is slightly larger than the diameter of the zona pellucida of the GV oocyte, and connecting it to a mouth suction silicone tube for use; the preparation method of the homemade glass needle is as follows: placing a glass capillary tube with an outer diameter of 1.00 mm, an inner diameter of 0.8 mm, and a length of about 10 cm in an outer flame of an alcohol lamp, heating it while rotating, and when the glass tube becomes soft, rapidly stretching it horizontally, cutting it with a grinding wheel, and truncating it, and observing under a stereomicroscope to confirm that the cross section of the needle tube is flat and smooth, the inner diameter of the drawn egg transfer needle is slightly larger than the diameter of the zona pellucida of the GV oocyte, and connecting it to a mouth suction silicone tube for use; Fix the upper end of the 300mm glass capillary on the clamp on the high-precision capillary vertical puller (PC-100), align it with the heating wire and tighten it, lift the sliding clamp below and fix it on the lower end of the capillary, adjust the temperature of the platinum wire solenoid of the needle puller to 69.2℃, then press the start button, wait for the glass capillary to be pulled into two required capillary needles, use a grinding wheel to cut or a needle forging instrument to obtain a glass needle with a diameter of 30~40μM.

2. The method according to claim 1, characterized in that The method further comprises digesting the zona pellucida of the oocyte and / or digesting the zona pellucida of the fertilized egg.

3. The method according to claim 1, characterized in that The method for extracting the nucleus of the GV stage oocyte is to use an egg transfer needle to blow 20-25 oocytes with the zona pellucida removed into the nuclear extraction solution, let it stand for 5-10 seconds, change to a glass needle with a diameter of about 30~40μM, suck the oocyte into the glass needle, and then slowly blow it into the sample cleaning solution droplet, quickly use a glass needle with a diameter of about 30~40μM to pick up the cell nuclei scattered in the cleaning droplet one by one, and blow them into a 200μL adsorption tube.

4. The method according to claim 1, characterized in that The method for extracting the pronucleus of the fertilized egg is to blow the fertilized egg with the zona pellucida removed into a drop of nuclear extraction liquid, let it stand for 5-10 seconds, change to a glass needle with a diameter of about 30~40μM, suck the fertilized egg into the glass needle, and then slowly blow it into the sample cleaning drop, and quickly use a glass needle with a diameter of about 30~40μM to pick up the pronuclei scattered in the cleaning drop.

5. The method according to claim 2, characterized in that The method for digesting the zona pellucida of oocytes is as follows: use an egg transfer needle to suck up 20-25 prepared GV stage oocytes, blow them into the acidic zona pellucida digestion solution for about 3-5 seconds, and the zona pellucida can be seen to be gradually digested. Immediately transfer the eggs with the zona pellucida removed to a drop of cleaning solution, and blow and wash them repeatedly several times; then transfer them to a new drop of cleaning solution, and blow and wash them repeatedly several times to fully remove the zona pellucida digestion acid components.

6. The method according to claim 2, characterized in that The method for digesting the zona pellucida of fertilized eggs is as follows: use an egg transfer needle to suck up to 10 fertilized egg embryos, blow them into the acidic zona pellucida digestion solution for about 3-5 seconds, and then immediately transfer the fertilized eggs with the zona pellucida removed to a drop of cleaning solution, and blow and wash them repeatedly several times; then transfer them to a new drop of cleaning solution and blow and wash them repeatedly several times.

7. The method according to claim 2 or 3, characterized in that: The preparation method of nuclear extract is as follows: buy commercial 10% Triton X-100 solution (Millipore, 648463), and dilute the 10% Triton X-100 solution 10,000 times with sample cleaning solution. The specific operation is as follows: add 990μL sample cleaning solution and 10μL 10% Triton X-100 solution to a 1.5mL EP tube, mix thoroughly, and prepare a 0.1% Triton X-100 solution; then take 10μL 0.1% Triton X-100 solution and add 990μL sample cleaning solution, mix thoroughly, and prepare a nuclear extract containing 0.001% Triton X-100 and 5‰ HSA.

8. The method according to claim 6 or 7, characterized in that The preparation method of acidic zona pellucida digestion solution is as follows: in a biological safety cabinet, add 250 μL of concentrated HCL to 50 mL of sample cleaning solution, invert it upside down dozens of times to mix it thoroughly, and prepare a 5‰ HCL solution, which is the acidic zona pellucida digestion solution.

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