A method for rapid extraction of the nucleus of GV-stage oocytes and pronuclei of zygotes

Abstract

The present invention relates to the field of biotechnology. Specifically, the present invention relates to a method for rapidly extracting the nuclei of germinal vesicle (GV)-stage oocytes and pronuclei of zygotes. This method uses a self-made egg transfer needle to aspirate oocytes or fertilized eggs, and then uses a self-made glass needle to aspirate the nuclei of GV-stage oocytes and pronuclei of zygotes from the nuclear extraction droplet. A relatively large number of nuclei of GV-stage oocytes or pronuclei of fertilized eggs can be obtained in a short time, and expensive equipment and professional technicians are not required. The obtained nuclei of GV-stage oocytes or pronuclei of fertilized eggs have high integrity and low contamination rate, and have great application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a method for rapidly extracting the nucleus of oocytes at the germinal vesicle (GV) stage and the pronucleus at the zygotic stage. Background Art

[0002] The oocyte nucleus and the two pronuclei (2PN) of fertilized eggs are both of extremely important status in reproductive biology and medical research. However, due to the rarity and preciousness of oocyte and fertilized egg samples, especially human samples are even rarer and more precious. And the research of transcriptomics and proteomics requires a certain amount of starting amounts of RNA and protein for detection. At present, the research on the cell nucleus of oocyte and embryo samples lags behind and is vacant.

[0003] Previously, the methods for extracting cell nuclei from other tissues or cell lines mainly separated cell nuclei by grinding / digestion and density gradient centrifugation. During the process of separating cell nuclei by the above methods, because the cell nuclei are relatively sticky and will stick to the pipette tips / wall of the tube and cause loss, at least a certain number level (at least tens of thousands or millions) of cells are required for starting the extraction of cell nuclei. For rare samples such as oocytes and embryos, even the lowest 10,000 cells are difficult to obtain (hundreds of mice are required, with high costs). Therefore, the previously existing methods for nuclear extraction from tissues and cell lines are not applicable to rare samples such as oocytes and embryos.

[0004] In the field of embryos, the existing nuclear transfer technology requires the use of a micromanipulator to extract cell nuclei. However, the technical requirements are high. Non-professional technicians need to practice for a long time (3 - 6 months) and it is difficult to quickly get started and master. Extracting cell nuclei using a micromanipulator requires operating one oocyte at a time and cannot operate dozens or even hundreds of oocytes batchwise simultaneously, which takes a long time. And the micromanipulator belongs to a high-end and precise instrument platform (tens to millions), and has high requirements for the laboratory platform. Not all laboratories have high-end micromanipulation systems.

[0005] Therefore, up to now, there is no method that can obtain a relatively large amount of GV-stage oocyte nuclei or pronuclear samples at the fertilized egg stage in a short 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 nucleus of oocytes at the GV stage and / or the pronucleus at the zygotic stage.

[0007] Specifically, the method includes sucking the GV-stage oocyte nucleus and / or fertilized egg from the zona pellucida digestive solution with a self-made egg transfer needle, and sucking the oocyte nucleus and / or zygotic pronucleus with a self-made glass needle. Among them, the preparation method of the self-made egg transfer needle is as follows: Place 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, heat it while rotating, and when the glass tube becomes soft, quickly stretch it horizontally, cut it with a grinding wheel, and truncate it. Observe and confirm under a stereomicroscope 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 it is used after connecting with a mouth-sucking silica gel tube; the preparation method of the self-made glass needle is as follows: Fix the upper end of a glass capillary with an outer diameter of 1.00 mm and an inner diameter of 0.8 mm on the clamp on the high-precision capillary vertical pulling instrument (PC-100), align it with the heating wire and then tighten it, lift the lower sliding clamp and fix it at the lower end of the capillary. Adjust the heat of the platinum wire solenoid of the pulling needle instrument to 69.2 °C, then press the start button and wait for the glass capillary to be pulled into two required capillary needles. Use a grinding wheel to truncate or a needle forging instrument to obtain a glass needle with a diameter of 30 - 40 μM.

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

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

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

[0011] In one embodiment, the method for digesting the zona pellucida of the oocyte is as follows: Use an egg transfer needle to suck 20 - 25 prepared GV-stage oocytes, blow them into the acidic zona pellucida digestive solution for about 3 - 5 seconds, and it can be seen that the zona pellucida is gradually digested. Immediately transfer the zona pellucida-removed eggs to a cleaning solution drop and blow and wash them several times; then transfer them to a new cleaning drop and blow and wash them several times to fully remove the components of the zona pellucida digestive acid solution.

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

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

[0014] In one embodiment, the method for preparing the acidic zona pellucida digestion solution is as follows: in a biosafety cabinet, add 250 μL of concentrated HCl to 50 mL of the sample washing solution, invert it up and down dozens of times until fully mixed to prepare a 5‰ HCl solution, which is the acidic zona pellucida digestion solution.

[0015] The present invention has the following remarkable progress compared with the prior art:

[0016] ① In terms of the requirements for instrument and equipment, the present invention can be realized only with an ordinary stereomicroscope (usually costing tens of thousands of yuan), and the requirements for the laboratory platform are relatively low. Other related reagents and consumables are relatively common and inexpensive.

[0017] ② In the present invention, a capillary glass needle with an inner diameter of 30 - 40 μM is used. During the preliminary test process, capillary glass needles with various inner diameters (90 μM, 60 μM, 50 μM, 40 μM, 30 μM, 20 μM) were tried. It was found through testing that the capillary glass needle with an inner diameter of 30 - 40 μM is most suitable for nuclear separation. On the one hand, it is reflected in that the glass needle of this caliber can just break the cell membrane by the frictional effect on the oocyte cell membrane without damaging the nuclear membrane (18 - 24 μM); on the other hand, it is convenient for the subsequent nuclear washing process, and the cytoplasm can be washed more cleanly, avoiding contamination of the nuclear sample. The present invention only requires the experimenter to master the most conventional and basic mouse superovulation and egg collection experimental operations. It is possible to use a capillary glass needle with a suitable caliber to separate the nucleus. Compared with the need to use a micromanipulator, the present invention has low requirements for experimental technicians and can be quickly mastered.

[0018] ③ During the process of nuclear separation in this patent, through repeated exploration and verification (testing the effects of different concentrations of 0.05%, 0.01%, 0.005%, and 0.001% respectively), it was confirmed that a low concentration of 0.001% Triton X-100 needs to be added. During the separation process, if the concentration of Triton X-100 is too high, the vitelline membrane will be extremely easy to break, and it may rupture before sample collection, resulting in the loss of embryonic cytoplasm, organelles, or pronuclei; but if the concentration is too low, the membrane permeability is insufficient, and the pronuclei cannot be released from the whole embryo. 0.001% Triton X-100 can increase the cell membrane permeability to a certain extent, making it easier to suck and break the cell membrane to release the nucleus.

[0019] ④ Nuclei are separated from multiple oocytes (20 - 30) simultaneously. On the one hand, when multiple cells are aspirated and blown with a capillary glass tube at the same time, the mutual extrusion between oocytes also contributes to the rupture of the cell membrane and the release of the nucleus; on the other hand, it improves the speed of nuclear separation and reduces the time. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The 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 to the present invention. In the drawings:

[0021] Figure 1 For the nucleus of GV-stage oocytes (left) and the extraction of pronuclei of fertilized eggs (right);

[0022] Figure 2 For immunofluorescence staining of the nucleus of GV-stage oocytes;

[0023] Figure 3 For Western Blot detection of cytoplasmic contamination of the nucleus samples of GV-stage oocytes. DETAILED DESCRIPTION OF THE INVENTION

[0024] The following describes the preferred embodiments of the present invention with reference to the 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.

[0025] Example 1 Method for Extracting the Nucleus of GV-Stage Oocytes

[0026] I. Prepare the capillary needles of glass tubes required for extracting the nucleus of GV-stage oocytes

[0027] 1. Prepare the egg transfer glass needle: Hold both ends of a glass capillary tube about 10 cm long (outer diameter: 1.00 mm, inner diameter: 0.8 mm) horizontally with both hands. Place the middle part of the glass tube in the outer flame of the alcohol lamp, heat it while rotating, and when the glass tube becomes soft, quickly stretch it horizontally, cut it with a grinding wheel, and truncate it. Observe whether the cross-section of the needle tube is flat and smooth under the stereomicroscope. Pull the inner diameter of the egg transfer needle between 90 and 100 μM by the above method, which is slightly larger than the diameter of the zona pellucida of mouse GV oocytes, and use it after connecting with the oral suction silica gel tube.

[0028] 2. Prepare the glass needles for taking the nuclei of GV-stage oocytes and the pronuclei of fertilized eggs:

[0029] 1) Needle pulling: Fix the upper end of the glass capillary tube (outer diameter: 1.00 mm, inner diameter: 0.8 mm) on the clamp on the high-precision capillary vertical pulling instrument (PC-100), align it with the heating wire and then tighten it. Lift the lower sliding clamp and fix it at the lower end of the capillary tube. Adjust the heat of the platinum wire solenoid of the needle pulling instrument to 69.2 °C. Then press the start button and wait for the glass capillary tube to be pulled into two required capillary needles.

[0030] 2) Needle breaking: Use a grinding wheel to truncate or a needle forging instrument to obtain a glass needle with a diameter of about 30 - 40 μM, and place it in the needle box for standby.

[0031] II. Prepare the reagents required for nuclear extraction

[0032] 1. Prepare the 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 250 μL of HSA to 50 mL of PBS solution in the ultra-clean bench, invert it up and down dozens of times until fully mixed, and prepare a sample cleaning solution containing 5‰ HSA. Filter the prepared solution with a 0.22 μM filter, dispense it into 1.5 mL EP tubes, store it in a 4 °C refrigerator, and the use validity period is 1 month.

[0033] 2. Prepare the acidic zona pellucida digestion solution: In the biosafety cabinet, add 250 μL of concentrated HCL to 50 mL of the sample cleaning solution, invert it up and down dozens of times until fully mixed, and prepare a 5‰ HCL solution. It is used for the digestion and removal of the outer zona pellucida of oocytes.

[0034] 3. Preparation of nuclear extraction buffer: Purchase a commercial 10% Triton X-100 solution (Millipore, 648463). Dilute the 10% Triton X-100 solution 10,000 times with the sample cleaning solution. The specific operation is as follows: In a 1.5 mL EP tube, add 990 μL of the sample cleaning solution and 10 μL of the 10% Triton X-100 solution, and mix well to prepare a 0.1% Triton X-100 solution; then take 10 μL of the 0.1% Triton X-100 solution and add it to 990 μL of the sample cleaning solution, and mix well to prepare a nuclear extraction buffer containing 0.001% Triton X-100 and 5‰ HSA.

[0035] III. Superovulation of female mice

[0036] Purchase 3-week-old female ICR strain mice from the company and raise them in a SPF-level barrier environment (free access to food and water, temperature, humidity, dark: light time = 12:12 hours). The mice should be kept in the barrier environment for at least one week before the experiment. Between 1 and 2 pm, intraperitoneally inject 5 IU of pregnant mare serum gonadotropin (PMSG) into 4-week-old ICR female mice to promote the development of oocytes into fully grown germinal vesicle (GV)-stage oocytes.

[0037] IV. Obtaining GV-stage oocytes

[0038] 1. Mouse euthanasia and tissue collection: Euthanize the female mice 44 - 48 hours after injecting the PMSG hormone and obtain intact and clean ovaries (avoiding adhesion of adipose tissue and lipid droplets during this process). Place the dissected ovaries in M2 medium (Sigma, MR-015-D) preheated to 37°C and containing 2.5 μM milrinone (MCE, HY-14252) for temporary storage until egg collection.

[0039] 2. Release of cumulus-oocyte complexes: Make 200 μL droplets with M2 medium containing 2.5 μM milrinone. Place approximately 6 ovaries in the culture droplets. Fix the ovaries with ophthalmic forceps in the left hand and use a 1 mL syringe needle to pierce the follicles on the surface of the ovaries under a stereomicroscope in the right hand to release the cumulus-oocyte complexes (COCs) into the culture medium.

[0040] 3. Removal of granulosa cells around GV oocytes: Repeatedly aspirate and expel the COCs mass using a glass needle with a diameter of approximately 80 μM to remove the granulosa cells around the GV-stage oocytes by mechanical force. Use 200 μL of M2 medium containing 2.5 μM milrinone to make approximately 6 - 7 culture drops in a 3.5 cm culture dish, with each culture drop containing approximately 30 μL of liquid. Add ~2 mL of mineral oil to the culture dish to completely cover the surface of the culture drops. Collect the GV-stage oocytes with the peripheral granulosa cells removed into the prepared culture dish, and place 20 - 25 GV oocytes in each drop.

[0041] The method for obtaining fertilized eggs is similar to the above method.

[0042] V. Nuclear extraction

[0043] 1. Digestion of the zona pellucida of oocytes: Aspirate 20 - 25 prepared GV-stage oocytes with an egg transfer needle and blow them into the acidic zona pellucida digestion solution for about 3 - 5 seconds. It can be seen that the zona pellucida is gradually digested. Immediately transfer the oocytes without zona pellucida to a cleaning solution drop and aspirate and expel them several times; then transfer them to a new cleaning drop and aspirate and expel them several times to fully remove the components of the zona pellucida digestion acid solution.

[0044] 2. Nuclear extraction of GV-stage oocytes: Use an egg transfer needle to blow 20 - 25 oocytes without zona pellucida into the nuclear extraction solution and let it stand for 5 - 10 seconds. Then change to a glass needle with a diameter of approximately 30 - 40 μM, aspirate the oocytes into the glass needle, and then slowly blow them into a sample cleaning solution drop. It can be seen that the oocyte membrane ruptures and the cytoplasm flows out, while the GV oocyte nucleus remains intact. Quickly use a glass needle with a diameter of approximately 30 - 40 μM to pick up the nuclei scattered in the cleaning drop one by one and aspirate and expel them into a 200 μL adsorption tube. One round of operation takes about 2 - 3 minutes and 20 - 25 GV oocyte nuclei can be obtained. If more GV-stage oocyte nuclei need to be collected, the above steps can be repeated until enough nuclear samples are collected. The bright-field image of the extracted oocyte nuclei can be seen Figure 1 (left).

[0045] 3. Digestion of the zona pellucida of fertilized eggs: Aspirate ~10 fertilized egg embryos with an egg transfer needle and blow them into the acidic zona pellucida digestion solution for about 3 - 5 seconds. Then immediately transfer the fertilized eggs without zona pellucida to a cleaning solution drop and aspirate and expel them several times; then transfer them to a new cleaning drop and aspirate and expel them several times.

[0046] 4. Pronucleus extraction of fertilized eggs: Blow the fertilized eggs without zona pellucida into a nuclear extraction solution drop and let it stand for 5 - 10 seconds. Then change to a glass needle with a diameter of approximately 30 - 40 μM, aspirate the fertilized eggs into the glass needle, and then slowly blow them into a sample cleaning solution drop. It can be seen that the cell membrane of the fertilized eggs ruptures and the cytoplasm flows out, while the pronucleus remains intact. Quickly use a glass needle with a diameter of approximately 30 - 40 μM to pick up the pronuclei scattered in the cleaning drop. The bright-field image of some of the extracted pronuclei of fertilized eggs can be seenFigure 1 (right).

[0047] Example 2 Immunofluorescence Detection of the Integrity of GV-stage Oocyte Nucleus Samples

[0048] 2.1 Digestion of the zona pellucida of oocytes: Aspirate 20 - 25 GV-stage mouse oocytes with a transfer pipette and blow them into the acidic zona pellucida digestion solution for about 3 - 5 seconds. Immediately afterwards, transfer the zona pellucida-free oocytes to a droplet of washing solution and blow and wash them several times; then transfer them to a new droplet of washing solution and blow and wash them several times.

[0049] 2.2 Extraction of GV-stage oocyte nuclei: Use a transfer pipette to blow 20 - 25 zona pellucida-free oocytes into a droplet of nuclear extraction solution and let them stand for 5 - 10 seconds. Replace the pipette with a glass needle with a diameter of about 30 - 40 μM, aspirate the oocytes into the glass needle, and then slowly blow them into a droplet of sample washing solution. It can be seen that the oocyte cell membrane ruptures and the cytoplasm flows out, while the GV oocyte nucleus remains intact. Quickly pick up the nuclei scattered in the washing droplet with a glass needle with a diameter of about 30 - 40 μM.

[0050] 2.3 Fixation of GV-stage oocyte nuclei: Make a droplet of 4% paraformaldehyde fixation solution about 10 μL on the bottom of a 35 mm glass-bottom confocal dish, and cover it with mineral oil to prevent the droplet from evaporating. Blow the GV-stage oocyte nuclei picked up by the glass needle into the 4% paraformaldehyde fixation solution droplet. The nuclei gradually settle and adhere to the bottom of the confocal dish and are fixed at room temperature for about 30 minutes.

[0051] 2.4 Staining with Wheat Germ Agglutinin (WGA) and Hoechst33342 and Observation under a Fluorescence Microscope. The specific steps are as follows:

[0052] 1) Preparation of the dye working solution: Dilute the WGA dye (Thermo Fisher, W32466) and Hoechst33342 (Invitrogen, 62249) dyes respectively according to the recommended working concentrations in the instructions using PBS solution to obtain a working solution Mix containing the two dyes.

[0053] 2) Staining: Use a transfer pipette to aspirate the liquid of the 4% paraformaldehyde fixation solution droplet and blow it into the working solution Mix liquid of the dyes, repeating several times until the droplet where the GV oocyte nucleus is located is completely replaced by the working solution Mix containing the two dyes WGA and Hoechst33342. Incubate in the dark at room temperature for 15 - 20 minutes.

[0054] 3) Washing: After staining, use a transfer pipette to aspirate the liquid of the working solution Mix of the dyes and blow it into the PBS liquid, repeating the operation several times until the droplet where the nucleus is located is completely replaced by the PBS liquid.

[0055] 4) Confocal microscopy imaging: Place the confocal culture dish under a confocal fluorescence microscope for observation and data acquisition. The imaging results are as Figure 2 shown. The nuclear membrane is intact without damage, and the morphology of the DNA chromatin region is normal, confirming that the method of extracting nuclei does not cause damage to the nuclei.

[0056] Example 3 Western Blot detection of cytoplasmic contamination in GV-stage oocyte nuclear samples

[0057] 3.1 Digestion of the zona pellucida of oocytes: Aspirate 20 - 25 GV-stage mouse oocytes with an egg transfer pipette and blow them into the acidic zona pellucida digestion solution for about 3 - 5 seconds. Then immediately transfer the zona pellucida-removed eggs to a droplet of washing solution and blow and wash them several times; then transfer them to a new droplet of washing solution and blow and wash them several times.

[0058] 3.2 Extraction of GV-stage oocyte nuclei: Use an egg transfer pipette to blow 20 - 25 zona pellucida-removed oocytes into a droplet of nuclear extraction solution and let them stand for 5 - 10 seconds. Then change to a glass needle with a diameter of about 30 - 40 μM, aspirate the oocytes into the glass needle, and slowly blow them into a droplet of sample washing solution. It can be seen that the oocyte membrane ruptures and the cytoplasm flows out, while the GV oocyte nuclei remain intact. Quickly pick up the nuclei scattered in the washing droplet with a glass needle with a diameter of about 30 - 40 μM and blow them onto the bottom of a 200 μL low-attachment EP tube and place them on ice temporarily. One operation takes about 2 - 3 minutes and about 20 - 25 GV-stage 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 multiple times to obtain more nuclear samples. At the same time, directly collect GV-stage oocyte samples after digesting the zona pellucida as a control.

[0059] The situation of collecting GV-stage oocyte samples is as follows:

[0060] Sample 1: GV-stage oocyte nuclei, N = 160

[0061] Sample 2: GV-stage oocytes, N = 100

[0062] Sample 3: GV-stage oocytes, N = 160

[0063] 3.3 Denaturation of GV oocyte nuclear proteins, the specific steps are as follows:

[0064] 1) After sample collection, add 2×SDS-PAGE protein loading buffer (Beyotime, P0015B) with a volume corresponding to the sample in the 200 μL EP tube and gently vortex for 5 - 10 seconds;

[0065] 2) Place the EP tube in a tabletop centrifuge pre-cooled to 4°C and centrifuge at 10000 rpm for 5 minutes;

[0066] 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.

[0067] 3.4 Western Blot detection of the GV oocyte nucleus is as follows:

[0068] 1) Loading and gel electrophoresis: Add the prepared samples 1, 2, and 3 into the polyacrylamide gel wells, add protein marker to the lanes on both sides of the samples, and then separate the proteins according to their molecular weights by electrophoresis;

[0069] 2) Membrane transfer: Transfer the separated proteins from the gel to the PVDF membrane;

[0070] 3) Blocking: Incubate the PVDF membrane with QuickBlock™ Western Blocking Buffer (Beyotime, P0252-100ml) at room temperature for about 1 hour to prevent non-specific antibody binding;

[0071] 4) Primary antibody incubation: Use Anti-GAPDH antibody (Abcam, ab8245) as the cytoplasmic internal reference and Anti-LaminA+C antibody (Abcam, ab238303) as the nuclear internal reference, and perform corresponding dilutions according to the recommended concentrations in the instructions. Cut the PVDF membrane according to the positions of GAPDH and LaminA+C proteins, and add it to the corresponding antibodies and incubate overnight at 4°C;

[0072] 5) Washing: Wash the PVDF membrane multiple times with 1× washing buffer TBST to remove unbound primary antibody;

[0073] 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;

[0074] 7) Washing: Wash the PVDF membrane multiple times with 1× washing buffer TBST to remove unbound secondary antibody;

[0075] 8) Chemiluminescent color development: Drop the developing solution on the membrane, perform chemiluminescent color development, expose, and observe the target bands. See the Western Blot results in Figure 3 , no GAPDH protein band was detected in the oocyte nucleus sample, indicating that there is no cytoplasmic contamination in the oocyte nucleus sample, confirming that the purity of the extracted cell nucleus by this method is high and there is no cytoplasmic contamination.

[0076] Obviously, those skilled in the art can make various modifications and variations 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 equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. A method for rapidly extracting the nucleus of mouse GV-stage oocytes and / or pronuclei at the zygotic stage, characterized in that, The method includes sucking the GV-stage oocyte nucleus and / or fertilized egg with a self-made egg transfer needle from the zona pellucida digestion solution, and then sucking the oocyte nucleus and / or zygotic pronucleus with a self-made glass needle in the nuclear extraction solution droplet. Among them, the preparation method of the self-made egg transfer needle is as follows: Place 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, heat it while rotating, and when the glass tube becomes soft, quickly stretch it horizontally, cut it with a grinding wheel, and truncate it. Observe and confirm under a stereomicroscope 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. After connecting it with a mouth-sucking silica gel tube, it can be used; the preparation method of the self-made glass needle is as follows: Fix the upper end of a glass capillary with an outer diameter of 1.00 mm and an inner diameter of 0.8 mm on the clip on the high-precision capillary vertical puller of model PC-100, align it with the heating wire and then tighten it, lift the lower sliding clip and fix it at the lower end of the capillary. Adjust the heat of the platinum wire solenoid of the needle puller to 69.2 °C, and then press the start button and wait for the glass capillary to be pulled into two required capillary needles. Use a grinding wheel to truncate or a needle forging instrument to obtain a glass needle with a diameter of 30-40 μM. Among them, The preparation method of the nuclear extraction solution is as follows: Purchase a commercial 10% Triton X-100 solution, and dilute the 10% Triton X-100 solution 10,000 times with a sample cleaning solution. The specific operation is as follows: In a 1.5 mL EP tube, add 990 μL of the sample cleaning solution and 10 μL of the 10% Triton X-100 solution, mix well to prepare a 0.1% Triton X-100 solution; then take 10 μL of the 0.1% Triton X-100 solution and add it to 990 μL of the sample cleaning solution, mix well to prepare a nuclear extraction solution containing 0.001% Triton X-100 and 5‰ HSA; The preparation method of the zona pellucida digestion solution is as follows: In a biosafety cabinet, add 250 μL of concentrated HCl to 50 mL of the sample cleaning solution, invert it up and down dozens of times until fully mixed to prepare a 5‰ HCl solution, which is the acidic zona pellucida digestion solution.

2. The method according to claim 1, wherein The method also includes digesting the zona pellucida of the oocyte and / or digesting the zona pellucida of the fertilized egg.

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

4. The method according to claim 1, wherein The method for extracting the pronucleus of the fertilized egg is as follows: Blow the zona pellucida-removed fertilized egg into the nuclear extraction solution droplet, let it stand for 5-10 seconds, change to a glass needle with a diameter of about 30-40 μM, suck the fertilized egg into the glass needle, and then slowly blow it into a sample cleaning solution droplet. Quickly use a glass needle with a diameter of about 30-40 μM to pick up the pronuclei scattered in the cleaning droplet.

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

6. The method according to claim 2, wherein, The method for digesting the zona pellucida of fertilized eggs is as follows: aspirate ~10 fertilized egg embryos with an egg transfer needle, blow them into the acidic zona pellucida digestion solution for about 3 - 5 seconds. Subsequently, immediately transfer the zona pellucida-removed fertilized eggs to a droplet of washing solution and blow and wash them several times repeatedly; then transfer them to a new droplet of washing solution and blow and wash them several times repeatedly.

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