CN102127521B - Method for removing mouse oocyte nuclei by adopting zona pellucida solution cavity method - Google Patents
Method for removing mouse oocyte nuclei by adopting zona pellucida solution cavity method Download PDFInfo
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Abstract
The invention discloses a method for removing mouse oocyte nuclei by adopting a zona pellucida solution cavity method. The method comprises the following steps of: performing central slight dripping and peripheral slight dripping in a plastic utensil; making an acid Tyrode's solution react with a zona pellucida under a microscope; forming a pore on the zona pellucida 1-6 seconds later by dissolving till the zona pellucida becomes thin and soft and cytoplasm protrudes outwards; absorbing a first polar body and surrounding cytoplasm out; releasing an oocyte; and denucleating and observing integrality by adopting trypan blue dyeing, wherein the denucleation success rate is over 99.1 percent. In the preparation method, used equipment is simple and the denucleation operation can be completed under a micromanipulator; a reagent has the advantages of simple components, easiness of preparation, low cost, low difficulty of operation, easiness of comprehension, soft denucleation operation, small mechanical damage, quick denucleation and high efficiency; the average denucleation time of each oocyte is 1-6 seconds, the survival rate of each denucleated oocyte is over 97 percent and the success rate of denucleation is over 99 percent; and the method is suitable for researching mouse nucleus transplantation in a Piezo-free laboratory and early events relevant to embryonic development.
Description
Technical field
The present invention relates to a kind of method of removing oocyte of mouse nuclear, specifically a kind of method that adopts zona pellucida solution cavity method to remove oocyte of mouse nuclear.
Background technology
Development along with biotechnology, the mammal body-cell neucleus transplanting technology is as an irreplaceable technology platform producing the Disciplinary Frontiers such as transgenic animal, making galactophore biological reactor, therapeutic cloning, although obtained huge progress sheep, ox, pig and mouse, but because the whole technical system of body-cell neucleus transplanting relates to numerous links, and links inefficiency, finally cause the overall efficiency of somatic cell clone technique lower, also do not surpass 10%.Making up reconstituted embryo by micrurgy is the classical way that generally adopts in the nuclear transplantation research, wherein the stoning of ovocyte is one of important step in the nuclear transplantation, also be the critical limitation factor that success is finished nuclear transplantation, be directly connected to the success or failure of nuclear transplantation experiment.A kind of fast and accurately pitting method will can improve the whole efficiency of nuclear transfer technology undoubtedly.The mouse conduct is the laboratory animal of commonly using the most, yet the characteristic of oocyte of mouse and domestic animal (ox, sheep and pig) are widely different.The diameter less of oocyte of mouse, zona pellucida is thin and soft, and this specific character increases oocyte of mouse difficulty when the micrurgy stoning.
In traditional nuclear transfer technology, mainly be to obtain recipient cytoplasm by the chromatin that the micrurgy mechanicalness is removed in the ovocyte, but exist expend time in long, to shortcomings such as cell injury are large, and the relative position of the 1st polar body and nuclear easily changes, so that the stoning rate is lower.At present, oocyte of mouse stoning over-borrowing helps the Piezo device, and the stoning rate is up to 99%, but this method needs expensive plant and instrument, simultaneously the researcher is had very high technical requirements, and most of laboratories are difficult for realizing.And used needle tubing can not the sucking-off polar body, and the electricity that the existence of polar body will affect donorcells and cytosome merges.In addition, also have chemical stoning method, zona pellucida perforation extruding stoning method, the zona pellucida ground changes flat mouth pin two step stoning methods etc.Different pitting methods has relative merits separately, and is different on operating time, stoning success ratio, cost cost and degree of injury.The two-step approach complex operation step, the length that expends time in has a negative impact to ovocyte.The perforation extrusion process very easily causes cytomorphosis, and kytoplasm dissipates.And the pitting method technical requirements that has is very high, and method is difficult for grasping unfavorable popularization.
Summary of the invention
The present invention is mainly for the problems referred to above, and provides a kind of easy and simple to handle, and elapsed time is few, and the damage that cell membrane causes is little, and is with low cost, the method that the stoning success ratio is high.
The present invention is for addressing the above problem, and the technical scheme that adopts is:
Step 1: choose basic acid Tai Shi solution, for subsequent use;
Step 2: the improvement of acid Tai Shi solution: utilizing concentration is the pH value of 36% HCl adjustment of acidity Tai Shi solution, and the millipore filter of rear usefulness 0.22 μ m filters, and is frozen, for subsequent use in-20 ℃;
Step 3: damage droplet preparation: be in the plastic ware of 35mm in specification, equalization at the bottom of the ware is divided into four zones, in each zone, make the micrurgy liquid droplet of 1 30 μ l, title center droplet; Drip micrurgy liquid and 1 acid Tai Shi solution droplet of 2 20 μ l in each center droplet periphery, form peripheral droplet, rear top one deck mineral oil that covers;
Step 4: ovocyte places droplet: the ovocyte of getting the sexually matured mouse M II phase places the peripheral micrurgy liquid droplet of plastic ware, under stereoscopic microscope, wash 5 times with inhaling the ovum pin, put into the center droplet that is coated with mineral oil, make and contain at least 15 ovocytes that contain the 1st polar body in each center droplet;
Step 5: adjust microneedle: place inversion to differ on the micrurgy platform of fluorescent microscope plastic ware, fixed tube peace lancet is installed on the fixed link of micrurgy instrument, reconcile spiral, in the visual field of inverted microscope, observe the position that ovocyte, fixed tube peace lancet is in the same level line;
Step 6: stoning: in the visual field, find ovocyte, fixed tube peace lancet, ovocyte is placed the fixed tube front end, utilize the flat mouth pin to draw acid Tai Shi solution, after the flat mouth pin is moved in the center droplet that contains ovocyte, utilize the flat mouth pin to adjust the position of the 1st polar body, make the pin mouth of flat mouth pin be positioned at the 1st polar body zona pellucida front end, make fixed tube, the ovocyte first polar body, flat mouth pin pin mouth is in the same level line position, blow out acid Tai Shi solution and zona reaction in the flat mouth needle tubing, to zona pellucida attenuation deliquescing, end during the kytoplasm convex, open microscopical fluorescence with the indicator cells nuclear location, convolution flat mouth needle injection makes it produce negative pressure, sucking-off the 1st polar body and kytoplasm on every side thereof, stoning finishes;
Step 7: ovocyte activity identification after the stoning: will go the ovocyte of nuclear to place 0.3% Trypan Blue liquid, and dye the rear M that uses 3-4 minutes
2Liquid washing 4-6 times, inspection dyeing situation under stereoscopic microscope, the indigo plant person of dying is judged to be death, and the tinter is not judged to be survival.
Described acid Tai Shi solution is by NaCl, KCl, CaCl
2H
2O, MgCl
2H
2O, glucose, Polyvinylpyrolidone (PVP) and ultrapure water form, and the weight percent of each composition is NaCl 0.8%, KCl 0.02%, CaCl
2H
2O 0.024%, MgCl
2H
2O 0.01%, glucose 0.1%, Polyvinylpyrolidone (PVP) 0.4%, surplus are ultrapure water.
Described ultrapure water is for the conducting medium in the removal water, not from colloidalmaterial, gas and the organism separated, and its resistivity is greater than 18M Ω * cm.
Described M
2The main component of liquid is: add the NaCl of 0.5533g in the ultrapure water of 10ml, the KCl of 0.0356g, the 4-hydroxyethyl piperazine ethanesulfonic acid of 0.4969g, 0.0036g Sodium.alpha.-ketopropionate, 0.261g Sodium.alpha.-hydroxypropionate, the bovine serum albumin of 0.4g, surplus is ultrapure water.
Described 0.3% Trypan Blue liquid is dissolved in the M of 10ml for taking by weighing the 0.03g trypan blue with it
2In the liquid, the millipore filter filtration sterilization of 0.22 μ m ,-20 ℃ are frozen for subsequent use.
Described micrurgy liquid is the M on basis
2Liquid adds the cytochalasin B, 5 μ g/ml Hoechst 33342,10% foetal calf serum, 0.01% polyvinyl alcohol of 5 μ g/ml.
Foetal calf serum should be taken from caesarean tire ox, it is the natural medium of consumption maximum in the cell cultures, contain the necessary nutritions of Growth of Cells such as various plasma proteinss, polypeptide, fat, carbohydrate, somatomedin, hormone, inorganics, growth has very important to the zooblast growth in vitro.
Hoechst 33342 is a kind of blue fluorescent dyes that can permeates cell membranes, and is lower to the toxicity of cell, is usually used in apoptosis and detects, and detects with fluorescence microscope or flow cytometer after the dyeing.
Beneficial effect of the present invention
1. device simple: need not expensive Piezo device, under the micrurgy instrument, can finish the stoning operation;
2. reagent (acid Tai Shi solution) composition is simple: the overwhelming majority is inorganic reagent, is easy to buy, and configuration is simple, and is with low cost;
3. operation easier is low, easily grasp: only need the kernel removing needle mouth is aimed at zona pellucida, discharge gently a little acid Tai Shi solution, it is dissolved a small holes, again kernel removing needle is taken advantage of a situation and insert this hole sucking-off polar body and nucleus gets final product, operation easier is more much smaller than other pitting method, and the tyro just can grasp very soon;
4. stoning operation is soft, and physical abuse is little: can finish the stoning damage under the relative rest state of ovocyte, the physical abuse of when having overcome other pitting method with ovocyte crimp stoning cytoskeleton and cytolemma having been brought;
5. stoning is fast, and efficient is high: on average the used time of each oocyte enucleation is 1-6 seconds, and survival rate is all more than 97% after the stoning of ovocyte, and the stoning success ratio is more than 99%;
6. stoning liquid cytotoxicity is little, and surge capability is strong: stoning operates in M
2Carry out in the liquid, need not add special-purpose stoning reagent, cytotoxicity is little; In addition, M
2The buffer composition of the liquid the inside unnecessary H in the acid Tai Shi solution that can neutralize
+, acute variation does not occur in the pH value of stablizing operation liquid in the After Enucleation, thus protection ovocyte activity is avoided the infringement that potential of hydrogen changes.
Description of drawings
Fig. 1 micrurgy droplet figure;
Ovocyte figure before Fig. 2 zona pellucida solution cavity;
Ovocyte figure behind Fig. 3 zona pellucida solution cavity;
Fig. 4 enucleation oocyte figure;
Fig. 5 enucleation oocyte Trypan Blue figure.
Embodiment
Embodiment one
Step 1: choose basic acid Tai Shi solution, for subsequent use;
Step 2: the improvement of acid Tai Shi solution: utilizing concentration is that the pH value of 36% HCl adjustment of acidity Tai Shi solution is 1.5, and the millipore filter of rear usefulness 0.22 μ m filters, and is frozen, for subsequent use in-20 ℃;
Step 3: damage droplet preparation: be in the plastic ware of 35mm in specification, equalization at the bottom of the ware is divided into four zones, in each zone, make the micrurgy liquid droplet of 1 30 μ l, title center droplet; Drip micrurgy liquid and 1 acid Tai Shi solution droplet of 2 20 μ l in each center droplet periphery, form peripheral droplet, rear top one deck mineral oil that covers;
Step 4: ovocyte places droplet: the ovocyte of getting the sexually matured mouse M II phase places the peripheral micrurgy liquid droplet of plastic ware, under stereoscopic microscope, wash 5 times with inhaling the ovum pin, put into the center droplet that is coated with mineral oil, make and contain 17 ovocytes that contain the 1st polar body in each center droplet;
Step 5: adjust microneedle: place inversion to differ on the micrurgy platform of fluorescent microscope plastic ware, fixed tube peace lancet is installed on the fixed link of micrurgy instrument, reconcile spiral, in the visual field of inverted microscope, observe the position that ovocyte, fixed tube peace lancet is in the same level line;
Step 6: in the visual field, find ovocyte, fixed tube peace lancet, ovocyte is placed the fixed tube front end, utilizing the flat mouth pin to draw the pH value is 1.5 acid Tai Shi solution, after the flat mouth pin is moved in the center droplet that contains ovocyte, utilize the flat mouth pin to adjust the position of the 1st polar body, make the pin mouth of flat mouth pin be positioned at the 1st polar body zona pellucida front end, make fixed tube, the ovocyte first polar body, flat mouth pin pin mouth is in the same level line position, blow out acid Tai Shi solution and zona reaction in the flat mouth needle tubing, to zona pellucida attenuation deliquescing, end during the kytoplasm convex, open microscopical fluorescence with the indicator cells nuclear location, convolution flat mouth needle injection makes it produce negative pressure, sucking-off the 1st polar body and kytoplasm on every side thereof, stoning finishes;
Step 7: ovocyte activity identification after the stoning: will go the ovocyte of nuclear to place 0.3% Trypan Blue liquid, and dye the rear M that uses 4 minutes
2Liquid washing 6 times, inspection dyeing situation under stereoscopic microscope, the indigo plant person of dying is judged to be death, and the tinter is not judged to be survival, and the stoning success ratio is 99.8%, and survival rate is 97.3% after the stoning.
Embodiment two
Step 1: choose basic acid Tai Shi solution, for subsequent use;
Step 2: the improvement of acid Tai Shi solution: utilizing concentration is that the pH value of 36% HCl adjustment of acidity Tai Shi solution is 2.0, and the millipore filter of rear usefulness 0.22 μ m filters, and is frozen, for subsequent use in-20 ℃;
Step 3: damage droplet preparation: be in the plastic ware of 35mm in specification, equalization at the bottom of the ware is divided into four zones, in each zone, make the micrurgy liquid droplet of 1 30 μ l, title center droplet; Drip micrurgy liquid and 1 acid Tai Shi solution droplet of 2 20 μ l in each center droplet periphery, form peripheral droplet, rear top one deck mineral oil that covers;
Step 4: ovocyte places droplet: the ovocyte of getting the sexually matured mouse M II phase places the peripheral micrurgy liquid droplet of plastic ware, under stereoscopic microscope, wash 5 times with inhaling the ovum pin, put into the center droplet that is coated with mineral oil, make and contain 16 ovocytes that contain the 1st polar body in each center droplet;
Step 5: adjust microneedle: place inversion to differ on the micrurgy platform of fluorescent microscope plastic ware, fixed tube peace lancet is installed on the fixed link of micrurgy instrument, reconcile spiral, in the visual field of inverted microscope, observe the position that ovocyte, fixed tube peace lancet is in the same level line;
Step 6: in the visual field, find ovocyte, fixed tube peace lancet, ovocyte is placed the fixed tube front end, utilizing the flat mouth pin to draw the pH value is 2.0 acid Tai Shi solution, after the flat mouth pin is moved in the center droplet that contains ovocyte, utilize the flat mouth pin to adjust the position of the 1st polar body, make the pin mouth of flat mouth pin be positioned at the 1st polar body zona pellucida front end, make fixed tube, the ovocyte first polar body, flat mouth pin pin mouth is in the same level line position, blow out acid Tai Shi solution and zona reaction in the flat mouth needle tubing, to zona pellucida attenuation deliquescing, end during the kytoplasm convex, open microscopical fluorescence with the indicator cells nuclear location, convolution flat mouth needle injection makes it produce negative pressure, sucking-off the 1st polar body and kytoplasm on every side thereof, stoning finishes;
Step 7: ovocyte activity identification after the stoning: will go the ovocyte of nuclear to place 0.3% Trypan Blue liquid, and dye the rear M that uses 3.5 minutes
2Liquid washing 5 times, inspection dyeing situation under stereoscopic microscope, the indigo plant person of dying is judged to be death, and the tinter is not judged to be survival, and the stoning success ratio is 99.3%, and survival rate is 97.4% after the stoning.
Embodiment three
Step 1: choose basic acid Tai Shi solution, for subsequent use;
Step 2: the improvement of acid Tai Shi solution: utilizing concentration is that the pH value of 36% HCl adjustment of acidity Tai Shi solution is 2.5, and the millipore filter of rear usefulness 0.22 μ m filters, and is frozen, for subsequent use in-20 ℃;
Step 3: damage droplet preparation: be in the plastic ware of 35mm in specification, equalization at the bottom of the ware is divided into four zones, in each zone, make the micrurgy liquid droplet of 1 30 μ l, title center droplet; Drip micrurgy liquid and 1 acid Tai Shi solution droplet of 2 20 μ l in each center droplet periphery, form peripheral droplet, rear top one deck mineral oil that covers;
Step 4: ovocyte places droplet: the ovocyte of getting the sexually matured mouse M II phase places the peripheral micrurgy liquid droplet of plastic ware, under stereoscopic microscope, wash 5 times with inhaling the ovum pin, put into the center droplet that is coated with mineral oil, make and contain 15 ovocytes that contain the 1st polar body in each center droplet;
Step 5: adjust microneedle: place inversion to differ on the micrurgy platform of fluorescent microscope plastic ware, fixed tube peace lancet is installed on the fixed link of micrurgy instrument, reconcile spiral, in the visual field of inverted microscope, observe the position that ovocyte, fixed tube peace lancet is in the same level line;
Step 6: in the visual field, find ovocyte, fixed tube peace lancet, ovocyte is placed the fixed tube front end, utilizing the flat mouth pin to draw the pH value is 2.5 acid Tai Shi solution, after the flat mouth pin is moved in the center droplet that contains ovocyte, utilize the flat mouth pin to adjust the position of the 1st polar body, make the pin mouth of flat mouth pin be positioned at the 1st polar body zona pellucida front end, make fixed tube, the ovocyte first polar body, flat mouth pin pin mouth is in the same level line position, blow out acid Tai Shi solution and zona reaction in the flat mouth needle tubing, to zona pellucida attenuation deliquescing, end during the kytoplasm convex, open microscopical fluorescence with the indicator cells nuclear location, convolution flat mouth needle injection makes it produce negative pressure, sucking-off the 1st polar body and kytoplasm on every side thereof, stoning finishes;
Step 7: ovocyte activity identification after the stoning: will go the ovocyte of nuclear to place 0.3% Trypan Blue liquid, and dye the rear M that uses 3 minutes
2Liquid washing 4 times, inspection dyeing situation under stereoscopic microscope, the indigo plant person of dying is judged to be death, and the tinter is not judged to be survival, and the stoning success ratio is 99.1%, and survival rate is 97.2% after the stoning.
Claims (1)
1. one kind is adopted zona pellucida solution cavity method to remove the method that oocyte of mouse is examined, and it is characterized in that: pitting method is:
Step 1: choose basic acid Tai Shi solution, for subsequent use;
Step 2: the improvement of acid Tai Shi solution: utilizing concentration is the pH value of 36% HCl adjustment of acidity Tai Shi solution, and the millipore filter of rear usefulness 0.22 μ m filters, and is frozen, for subsequent use in-20 ℃;
Step 3: damage droplet preparation: be in the plastic ware of 35mm in specification, equalization at the bottom of the ware is divided into four zones, in each zone, make the micrurgy liquid droplet of 1 30 μ l, title center droplet; Drip micrurgy liquid and 1 acid Tai Shi solution droplet of 2 20 μ l in each center droplet periphery, form peripheral droplet, rear top one deck mineral oil that covers;
Step 4: ovocyte places droplet: the ovocyte of getting the sexually matured mouse M II phase places the peripheral micrurgy liquid droplet of plastic ware, under stereoscopic microscope, wash 5 times with inhaling the ovum pin, put into the center droplet that is coated with mineral oil, make and contain at least 15 ovocytes that contain the 1st polar body in each center droplet;
Step 5: adjust microneedle: place inversion to differ on the micrurgy platform of fluorescent microscope plastic ware, fixed tube peace lancet is installed on the fixed link of micrurgy instrument, reconcile spiral, in the visual field of inverted microscope, observe the position that ovocyte, fixed tube peace lancet is in the same level line;
Step 6: stoning: in the visual field, find ovocyte, fixed tube peace lancet, ovocyte is placed the fixed tube front end, utilize the flat mouth pin to draw acid Tai Shi solution, after the flat mouth pin is moved in the center droplet that contains ovocyte, utilize the flat mouth pin to adjust the position of the 1st polar body, make the pin mouth of flat mouth pin be positioned at the 1st polar body zona pellucida front end, make fixed tube, the ovocyte first polar body, flat mouth pin pin mouth is in the same level line position, blow out acid Tai Shi solution and zona reaction in the flat mouth needle tubing, to zona pellucida attenuation deliquescing, end during the kytoplasm convex, open microscopical fluorescence with the indicator cells nuclear location, convolution flat mouth needle injection makes it produce negative pressure, sucking-off the 1st polar body and kytoplasm on every side thereof, stoning finishes;
Step 7: ovocyte activity identification after the stoning: will go the ovocyte of nuclear to place 0.3% Trypan Blue liquid, and dye the rear M that uses 3-4 minutes
2Liquid washing 4-6 times, inspection dyeing situation under stereoscopic microscope, the indigo plant person of dying is judged to be death, and the tinter is not judged to be survival;
Described acid Tai Shi solution is by NaCl, KCl, CaCl
2H
2O, MgCl
2H
2O, glucose, Polyvinylpyrolidone (PVP) and ultrapure water form, and the weight percent of each composition is NaCl 0.8%, KCl 0.02%, CaCl
2H
2O 0.024%, MgCl
2H
2O 0.01%, glucose 0.1%, Polyvinylpyrolidone (PVP) 0.4%, surplus are ultrapure water;
The pH value of the acid Tai Shi solution after the described improvement is 1.5 or 2.0;
Described micrurgy liquid is the M on basis
2Liquid adds the cytochalasin B, 5 μ g/ml Hoechst 33342,10% foetal calf serum, 0.01% polyvinyl alcohol of 5 μ g/ml;
Described M
2The composition of liquid is: in the ultrapure water of 10ml, add the NaCl of 0.5533g, and the KCl of 0.0356g, the 4-hydroxyethyl piperazine ethanesulfonic acid of 0.4969g, the Sodium.alpha.-ketopropionate of 0.0036g, the Sodium.alpha.-hydroxypropionate of 0.261g, the bovine serum albumin of 0.4g, surplus is ultrapure water.
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| CN102559752A (en) * | 2011-12-07 | 2012-07-11 | 西北农林科技大学 | Method for transfection and implantation of pre-embryo by plasmid DNA (deoxyribonucleic acid) or antisense oligonucleotide |
| CN104818314A (en) * | 2015-05-13 | 2015-08-05 | 上海海洋大学 | Transparent liquid for rapidly identifying oocyte nuclei of shrimps and crabs |
| CN111004775B (en) * | 2020-01-08 | 2023-05-02 | 中国医学科学院医学生物学研究所 | Method for physically removing oocyte zona pellucida |
| CN114075576A (en) * | 2021-11-02 | 2022-02-22 | 上海交通大学医学院附属第九人民医院 | Spindle nucleoplasm replacement method for reducing residual mitochondria |
| CN116004524B (en) * | 2023-03-06 | 2024-06-18 | 安徽农业大学 | Method for rapidly removing oocyte zona pellucida |
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| CN101914487A (en) * | 2010-01-22 | 2010-12-15 | 新疆维吾尔自治区畜牧科学院中国—澳大利亚绵羊育种研究中心 | Serum-free separating and culturing method for sheep embryo stem cell |
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| CN101914487A (en) * | 2010-01-22 | 2010-12-15 | 新疆维吾尔自治区畜牧科学院中国—澳大利亚绵羊育种研究中心 | Serum-free separating and culturing method for sheep embryo stem cell |
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| 卢晟盛等.在成熟过程中添加牛血清和猪***对猪***核成熟及体外受精后早期胚胎发育的影响.《黑龙江畜牧兽医》.2002,(第8期),1-3. * |
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