CN102127521A - 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
- Publication number
- CN102127521A CN102127521A CN 201010604996 CN201010604996A CN102127521A CN 102127521 A CN102127521 A CN 102127521A CN 201010604996 CN201010604996 CN 201010604996 CN 201010604996 A CN201010604996 A CN 201010604996A CN 102127521 A CN102127521 A CN 102127521A
- Authority
- CN
- China
- Prior art keywords
- ovocyte
- droplet
- zona pellucida
- lancet
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
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, a kind of specifically method that adopts zona pellucida solution cavity method to remove oocyte of mouse nuclear.
Background technology
Along with development of biology, the mammal body-cell neucleus transplanting technology is as producing transgenic animal, making an irreplaceable technology platform in fields, forward position such as galactophore biological reactor, therapeutic cloning, though on sheep, ox, pig and mouse, obtained huge progress, but because the whole technique system of body-cell neucleus transplanting relates to numerous links, and each Link Efficiency is low, 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 a critical limitation factor that can complete successfully nuclear transplantation, be directly connected to the success or failure of nuclear transplantation experiment.A kind of pitting method fast and accurately will can improve the whole efficiency of nuclear transfer technology undoubtedly.The mouse conduct is a laboratory animal the most commonly used, yet the characteristic of oocyte of mouse and domestic animal (ox, sheep and pig) are widely different.The diameter of oocyte of mouse is less relatively, and 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 have long, the shortcoming such as the pair cell damage is big that expends time in, and the relative position of the 1st polar body and nuclear changes easily, 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 influence donorcells and cytosome merges.In addition, also have chemical stoning method, zona pellucida perforation extruding stoning method, the zona pellucida ground changes flat lancet 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 primarily aimed at 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 is adopted is:
Step 1: choose basic acid Tai Shi solution, standby;
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 filter with the millipore filter of 0.22 μ m the back, frozen, standby in-20 ℃;
Step 3: the preparation of damage droplet: in specification is in the plastic ware of 35mm, and equalization at the bottom of the ware is divided into four zones, makes the micrurgy liquid droplet of 1 30 μ l in each zone, claims the center droplet; Micrurgy liquid and 1 acid Tai Shi solution droplet at 2 20 μ l of each center droplet periphery dropping form peripheral droplet, cover one deck mineral oil above the back;
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 at least;
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 ovocyte, fixed tube peace lancet and be in same horizontal position;
Step 6: stoning: in the visual field, find ovocyte, fixed tube peace lancet, ovocyte is placed the fixed tube front end, utilize flat lancet to draw acid Tai Shi solution, after flat lancet is moved in the center droplet that contains ovocyte, utilize flat lancet to adjust the position of the 1st polar body, make the pin mouth of flat lancet be positioned at the 1st polar body zona pellucida front end, make fixed tube, the ovocyte first polar body, flat lancet pin mouth is in same sea line position, blow out acid Tai Shi solution and zona reaction in the flat lancet pipe, to zona pellucida attenuation deliquescing, kytoplasm ends when evagination, open microscopical fluorescence with the indicator cells nuclear location, the flat lancet syringe that circles round 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 nuclear ovocyte place 0.3% trypan blue staining fluid, dyeed 3-4 minutes, after use M
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 are formed, 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 removing conducting medium in the water, not dissociative colloidalmaterial, gas and organism, 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 a ultrapure water.
Described 0.3% trypan blue staining fluid 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 standby.
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 cell growth 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 the toxicity of pair cell is lower, is usually used in apoptosis and detects, and the dyeing back is detected with fluorescence microscope or flow cytometer.
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 an 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 a little acid Tai Shi solution gently, 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 very fast grasp;
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 that pair cell skeleton and cytolemma brought when having overcome other pitting method with ovocyte crimp stoning;
5. stoning is fast, the efficient height: on average each used time of ovocyte stoning 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 take place in the pH value of stablizing operation liquid in the stoning process, 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 colored graph.
Embodiment
Embodiment one
Step 1: choose basic acid Tai Shi solution, standby;
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 filter with the millipore filter of 0.22 μ m the back, frozen, standby in-20 ℃;
Step 3: the preparation of damage droplet: in specification is in the plastic ware of 35mm, and equalization at the bottom of the ware is divided into four zones, makes the micrurgy liquid droplet of 1 30 μ l in each zone, claims the center droplet; Micrurgy liquid and 1 acid Tai Shi solution droplet at 2 20 μ l of each center droplet periphery dropping form peripheral droplet, cover one deck mineral oil above the back;
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 ovocyte, fixed tube peace lancet and be in same horizontal position;
Step 6: in the visual field, find ovocyte, fixed tube peace lancet, ovocyte is placed the fixed tube front end, utilizing flat lancet to draw the pH value is 1.5 acid Tai Shi solution, after flat lancet is moved in the center droplet that contains ovocyte, utilize flat lancet to adjust the position of the 1st polar body, make the pin mouth of flat lancet be positioned at the 1st polar body zona pellucida front end, make fixed tube, the ovocyte first polar body, flat lancet pin mouth is in same sea line position, blow out acid Tai Shi solution and zona reaction in the flat lancet pipe, to zona pellucida attenuation deliquescing, kytoplasm ends when evagination, open microscopical fluorescence with the indicator cells nuclear location, the flat lancet syringe that circles round 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 nuclear ovocyte place 0.3% trypan blue staining fluid, dyeed 4 minutes, after use M
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, standby;
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 filter with the millipore filter of 0.22 μ m the back, frozen, standby in-20 ℃;
Step 3: the preparation of damage droplet: in specification is in the plastic ware of 35mm, and equalization at the bottom of the ware is divided into four zones, makes the micrurgy liquid droplet of 1 30 μ l in each zone, claims the center droplet; Micrurgy liquid and 1 acid Tai Shi solution droplet at 2 20 μ l of each center droplet periphery dropping form peripheral droplet, cover one deck mineral oil above the back;
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 ovocyte, fixed tube peace lancet and be in same horizontal position;
Step 6: in the visual field, find ovocyte, fixed tube peace lancet, ovocyte is placed the fixed tube front end, utilizing flat lancet to draw the pH value is 2.0 acid Tai Shi solution, after flat lancet is moved in the center droplet that contains ovocyte, utilize flat lancet to adjust the position of the 1st polar body, make the pin mouth of flat lancet be positioned at the 1st polar body zona pellucida front end, make fixed tube, the ovocyte first polar body, flat lancet pin mouth is in same sea line position, blow out acid Tai Shi solution and zona reaction in the flat lancet pipe, to zona pellucida attenuation deliquescing, kytoplasm ends when evagination, open microscopical fluorescence with the indicator cells nuclear location, the flat lancet syringe that circles round 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 nuclear ovocyte place 0.3% trypan blue staining fluid, dyeed 3.5 minutes, after use M
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, standby;
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 filter with the millipore filter of 0.22 μ m the back, frozen, standby in-20 ℃;
Step 3: the preparation of damage droplet: in specification is in the plastic ware of 35mm, and equalization at the bottom of the ware is divided into four zones, makes the micrurgy liquid droplet of 1 30 μ l in each zone, claims the center droplet; Micrurgy liquid and 1 acid Tai Shi solution droplet at 2 20 μ l of each center droplet periphery dropping form peripheral droplet, cover one deck mineral oil above the back;
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 ovocyte, fixed tube peace lancet and be in same horizontal position;
Step 6: in the visual field, find ovocyte, fixed tube peace lancet, ovocyte is placed the fixed tube front end, utilizing flat lancet to draw the pH value is 2.5 acid Tai Shi solution, after flat lancet is moved in the center droplet that contains ovocyte, utilize flat lancet to adjust the position of the 1st polar body, make the pin mouth of flat lancet be positioned at the 1st polar body zona pellucida front end, make fixed tube, the ovocyte first polar body, flat lancet pin mouth is in same sea line position, blow out acid Tai Shi solution and zona reaction in the flat lancet pipe, to zona pellucida attenuation deliquescing, kytoplasm ends when evagination, open microscopical fluorescence with the indicator cells nuclear location, the flat lancet syringe that circles round 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 nuclear ovocyte place 0.3% trypan blue staining fluid, dyeed 3 minutes, after use M
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 (3)
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, standby;
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 filter with the millipore filter of 0.22 μ m the back, frozen, standby in-20 ℃;
Step 3: the preparation of damage droplet: in specification is in the plastic ware of 35mm, and equalization at the bottom of the ware is divided into four zones, makes the micrurgy liquid droplet of 1 30 μ l in each zone, claims the center droplet; Micrurgy liquid and 1 acid Tai Shi solution droplet at 2 20 μ l of each center droplet periphery dropping form peripheral droplet, cover one deck mineral oil above the back;
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 at least;
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 ovocyte, fixed tube peace lancet and be in same horizontal position;
Step 6: stoning: in the visual field, find ovocyte, fixed tube peace lancet, ovocyte is placed the fixed tube front end, utilize flat lancet to draw acid Tai Shi solution, after flat lancet is moved in the center droplet that contains ovocyte, utilize flat lancet to adjust the position of the 1st polar body, make the pin mouth of flat lancet be positioned at the 1st polar body zona pellucida front end, make fixed tube, the ovocyte first polar body, flat lancet pin mouth is in same sea line position, blow out acid Tai Shi solution and zona reaction in the flat lancet pipe, to zona pellucida attenuation deliquescing, kytoplasm ends when evagination, open microscopical fluorescence with the indicator cells nuclear location, the flat lancet syringe that circles round 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 nuclear ovocyte place 0.3% trypan blue staining fluid, dyeed 3-4 minutes, after use M
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.
2. a kind of method that adopts zona pellucida solution cavity method to remove oocyte of mouse nuclear according to claim 1, it is characterized in that: described acid Tai Shi solution is by NaCl, KCl, CaCl
2H
2O, MgCl
2H
2O, glucose, Polyvinylpyrolidone (PVP) and ultrapure water are formed, 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.
3. a kind of method that adopts zona pellucida solution cavity method to remove oocyte of mouse nuclear according to claim 1, it is characterized in that: the pH value of the acid Tai Shi solution after the described improvement is 1.5 or 2.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010604996 CN102127521B (en) | 2010-12-25 | 2010-12-25 | Method for removing mouse oocyte nuclei by adopting zona pellucida solution cavity method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010604996 CN102127521B (en) | 2010-12-25 | 2010-12-25 | Method for removing mouse oocyte nuclei by adopting zona pellucida solution cavity method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102127521A true CN102127521A (en) | 2011-07-20 |
| CN102127521B CN102127521B (en) | 2013-02-06 |
Family
ID=44265796
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010604996 Expired - Fee Related CN102127521B (en) | 2010-12-25 | 2010-12-25 | Method for removing mouse oocyte nuclei by adopting zona pellucida solution cavity method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102127521B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111004775A (en) * | 2020-01-08 | 2020-04-14 | 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2010
- 2010-12-25 CN CN 201010604996 patent/CN102127521B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (4)
| Title |
|---|
| 《内分泌生殖生理学实验技术方法及其进展》 20100331 朱妙章等 第二篇 生殖生理学实验技术 第十四章 女（雌）性生殖实验技术 第七节 克隆－细胞核移植 第四军医大学出版社 162-163 1-3 , * |
| 《江苏农业学报》 20100630 剧世强等 猪卵母细胞第1极体与核相对位置变化对去核率的影响 526-531 1-3 第26卷, 第3期 * |
| 《现代生物技术与畜牧业》 20021231 李丽立等 第四章 二、小鼠核移植方法（四）核移植 科学出版社 56-57 1-3 , * |
| 《黑龙江畜牧兽医》 20021231 卢晟盛等 在成熟过程中添加牛血清和猪卵泡液对猪卵母细胞核成熟及体外受精后早期胚胎发育的影响 1-3 1-3 , 第8期 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111004775A (en) * | 2020-01-08 | 2020-04-14 | 中国医学科学院医学生物学研究所 | Method for physically removing oocyte zona pellucida |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102127521B (en) | 2013-02-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102127521B (en) | Method for removing mouse oocyte nuclei by adopting zona pellucida solution cavity method | |
| Voiculescu et al. | Spatially and temporally controlled electroporation of early chick embryos | |
| CN104130973B (en) | For the method for Ovine Oocytes in Vitro Maturation and pretreatment fluid and test kit | |
| CN113621553B (en) | Fugu bifidus ovary tissue cell line and application thereof | |
| CN102296090B (en) | Method for processing bovine somatic cell cloned embryos constructed on basis of somatic cell nuclear transplantation | |
| CN103451148A (en) | Human normal bronchial epithelial cell and preliminary isolated culture and subculture methods and purposes thereof | |
| Yong et al. | Sperm movement in the ooplasm, dithiothreitol pretreatment and sperm freezing are not required for the development of porcine embryos derived from injection of head membrane-damaged sperm | |
| Yong et al. | Production of a transgenic piglet by a sperm injection technique in which no chemical or physical treatments were used for oocytes or sperm | |
| CN107227298A (en) | A kind of clone embryos treatment fluid and its application method and the purposes of the treatment fluid | |
| Kumlehn et al. | Monitoring individual development of isolated wheat zygotes: a novel approach to study early embryogenesis | |
| CN107937444A (en) | The method of somatic cell clone dog | |
| Sereni et al. | Freezing spermatozoa obtained by testicular fine needle aspiration: a new technique | |
| US9074183B2 (en) | Method and apparatus for regulating optimum flow of semen and separating motile sperms | |
| CN104004706A (en) | Pseudosciaena crocea brain cell line and establishing method thereof | |
| CN108823067B (en) | Cell enucleation micromanipulation needle and cell enucleation method | |
| CN109512469A (en) | A method of uterine cavity liquid excretion body is extracted from pig living body | |
| TWI656214B (en) | Method for cleaning the surface of eggs or fertilized eggs | |
| CN103966357A (en) | Fast quantitative detection method of apostichopus japonicas coelomic fluid virus | |
| Bing et al. | Fertilization in vitro | |
| CN101451125A (en) | Method for culturing transgene rabbit adult fibroblast cloning embryo | |
| CN110184233B (en) | Oocyte in-vitro maturation culture solution additive and application thereof | |
| CN102450248A (en) | Sperm freezing method and freezing liquid thereof | |
| CN220799765U (en) | Freezing pole support that carries with information storage function | |
| CN216192264U (en) | Embryo biopsy needle assembly | |
| CN217536026U (en) | Special temperature return box for cell culture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130206 Termination date: 20131225 |