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Method for removing mouse oocyte nuclei by adopting zona pellucida solution cavity method

A technology for oocytes and zona pellucida, which is applied in the field of removing mouse oocyte nuclei by using the zona pellucida cave method, which can solve the problem of operation time, enucleation success rate, cost and damage degree, high technical requirements for enucleation methods, and oocyte Eliminate the adverse effects of mother cells and other issues, and achieve the effects of easy purchase, simple equipment, and small mechanical damage

Inactive Publication Date: 2011-07-20
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different enucleation methods have their own advantages and disadvantages, and differ in operation time, enucleation success rate, cost and damage degree
The two-step method is cumbersome and time-consuming, and has adverse effects on oocytes
The perforation extrusion method can easily lead to cell deformation and cytoplasm loss
Moreover, some enucleation methods have high technical requirements, are difficult to master, and are unfavorable for popularization.

Method used

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  • Method for removing mouse oocyte nuclei by adopting zona pellucida solution cavity method
  • Method for removing mouse oocyte nuclei by adopting zona pellucida solution cavity method
  • Method for removing mouse oocyte nuclei by adopting zona pellucida solution cavity method

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Experimental program
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Effect test

Embodiment 1

[0033] Step 1: Select the basic acidic Tyrode solution for later use;

[0034] Step 2: Improvement of the acidic Tyrode solution: use 36% HCl to adjust the pH value of the acidic Tyrode solution to 1.5, then filter it with a 0.22 μm microporous filter, and freeze it at -20°C for future use;

[0035] Step 3: Preparation of damage droplets: In a plastic dish with a specification of 35 mm, divide the bottom of the dish into four areas equally, and make a 30 μl micromanipulation liquid droplet in each area, called the central droplet; Add two 20 μl micromanipulation fluid and one acidic Tyrode solution droplet to the periphery of each central droplet to form a peripheral droplet, which is then covered with a layer of mineral oil;

[0036] Step 4: Place oocytes in microdrops: take oocytes of sexually mature mice at the MⅡ stage and place them in micromanipulation solution microdrops around the plastic dish, and wash 5 times with egg suction needles under a stereo microscope , put ...

Embodiment 2

[0041] Step 1: Select the basic acidic Tyrode solution for later use;

[0042] Step 2: Improvement of the acidic Tyrode solution: use 36% HCl to adjust the pH value of the acidic Tyrode solution to 2.0, then filter it with a 0.22 μm microporous filter, and freeze it at -20°C for future use;

[0043] Step 3: Preparation of damage droplets: In a plastic dish with a specification of 35 mm, divide the bottom of the dish into four areas equally, and make a 30 μl micromanipulation liquid droplet in each area, called the central droplet; Add two 20 μl micromanipulation fluid and one acidic Tyrode solution droplet to the periphery of each central droplet to form a peripheral droplet, which is then covered with a layer of mineral oil;

[0044] Step 4: Place oocytes in microdrops: take oocytes of sexually mature mice at the MⅡ stage and place them in micromanipulation solution microdrops around the plastic dish, and wash 5 times with egg suction needles under a stereo microscope , put ...

Embodiment 3

[0049] Step 1: Select the basic acidic Tyrode solution for later use;

[0050] Step 2: Improvement of the acidic Tyrode solution: use 36% HCl to adjust the pH value of the acidic Tyrode solution to 2.5, then filter it with a 0.22 μm microporous filter, store it at -20°C for future use;

[0051] Step 3: Preparation of damage droplets: In a plastic dish with a specification of 35 mm, divide the bottom of the dish into four areas equally, and make a 30 μl micromanipulation liquid droplet in each area, called the central droplet; Add two 20 μl micromanipulation fluid and one acidic Tyrode solution droplet to the periphery of each central droplet to form a peripheral droplet, which is then covered with a layer of mineral oil;

[0052] Step 4: Place oocytes in microdrops: take oocytes of sexually mature mice at the MⅡ stage and place them in micromanipulation solution microdrops around the plastic dish, and wash 5 times with egg suction needles under a stereo microscope , put into ...

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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 [0001] The invention relates to a method for removing the mouse oocyte nucleus, in particular to a method for removing the mouse oocyte nucleus by using the zona pellucida cave method. Background technique [0002] With the development of biotechnology, mammalian somatic cell nuclear transfer technology is an irreplaceable technology platform in frontier fields such as production of transgenic animals, production of breast bioreactors, and therapeutic cloning. Although it has been achieved in sheep, cattle, pigs, and mice However, because the whole technical system of somatic cell nuclear transfer involves many links, and the efficiency of each link is low, the overall efficiency of somatic cell cloning technology is finally low, which has not exceeded 10%. The construction of recombinant embryos by micromanipulation is a classic method commonly used in nuclear transfer research. The enucleation of oocytes is one of the important steps in nuclear transfer, an...

Claims

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Application Information

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IPC IPC(8): C12N5/075
Inventor 张玉玲刘凤军翟小伟陈晓丽付祥龙禹学礼靳丽军
Owner HENAN UNIV OF SCI & TECH
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