A kind of method of preimplantation without zona pellucida embryo aggregation and in vitro culture

A technology of in vitro culture and zona pellucida, which is applied in the fields of developmental biology and embryo engineering, can solve problems such as not easy to remove, affect embryo development, and take a long time, so as to reduce adverse side effects of embryo development, increase the efficiency of blastocyst development, and improve The effect of blastocyst development rate

Active Publication Date: 2021-10-26
南京市江宁医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, when using the scalding method to make dimples, it is necessary to heat the needle with an alcohol flame before making every dimple, which takes a long time, and bubbles are easy to appear after covering the culture medium, which is not easy to remove, and the plastic may be hot after scalding. produce some toxic substances
However, using the indentation method to make small dimples requires higher operating techniques, and plastic debris may appear, which will affect embryonic development.

Method used

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  • A kind of method of preimplantation without zona pellucida embryo aggregation and in vitro culture
  • A kind of method of preimplantation without zona pellucida embryo aggregation and in vitro culture
  • A kind of method of preimplantation without zona pellucida embryo aggregation and in vitro culture

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0027] (2) Preparation of Diploid (2N) Embryos

[0028] 1. Preparation of 2N embryos from parthenogenetic activation

[0029] Parthenogenetic activation of rodent oocytes, the recovered oocytes in the MII stage were placed in activation solution [containing 5 µg / mL CB (sigma), 10mM SrCl 2 (sigma) Ca-free 2+ , Mg 2+ M16 (sigma) culture medium] at 37 °C, 6% CO 2 , 5% O 2 , 89% N 2 Cultured in a three-gas incubator for 6-10 hr; livestock parthenogenetic activation, the recovered oocytes in the MII stage were first treated in M16 solution containing 10 μM ionomycin (sigma) for 5 minutes, and then in the solution containing 2 μM 6-dimethylaminopurina (6-DMAP, sigma), 5 μg / mL CB in M16 solution, at 37°C, 6% CO 2 , 5% O 2 , 89%N 2 Cultivate in a three-gas incubator for 4-6 hours; double pronuclei can be seen in the activated oocytes. Then place the activated oocytes above in M16 or G1 droplets at 37°C, 6% CO 2 , 5% O 2 , 89% N 2 Culture in a three-gas incubator for 2-3.5 ...

Embodiment 1

[0041] Example 1 Aggregation and in vitro culture of mouse preimplantation fertilized embryos without zona pellucida

[0042] 1 Materials and methods

[0043] 1.1 Reagents: The reagents used in this experiment are all embryo grade reagents, unless otherwise stated, are all Sigma products.

[0044]1.2 Method

[0045] 1.2.1 Experimental grouping:

[0046] The experiment was divided into experimental group and control group, respectively:

[0047] Experimental group: cultured by the gel microdrop hole method, that is, the method of the present invention, after heating and melting 1% low-melting point agarose gel, 1-3 μL agarose micro-droplets are made, and the tiny Make small holes in the block ( figure 2 a), the detailed method is shown in the following "1.2.4 Preparation of gel droplet holes".

[0048] Control group Ⅰ: cultivated by the indentation method, that is, using a sharp instrument (ophthalmological tweezers) to press small depressions on the bottom of the culture...

Embodiment 2

[0068] Example 2 Polymerization and in vitro culture of mouse preimplantation parthenogenetic embryos without zona pellucida

[0069] 1 Materials and methods

[0070] 1.1 Reagents All reagents used in this experiment are embryo grade reagents, unless otherwise stated, are all Sigma products.

[0071] 1.2 Method

[0072] 1.2.1 Experimental grouping, same as Example 1.

[0073] The experiment was divided into experimental group and control group, respectively:

[0074] Experimental group: cultured by the gel microdrop hole method, that is, the method of the present invention, after heating and melting 1% low-melting point agarose gel, 1-3 μL agarose micro-droplets are made, and the tiny Make small holes in the block ( figure 2 a), the detailed method is shown in the following "1.2.4 Preparation of gel droplet holes".

[0075] Control group Ⅰ: cultivated by the indentation method, that is, using a sharp instrument (ophthalmological tweezers) to press small depressions on th...

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Abstract

The invention discloses a method for aggregation and in vitro culture of animal embryos without zona pellucida before implantation, and belongs to the technical fields of embryo engineering and developmental biology. In the present invention, after removing the zona pellucida, animal embryos at the 2-8 cell stage are placed in low-melting point agarose microdrop holes covered with embryo-grade mineral oil and G1 or M16 culture fluid for polymerization, at 37 ° C, 6% CO2, 5% O2, 89% N2 in a three-gas incubator for in vitro culture and develop into blastocysts. In the present invention, the fertilized embryos without zona pellucida and parthenogenetic embryos of mice before implantation were aggregated and cultured in vitro, and it was found that the development rate of blastocysts reached 89.47% and 82.14% respectively by using the gel micro-droplet method of the present invention. Significantly higher than the existing technology. The method is suitable for the aggregated embryos of animal pre-implantation embryos without zona pellucida and its in vitro culture.

Description

technical field [0001] The invention relates to a method for aggregation and in vitro culture of embryos without zona pellucida before implantation, and belongs to the technical fields of embryo engineering and developmental biology. Background technique [0002] A chimera is an embryo formed by combining two or more embryos or cells with different genetic traits. There are two main methods of making chimeras: blastocyst injection and polymerization. The polymerization method is simple to operate and does not require special equipment, but its success rate is lower than that of the blastocyst injection method. The reason may be that the polymerization method mainly relies on manual operation, and the technical and operational aspects of each link are very strong, such as the removal of the zona pellucida, the conditions of embryo polymerization, the culture medium of the aggregated embryos, and the culture conditions of embryos without zona pellucida, etc. There are many in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/073
CPCC12N5/0604C12N2500/02C12N2533/76
Inventor 陈建泉陆颖菲赵承承周宇居蓉
Owner 南京市江宁医院
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