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Method for polymerization and in-vitro culture of embryo without zona pellucida before implantation

A technology of in vitro culture and zona pellucida, which is applied in the field of embryo engineering and developmental biology, 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: 2020-06-12
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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  • Method for polymerization and in-vitro culture of embryo without zona pellucida before implantation
  • Method for polymerization and in-vitro culture of embryo without zona pellucida before implantation
  • Method for polymerization and in-vitro culture of embryo without zona pellucida before implantation

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 5μg / 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 6-10hr; livestock parthenogenetic activation, the recovered oocytes in the MII stage were first treated in M16 solution containing 10μM ionomycin (sigma) for 5 minutes, and then treated in M16 solution containing 2μM 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 above-mentioned activated oocytes in M16 or G1 droplets, at 37°C, 6% CO 2 , 5%O 2 , 89%N 2 Cultured in a three-gas incubato...

Embodiment 1

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

[0042] 1 Materials and methods

[0043] 1.1 Reagents: All reagents used in this experiment are embryo-grade reagents, which are all Sigma products unless otherwise specified.

[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, i.e. the method of the present invention, after adopting 1% low-melting point agarose gel to heat and melt, to make 1-3 μ L agarose micro-droplets, and the tiny blocks after solidification Make small holes on ( 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 o...

Embodiment 2

[0069] Example 2 Polymerization and in vitro culture of mouse preimplantation parthenogenetic embryos without zona pellucida

[0070] 1 Materials and methods

[0071] 1.1 Reagents All reagents used in this experiment are embryo grade reagents, unless otherwise stated, are all Sigma products.

[0072] 1.2 Method

[0073] 1.2.1 Experimental grouping, same as Example 1.

[0074] The experiment was divided into experimental group and control group, respectively:

[0075] Experimental group: cultured by the gel microdrop hole method, i.e. the method of the present invention, after adopting 1% low-melting point agarose gel to heat and melt, to make 1-3 μ L agarose micro-droplets, and the tiny blocks after solidification Make small holes on ( figure 2 a), the detailed method is shown in the following "1.2.4 Preparation of gel droplet holes".

[0076] Control group Ⅰ: cultivated by the indentation method, that is, using a sharp instrument (ophthalmological tweezers) to press sma...

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Abstract

The invention discloses a method for polymerization and in-vitro culture of embryo without zona pellucida before implantation, and belongs to the technical field of embryo engineering and developmental biology. The method comprises the following steps: removing a zona pellucida from an embryo in a 2-8 cell period, putting the embryo into a low-melting-point agarose microdroplet hole covered with embryo-grade mineral oil and a G1 or M16 culture solution for polymerization, and performing in-vitro culture in a tri-gas incubator with 6% CO2, 5% O2 and 89% N2 at 37 DEG C to develop a blastocyst. According to the invention, polymerization and in-vitro culture are respectively carried out on non-zona pellucida fertilized embryos and parthenogenetic embryos of mice before implantation, and it isfound that the blastocyst development rates respectively reach 89.47% and 82.14%, which are obviously higher than those in the prior art, by adopting the gel microdroplet hole method disclosed by theinvention. The method is not only suitable for polyembryos of embryos without zona pellucida before implantation of animals and in-vitro culture of the polyembryos, but also suitable for polyembryos of embryos without zona pellucida before implantation of human beings and in-vitro culture of the polyembryos.

Description

technical field [0001] The invention relates to a method for the 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 man...

Claims

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Application Information

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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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