阅读说明：本技术 一种提高修正的人三原核受精卵体外培养囊胚发育率的方法 (Method for improving development rate of corrected human triprogenic fertilized egg in vitro culture blastocyst ) 是由 范勇 朱海瑛 于 2019-09-19 设计创作，主要内容包括：本发明公开了一种提高修正的人三原核受精卵体外培养囊胚发育率的方法,其特征在于,包括以下步骤：(1)提供修正的人三原核受精卵；(2)显微注射KDM4A mRNA到所述修正的人三原核受精卵胞质中；(3)体外培养经所述步骤(2)得到的修正的人三原核受精卵。本发明的方法操作简单,可显著提高修正的人三原核受精卵体外培养囊胚发育率。(The invention discloses a method for improving the development rate of a corrected blastocyst cultured in vitro of a human trinuclear fertilized egg, which is characterized by comprising the following steps of: (1) providing a modified human tri-pronuclear fertilized egg; (2) microinjecting KDM4A mRNA into the modified human tri-nuclear zygote cytoplasm; (3) culturing the corrected human tri-pronucleus fertilized egg obtained in the step (2) in vitro. The method is simple to operate, and can obviously improve the development rate of the corrected human trinuclear fertilized eggs in vitro culture blastocysts.)

1. A method for improving the development rate of a corrected blastocyst cultured in vitro from a human trinuclear fertilized egg, which is characterized by comprising the following steps:

(1) providing a modified human tri-pronuclear fertilized egg;

(2) microinjecting KDM4A mRNA into the modified human tri-nuclear zygote cytoplasm;

(3) culturing the corrected human tri-pronucleus fertilized egg obtained in the step (2) in vitro.

2. The method for increasing the developmental rate of a corrected blastocyst of a human tri-pronuclear fertilized egg as claimed in claim 1, wherein the step (1) is specifically: and (4) removing pronucleus which is far away from the second diode and has larger diameter from the human tri-pronuclear fertilized egg by micromanipulation to form the corrected human tri-pronuclear fertilized egg.

3. The method for improving the developmental rate of blastocysts cultured in vitro from modified human tri-pronuclear fertilized egg as claimed in claim 2, wherein the in vitro operating solution for removing the pronuclei from human tri-pronuclear fertilized egg in step (1) is G-mops solution added with 8-12 μ G/ml cytochalasin.

4. The method for increasing the developmental rate of blastocysts cultured in vitro from modified human tri-pronuclear fertilized egg as claimed in claim 3, wherein the in vitro working solution for removing the pronuclei from human tri-pronuclear fertilized egg in step (1) is G-mops solution supplemented with 10 μ G/ml cytochalasin.

5. The method for improving the developmental rate of blastocysts cultured in vitro from modified human tri-pronuclear fertilized eggs according to claim 1, wherein the injection concentration of KDM4A mRNA in step (2) is 800-1200 ng/μ L.

6. The method for increasing the development rate of blastocysts cultured in vitro from modified human tri-pronuclear fertilized eggs according to claim 5, wherein said KDM4A mRNA is injected at a concentration of 1000ng/μ L in step (2).

7. The method according to claim 1, wherein the KDM4A mRNA is injected in an amount of 1-2pL for each modified human tri-pronuclear fertilized egg in the step (2).

8. The method of claim 1, wherein the in vitro culture is a staged in vitro culture.

9. The method of claim 8, wherein the segmental in vitro culture is specifically: culturing the modified human trinuclear fertilized eggs obtained in the step (2) in vitro for the first 72h in a G-1 microdroplet culture solution, and culturing the fertilized eggs in a G-2 microdroplet culture solution after 72 h.

Technical Field

The invention relates to a method for improving the development rate of a corrected human trinuclear fertilized egg in-vitro culture blastocyst.

Background

A modified human tri-nuclear fertilized egg (ch3PN) refers to a fertilized egg in which one male pronucleus in a tri-nuclear zygote (3PN) is removed and restored to a diploid. The 3PN zygote is a clinically common type of abnormal fertilization (about 10%), and is clinically rejected as a transplant embryo for pregnancy because of early embryo development retardation/death and abortion due to chromosomal ploidy disorder.

The histone demethylases KDM4 family can regulate the transcriptional repression or activation of genes by removing lysine residues on histone H3/H4. The Zhang Y research team discovers that the H3K9me3 modification in the ZGA period can be obviously reduced by over-expressing histone demethylase KDM4A in a human somatic cell nuclear transfer embryo, and the in vitro blastocyst rate is improved from 4.2% to 26.8%. The Sun Q team optimizes the cynomolgus monkey nuclear transplantation system by over-expressing KDM4D, and realizes cloning of non-human primates internationally for the first time.

Although the corrected human triprogenic fertilized egg solves the problem of chromosomal aneuploidy to some extent, the low blastocyst development rate makes it still not used for pregnancy transplantation.

Disclosure of Invention

Based on the above, the present invention aims to provide a method for improving the development rate of corrected human trinuclear fertilized eggs in vitro blastocyst, in order to solve the problem of low development rate of corrected human trinuclear fertilized eggs in vitro blastocyst.

In order to achieve the purpose, the specific technical scheme of the invention is as follows:

a method for improving the development rate of a corrected blastocyst of a human trinuclear fertilized egg cultured in vitro comprises the following steps:

(2) providing a modified human tri-pronuclear fertilized egg;

(2) microinjecting KDM4A mRNA into the modified human tri-nuclear zygote cytoplasm;

(3) culturing the corrected human tri-pronucleus fertilized egg obtained in the step (2) in vitro.

In one embodiment, the step (1) is specifically: and (4) removing pronucleus which is far away from the second diode and has larger diameter from the human tri-pronuclear fertilized egg by micromanipulation to form the corrected human tri-pronuclear fertilized egg.

In one embodiment, in the step (1), the in vitro operation solution for removing the pronuclei in the human tri-pronucleus fertilized egg is a G-mops solution added with 8-12 μ G/ml Cytochalasin.

In one embodiment, in step (1), the in vitro operating solution for removing pronuclei from human tri-pronuclear fertilized eggs is G-mops solution added with 10. mu.g/ml Cytochalasin.

In one embodiment, in the step (2), the injection concentration of the KDM4A mRNA is 800-1200 ng/μ L.

In one embodiment, in step (2), the injection concentration of KDM4A mRNA is 1000 ng/. mu.L.

In one embodiment, in the step (2), the KDM4A mRNA is injected in an amount of 1-2pL for each modified human tri-pronuclear fertilized egg.

In one embodiment, the in vitro culture is a segmented in vitro culture.

In one embodiment, the segmented in vitro culture is specifically: culturing the modified human trinuclear fertilized eggs obtained in the step (2) in vitro for the first 72h in a G-1 microdroplet culture solution, and culturing the fertilized eggs in a G-2 microdroplet culture solution after 72 h.

Based on the technical scheme, the invention has the following beneficial effects:

the method for improving the development rate of the corrected human trinuclear fertilized eggs in vitro culture blastocysts is simple to operate, the development rate of the corrected human trinuclear fertilized eggs in vitro culture blastocysts can be obviously improved, and the development rate of the blastocysts of the corrected human trinuclear fertilized eggs in vitro culture can be improved to 22.47% from 7.88%.

Drawings

FIG. 1 is a schematic diagram of a human KDM4A mRNA vector;

FIG. 2 is a drawing of a micromanipulation system of a modified human tri-pronuclear fertilized egg (ch3 PN);

FIG. 3 is a diagram showing the development process of a modified human tri-pronuclear fertilized egg (ch3 PN).

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.