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Method for preparing single blastomere chromosome

A blastomere and chromosome technology, applied in the field of preparing single blastomere chromosomes, can solve the problems of difficult PGD analysis, low chromosome efficiency of single embryo cells, and low quality of mitotic phase, and achieve high quality, good quality and clear shape. Effect

Inactive Publication Date: 2006-12-27
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the efficiency of preparing the chromosomes of single embryonic cells by conventional cytogenetic methods is low, and the division phases obtained are also difficult for subsequent PGD analysis due to low quality.

Method used

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  • Method for preparing single blastomere chromosome
  • Method for preparing single blastomere chromosome
  • Method for preparing single blastomere chromosome

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Obtaining Human Blastomere Chromosomes Using Mouse Pronuclear Stage Fertilized Eggs as Cytoplasmic Recipients

[0028] 4-week-old female mice were first injected with 9.0 IU of pregnant horse serum, 48 hours later injected with 9.0 IU of chorionic gonadotropin (HCG), and housed with reproductive male mice. The female mice were sacrificed 24 hours after the injection of HCG, and the oviducts were separated, and the fertilized eggs at the pronuclear stage were washed out with human tubal fluid (mHTF) containing 10% bovine serum albumin (BSA), and the fertilized eggs were removed with 80IU / ml hyaluronidase cumulus.

[0029]The fertilized eggs were first placed in mHTF containing 0.3M sucrose, 0.9 μg / mL cytochalasin D and 0.2 μg / mL colcemid for 8 minutes, and then the pronuclear stage fertilized eggs were enucleated using a micromanipulator. The egg cells were fixed with an egg-holding needle with an inner diameter of 120 μm, and a hole was punched at the 3 o’cl...

Embodiment 2

[0035] Example 2: Obtaining a single human blastomere chromosome when abnormally fertilized human pronuclear zygotes were used as cytoplasmic recipients

[0036] Human abnormal fertilized eggs discarded by the human assisted reproductive laboratory were collected, including single prokaryotic eggs and polyprokaryotic eggs with more than three prokaryotic eggs. After enucleation, the method described in Example 1 was used to prepare a single human blastomere chromosome. Using DAPI staining (DAPI content 0.5μg / mL, staining for 5 minutes) to obtain a single human blastomere chromosome see figure 1 , using Gissam's staining (5% Gissam's staining for 5 minutes) to obtain single human blastomere chromosomes see figure 2 , to obtain the karyotype distribution map of a single human blastomere, see image 3 , Figure 4 .

[0037] Depend on Figure 1~4 It can be seen that by using the method of the present invention, the obtained single human blastomere chromosomes have very clear ...

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Abstract

The invention provides a method for preparing individual blastomere chromosome, and the said method is as follows: removing germ hill from normal fertilized ovum in prokaryotic period and performing enucleation to obtain cell plastid, in addition selecting individual human blastomere by biopsy, performing electric anabranch to the said blastomere and the cell plastid to obtain heterocaryon, introducing the prematured chromosome to conglomerate to shape with the obtained heterocaryon, then performing low permeability to the introduced heterocaryon, afterwards fixing and colouring the heterocaryon, finally obtaining the said individual blastomere chromosome. The invention obtains the method for preparing individual blastomere chromosome with high efficiency and fine quality by further improving and perfecting the electric anabranch solution, chromosome evoked solution, low permeability solution and mounting step, the prepared chromosome is of intelligible shape and of high quality, the method connected with G-bandings or chromosome paint-on and probe FISH technology can overcome the shortage existed in present interphase nucleus FISH technology and can make the PGD diagnostic range extended to the whole chromosome complement.

Description

(1) Technical field [0001] The invention relates to a method for preparing a single blastomere chromosome, which belongs to the field of preimplantation genetic diagnosis of chromosomal diseases. (2) Background technology [0002] Chromosomal diseases are a large class of serious genetic diseases. Severe cases die early in the embryo and cause spontaneous abortion. Even if a small number of cases with mild symptoms survive and give birth, they are often accompanied by congenital multiple deformities and abnormalities in growth, intelligence, sexual development or reproductive abnormalities. . Chromosomal diseases are extremely harmful to humans, but there is no good treatment strategy. At present, genetic counseling and prenatal diagnosis are mainly used to prevent them. However, genetic counseling is only focused on health education, lacking targeted means, and prenatal diagnosis is a traumatic means, and once the diagnosis is made, unintentional abortion is required to te...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/12
Inventor 徐晨明黄荷凤
Owner ZHEJIANG UNIV
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