Method for improving compatibility and development rate of interracial nuclear transfer embryo nucleoplasm

A technology of nuclear transfer and development rate, which is applied in the field of embryo engineering and developmental biology, and can solve the problems of low embryo development rate or normal development rate

Inactive Publication Date: 2012-03-21
SHANGHAI TRANSGENIC RES CENT
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Problems solved by technology

Such long-term subcultured cells in vitro are used as donor cells, resultin

Method used

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  • Method for improving compatibility and development rate of interracial nuclear transfer embryo nucleoplasm
  • Method for improving compatibility and development rate of interracial nuclear transfer embryo nucleoplasm
  • Method for improving compatibility and development rate of interracial nuclear transfer embryo nucleoplasm

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preparation example Construction

[0046] 6. Preparation of Human Oocyte Plastids

[0047] The undivided eggs (abandoned eggs) were collected after 24 hours of in vitro fertilization, and were transported to the somatic cell nuclear transfer operation laboratory in a 37°C warm state. After washing with PBS, a part of the eggs were taken, and the state and position of the nuclei were observed under a fluorescence microscope. Use protease to digest and remove the zona pellucida of human eggs, then place the eggs without the zona pellucida on a micromanipulator, use a microinjection needle to divide the eggs with the zona pellucida off into several ooplasmic bodies, and place them under a fluorescent microscope Observe, pick out the cytoplast containing the nucleus (chromosome), and the remaining cytoplast without nucleus can be used for cytoplasm supplementation.

[0048] 7. Preparation of nuclear transfer embryos composed of three parts: human oocyte plastid-human cell-goat enucleated egg

[0049] Place th...

Embodiment 1

[0059] Effects of Human Egg Cytoplasm Supplementation on the Development of Human Nucleus-Enucleated Goat Egg Reconstructed Embryo

[0060] 1 Materials and methods:

[0061] 1.1.2 Reagents

[0062] All embryo-grade reagents are Sigma products unless otherwise stated.

[0063] 1.2 Method

[0064] 1.2.1 Collection of donor oocytes

[0065] Dairy goats were routinely superovulated with FSH, and 29-30 hours after LRH injection, the oocytes were recovered by surgery, and the oviducts were washed with F-10 (Gibco) culture medium containing 5% calf serum (FCS, Gibco) . Oocytes are collected and hyaluronidase removes attached granulosa cells. The collected oocytes were cultured in M16 solution, 37.5°C, 5% carbon dioxide incubator.

[0066] 1.2.2 Donor cells

[0067] Cut human foreskin tissue or aborted fetal skin into 1cm 2 Tissue blocks, or digested into single cells with trypsin, cultured in DMEM medium (plus 10-20% fetal calf serum, FCS), passaged, and genetical...

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Abstract

The invention provides a method for improving compatibility and development rate of interracial nuclear transfer embryo nucleoplasm. The method includes the steps: (a) a cell nucleus of a nuclear donor cell is moved into a perivitelline space of an enucleated oocyte of a non-human mammal and integrated by means of electric stimulation, so that a reconstructed ovum is formed, (b) a human oocyte is moved into the reconstructed ovum in the step (a), and (c) the reconstructed ovum in the step (b) is electrically stimulated so as to form a reconstructed ovum composed of the cell nucleus of a human cell, the enucleated oocyte of the non-human mammal and the human oocyte. The embryo development rate can be increased remarkably by the method.

Description

technical field [0001] The invention relates to the fields of embryo engineering and developmental biology, and more specifically, relates to a method for improving the nuclear-cytoplasmic compatibility and developmental rate of inter-race nuclear transfer embryos. Background technique [0002] Nuclear transfer (Nuclear transfer, NT) is the most effective method to study many important issues in the nuclear-cytoplasmic interaction. Fully developed individuals have been obtained in amphibians by using the method of transferring nuclei of different degrees of differentiation into enucleated oocytes. During the last two decades, nuclear transfer methods have developed rapidly in mammals and have begun to be used in production. [0003] A large number of results show that among animals of the same species, the middle stage II oocyte / fertilized egg is used as the recipient cell (donor cell), and cells with different degrees of differentiation are used as the donor cell. The reco...

Claims

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Application Information

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IPC IPC(8): C12N15/877C12N5/10C12N5/0735
Inventor 成国祥冯云陈建泉沙红英
Owner SHANGHAI TRANSGENIC RES CENT
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