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Stem cell based mammal artificial embryo constructing and animal cloning breeding method and model thereof

A mammal and stem cell technology, applied in the new field of animal and human reproductive bioengineering, can solve the problems of complex production process, high cost and limited production of cloned animals, and achieve extremely low production cost, important application value, and easy operation.

Inactive Publication Date: 2017-05-31
INNER MONGOLIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limited technical level of cloning at present, the production process of cloned embryos based on nuclear transfer is complicated, the production efficiency of cloned animals after embryo transfer is still very low, and the production cost of each cloned animal is high, so it is difficult to carry out large-scale promotion application

Method used

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  • Stem cell based mammal artificial embryo constructing and animal cloning breeding method and model thereof
  • Stem cell based mammal artificial embryo constructing and animal cloning breeding method and model thereof
  • Stem cell based mammal artificial embryo constructing and animal cloning breeding method and model thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Horse Stem Cells - Artificial Embryo Construction:

[0043] 1) Preparation of stem cells: Same as the establishment of stem cell lines, first make a monolayer of vegetative cell layers (horse 32-34 day embryonic fibroblasts) treated with division arrest one week before culturing stem cells. The culture condition of vegetative cells was 5% CO 2 and 95% air, the composition of the culture medium is STO plus 20% serum. Take 8,000-10,000 cryopreserved horse embryonic stem cells that have been passaged for 16 times. After thawing, they are centrifuged with stem cell culture medium and placed in 2-3 4-well culture plates. They are allowed to proliferate for 3-4 days and form multiple 500- A stem cell cluster composed of 800 cells. The stem cell line is derived from embryonic stem cells (Embryonic SC), and does not carry any introduced genes for stem cell differentiation markers.

[0044] 2) Preparation of the zona pellucida of horse eggs: 24 hours before stem cells were in...

Embodiment 2

[0049] Mouse stem cells-artificial embryo construction:

[0050] 1) Preparation of stem cells: Same as the establishment of stem cell lines, a monolayer of vegetative cell layers (mouse 13.5-day embryonic fibroblasts) treated with division arrest was first prepared one week before stem cell culture. The culture condition of vegetative cells was 5% CO 2 and 95% air, the composition of the culture medium is STO plus 20% serum. Take 8,000-10,000 cryopreserved mouse embryonic stem cells that have been passaged 24 times. After thawing, they are centrifuged with stem cell culture medium and placed in 2-3 4-well culture plates to proliferate for 3-4 days and form multiple 500 cells. -A stem cell cluster composed of 800 cells. The stem cell line is the embryonic stem cell (Epiblast SC) embryonic stem cell (Embryonic SC) from the upper layer of the early embryo after implantation, carrying the Oct-4 gene green protein, and its differentiation status can be directly detected under a m...

Embodiment 3

[0056] Human Stem Cells - Artificial Embryo Construction:

[0057] 1) Preparation of stem cells: take 8000-10000 cryopreserved human embryonic stem cells that have been passaged 24 times, centrifuge them with stem cell culture medium after thawing, put them into 2-3 4-well culture plates, and let them proliferate for 3-4 days. And form multiple stem cell groups composed of 500-800 cells.

[0058] 2) Preparation of the zona pellucida of human eggs: 24 hours before stem cells are injected into the zona pellucida, according to the number of 12-15 cloned embryos produced, 15 discarded eggs are obtained from the test tube baby laboratory, and the zona pellucida is cut open by micromanipulation And aspirate the egg cytoplasm to make the empty zona pellucida.

[0059] 3) Stem cells are injected into the zona pellucida: the stem cells are injected into the zona pellucida when the stem cells form a cell cluster of 500-800 cells, treated with 0.5% trypsin to separate the stem cell clus...

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Abstract

Th invention discloses a stem cell based mammal artificial embryo constructing and animal cloning breeding method and a model thereof. With a mammal ovum zona pellucida or an artificially synthesized structure equivalent to the zona pellucida as a stem cell receptor of an artificial embryo, the stem cell receptor is implanted into stem cells, and differentiation of the stem cells is induced under an in-vitro condition, so that a mammal preimplantation embryo structure is obtained. Two key technical designs include differentiation capacity application of stem cell pluripotency to a primary embryo and early embryo differentiation inducing of a constructed stem cell-ovum zona pellucida complex. Four major technical steps include preparation of the stem cells, preparation of animal ovum or early embryo zona pellucida, artificial embryo construction and cell differentiation inducing, and animal cloning breeding. The method is expected to become an important technical means in the industrialization application animal cloning breeding technology in the future; and meanwhile, the method has an important medical application value.

Description

technical field [0001] The invention belongs to the new technology of animal and human reproductive bioengineering, and mainly relates to a stem cell-based mammalian artificial embryo construction-animal cloning breeding method and a model thereof. Background technique [0002] Since the cloned sheep were mostly born in the UK in 1997, animal cloning research has developed rapidly, and most experimental animals and livestock cloning have been successful, which has played a huge role in promoting the basic research of life sciences and the breeding and expansion of specific animal individuals. Cattle-based livestock cloning technology began to advance towards industrial application. However, due to the limited technical level of cloning at present, the production process of cloned embryos based on nuclear transfer is complicated, the production efficiency of cloned animals after embryo transfer is still very low, and the production cost of each cloned animal is high, so it is...

Claims

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Application Information

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IPC IPC(8): C12N5/073A01K67/027
Inventor 包斯琴吴宝江李喜和
Owner INNER MONGOLIA UNIVERSITY
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