Mice somatic cell nuclear transplantation method

Abstract

The invention discloses an optimized mice somatic cell nuclear transplantation method. According to the mice somatic cell nuclear transplantation method, three kinds of cells, including cumulus cell, fetal fibroblasts, and embryonic stem cell, are adopted to construct reconstructed embryos; the activation efficiencies of three reconstructed embryos in different activation mediums are compared, development efficiencies in different solutions are compared, and pregnancy efficiencies of different embryo transplantation methods are compared; it is concluded that calcium-free KSOM activation medium is a universal high efficiency activation medium, and the activation efficiency is about 93.5%; KSOM-AA culture medium is suitable for in vitro culture of the reconstructed embryos of cumulus cells and embryonic stem cells, and the best culture medium for fetal fibroblasts is alpha MEM culture medium; when the reconstructed embryos are at 2-cell period, nuclear transplanted animals can be obtained via transplantation of embryos through bilateral fallopian tubes, wherein for each fallopian tube, transplantation of 15 embryos is carried out. The optimized mice somatic cell nuclear transplantation method is capable of increasing reconstructed embryo cleavage rate, blastula development rate, cloning animal birth rate, and survival rate, so that the success rate of obtaining embryonic stem cells from the reconstructed embryos is increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mouse somatic cell nuclear transfer method. Background technique [0002] Somatic cell nuclear transfer refers to the technique of injecting somatic cell nuclei into the cytoplasm of enucleated oocytes to obtain reconstructed embryos, which eventually develop into offspring. Cell reprogramming can be achieved through nuclear transfer, cell fusion, specific transcription factors, and culture. However, at present, the only reprogramming method that can induce cells to redevelop into individuals is somatic cell nuclear transfer, and other methods can only be induced at the cellular, molecular or biochemical levels, and there are some shortcomings; However, since the establishment of somatic cell nuclear transfer technology, the cloning efficiency has been at a low level, only a few reconstituted embryos can develop to full-term birth, usually only 1-2% birth rate in mice, and most clo...

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Problems solved by technology

[0004] The in vitro culture of reconstituted embryos is one of the key factors for the development of cloned embryos. A good in vitro culture system for reconstituted embryos is very important for improving the cleavage rate of reconstituted embryos, the rate of blastocyst development, pregnancy and the birth of cloned animals; CZB, KSOM-AA and M16 are currently the most commonly used culture medium, both of which can cultivate fertilized eggs to the blastocyst stage, but the conditions suitable for in vitro culture of reconstituted embryos are not uniform, so a set of reconstituted embryo culture system is selected for further exploration. The developmental mechanism of reconstructed embryos has great research value and significance

[0005] Mouse embryo transfer technology is one of the essential key technologies in the production process of nuclear transfer animals. Oviduct fimbria transfer and uterine horn transfer are two technologies commonly used at present. Different, generally speaking, oviduct fimbria transplantation is used to prepare transgenic mice by pronuclear injection; uterine horn transplantation technology is used to produce blastomere hybrid fusion and chimera animals; but there is no unified method for transplanting reconstituted embryos by nuclear transfer. It is recognized that not only the methods used in different literatures are different, but also the birth efficiency of the same transplantation method is not consistent. Therefore, exploring a set of optimal transplantation schemes also has great research value and significance for further research on the developmental mechanism of nuclear transplantation animals.

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