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Method for improving pig cell nucleus transplantation efficiency

An animal body and embryo culture medium technology, applied in the fields of botanical equipment and methods, animal cells, vertebrate cells, etc., can solve the problem of low overall efficiency of pig somatic cell cloning, and achieve the increase of the number of blastocyst cells and the increase of blastocyst cells. efficiency and improve overall efficiency

Active Publication Date: 2017-05-17
南宁壮博生物科技有限公司
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AI Technical Summary

Problems solved by technology

Although after two decades of development, cloning technology has been relatively mature, and there is a set of overall technologies including oocyte collection, culture, donor cell isolation, culture, egg activation, embryo in vitro culture and embryo transfer, but so far, The overall efficiency of pig somatic cell cloning is still very low, about 1%

Method used

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  • Method for improving pig cell nucleus transplantation efficiency
  • Method for improving pig cell nucleus transplantation efficiency
  • Method for improving pig cell nucleus transplantation efficiency

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1, pig somatic cell nuclear transfer

[0044] 1. In vitro maturation culture of porcine oocytes

[0045] Take the pig ovary from the slaughterhouse, wash it three times with normal saline containing 100U / mL penicillin and 100U / mL streptomycin, and then use a 10mL disposable syringe and a 18-gauge needle to extract follicles with a diameter of 3-6mm Cumulus-oocyte complexes (COCs) wrapped with 3-5 layers of cumulus cells were selected from the liquid. COCs were rinsed twice with normal saline containing 100U / mL penicillin and 100U / mL streptomycin, then transferred to IVM solution, at 39°C, saturated humidity, 5% CO 2 Cultured in the incubator for 42h.

[0046] 2. Isolation and culture of porcine fetal fibroblasts

[0047] Pig fetal fibroblasts were prepared according to the following steps in turn:

[0048] 1. Take out the fetus from the uterus of pregnant sows at gestation day 35, wash with DPBS buffer solution containing 100U / mL penicillin and 100U / mL st...

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Abstract

The invention discloses a method for improving the pig cell nucleus transplantation efficiency. An embryo culture solution is provided and contains 50 nM of BIX-01294 and 0.5 nM of chaetocin. The embryo culture solution can be used for culturing a reconstructed embryo and / or preparing a kit used for culturing the reconstructed embryo. BIX-01294 and chaetocin with certain concentration are added to a PZM-3 culture solution for combined treatment of an early cloned embryo, the blastocyst rate and the blastocyst cell number of the cloned embryo can be remarkably increased, the development efficiency of the cloned embryo is remarkably improved, and the method has a better effect than single use of BIX-01294 or chaetocin for treatment. The method has great significance for improving the overall efficiency of a pig cell nucleus transplantation technology.

Description

technical field [0001] The invention relates to a method for improving the efficiency of pig somatic cell nuclear transplantation. Background technique [0002] The technology of somatic cell nuclear transfer was born in 1997, and it was first successfully used in sheep. The main method is to transfer fully differentiated donor cells into enucleated oocytes to form reconstructed embryos through micromanipulation and cell fusion technology, and then activate the reconstructed embryos for culture and transplantation, and finally obtain a complete individual. Because the genotype of the obtained individual is exactly the same as that of the donor cell, this technique is also called somatic cell cloning technique. Due to its unique technical advantages, somatic cell cloning technology has great application value in animal breeding, disease model construction, endangered animal protection, therapeutic cloning, and pharmaceutical protein production. Since the birth of the first ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/877C12N5/071
CPCC12N5/0602C12N15/8778C12N2500/30
Inventor 刘志国李奎牟玉莲郑新民毕延震
Owner 南宁壮博生物科技有限公司
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