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Method for improving pig nucleus transplantation efficiency

A technology of cell nucleus and donor cells, which is applied in the field of improving the efficiency of somatic cell nuclear transplantation, can solve problems such as not getting ideal results, and achieve the effect of improving efficiency and production efficiency

Inactive Publication Date: 2010-12-22
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
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AI Technical Summary

Problems solved by technology

Although this method has achieved a certain effect on the efficiency of somatic cell nuclear transfer in mice, it has not obtained ideal results in other species (Hochedlinger K, Jaenisch R. Nuclear transplantation: lessons from frogs and mice. 2002 CurrOpin Cell Biol. 14 (6 ): 741-748.; Enright BP, Kubota C, Yang X et al. Epigenetic characteristics and development of embryos cloned from donor cells treated by trichostatin A or 5-aza-2-deoxycytidine. 2003 Biol. Reprod.69: 896-901 .; Enright BP, Sung LY, Chang CC et al. Methylation and acetylation characteristics of cloned bovine embryos from donor cells treated with 5-aza-2-deoxycytidine. 2005 Biol Reprod.72: 944-948.)

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0023] The mature culture of embodiment 1 porcine oocyte

[0024] The ovaries are collected from the slaughterhouse and transported back to the laboratory with 37°C normal saline, and the cumulus-oocyte complexes are collected from the antral follicles with a diameter of 3-5mm, and the oocytes with more than three complete layers of cumulus cells are selected under a solid microscope The cells were used for maturation culture; the culture conditions for maturation culture were 38.5°C, 5% carbon dioxide, 95% air gas environment, and saturated humidity; after 42 hours of maturation culture, 0.5% hyaluronidase was used to remove cumulus cells, and the obtained MII stage Oocytes, as nuclear transfer recipients.

Embodiment 2

[0025] Obtaining and culturing of pig fetal fibroblasts of embodiment 2

[0026] The ear tissue of 35-day-old pig fetus was taken aseptically, and the tissue cells were separated by conventional 0.25% trypsin (Gibco) digestion method, DMEM (Gibco) was added with 20% fetal bovine serum for primary culture, and DMEM was added with 10% fetal bovine serum for subculture , the culture condition is 38.5°C, 5% carbon dioxide, 95% air gas environment, moderately saturated; 3-5 passages of contact-inhibited fetal fibroblasts are used for nuclear transfer, and 50, 30, and 10 nM CalyculinA are used respectively before nuclear transfer (sigma) Fetal fibroblasts were pretreated for 30, 20, and 10 min, and then digested with 0.25% trypsin into a single suspension cell, which was used as a nuclear transfer donor.

Embodiment 3

[0027] Example 3 Somatic cell nuclear transfer

[0028] Porcine somatic cell nuclear transfer was performed by the fusion method ( figure 1), the process is as follows: the spindle body and the first polar body of the MII egg were removed by blind suction, the somatic cells were injected under the zona pellucida, and made in close contact with the oocyte plasma membrane, and DC electric shock (1.2kv / cm, 30μs, 2 subpulse) to induce fusion of somatic cells and enucleated oocytoplasm to form reconstituted embryos and activate them, and culture them in PZM-3 culture medium under the culture conditions of 38.5°C, 5% CO2, and saturated humidity. The cleavage rate was calculated after 48 hours of culture, and the blastocyst rate was calculated after 146 hours. 10mg / L Hoechst 33342 was used to stain blastocysts for 5 minutes, and the number of blastocyst cells was observed under a fluorescent microscope; the tools, liquids, and instruments used were as follows: the inner diameter of ...

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Abstract

The invention discloses a method for improving pig nucleus transplantation efficiency, which belongs to the field of breeding or inseminating methods. The method comprises the following steps of: processing a donor cell by a small molecule medicament Calyculin A and inducing donor chromatin condensation, methylation rise and acetylation reduction, wherein a somatic nucleus expresses genetic modified zero resetting; then carrying out nucleus transplantation and constructing reconstructed embryos; and selecting optimal concentration of 10nM and the processing time of 30 minutes by an optimizing condition. The invention improves the ectogenesis blastocyst rate of a pig clone embryo by 9.7 percent, increases 23.78 blastomeres and lays the foundation for improving the production efficiency of clone animals.

Description

technical field [0001] The invention relates to a method for improving the efficiency of somatic cell nuclear transfer, in particular to a method for improving the efficiency of pig somatic cell nuclear transfer by pretreating donor cells with Calyculin A, and belongs to the field of breeding or fertilization methods. Background technique [0002] Since the birth of the cloned sheep "Dolly", somatic cell nuclear transfer technology has been successful in many mammals, but the cloning efficiency is still very low, averaging between 0.1% and 2.0%. In order to improve the efficiency of nuclear transfer, researchers have conducted a lot of research on nuclear transfer technical procedures, oocyte maturation quality, in vitro embryo culture conditions, screening and pretreatment of nuclear donor cells, etc. Although some progress has been made, nuclear transfer The maturity rate of embryo development is still very low (Li, J., Du, Y., et al. Chemically Assisted Handmade Enucleati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/877
Inventor 孔庆然刘忠华石永乾朱江王娟
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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