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Method for improving transferring efficiencies of pig somatic cell nucleuses

A cell nucleus and donor cell technology, applied in botany equipment and methods, biochemical equipment and methods, plant gene improvement, etc., can solve the problem that the developmental ability of pig cloned embryos has not been significantly improved, and achieve an increase in the number of cells in the blastocyst , Improving the efficiency of pig somatic cell nuclear transfer and improving the blastocyst rate

Active Publication Date: 2014-02-12
广东中芯种业科技有限公司
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AI Technical Summary

Problems solved by technology

This method has a certain effect on improving the efficiency of somatic cell nuclear transfer in mice, but does not significantly improve the developmental ability of pig cloned embryos

Method used

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  • Method for improving transferring efficiencies of pig somatic cell nucleuses
  • Method for improving transferring efficiencies of pig somatic cell nucleuses
  • Method for improving transferring efficiencies of pig somatic cell nucleuses

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Experimental program
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Embodiment Construction

[0022] Experiments without specific methods were performed according to the methods described in common somatic cell cloning techniques.

[0023] 1: In vitro maturation culture of porcine oocytes

[0024] Pig ovaries are collected from the slaughterhouse, placed in normal saline and sent back to the laboratory. The fluid in the 2-6mm diameter follicle is aspirated with a syringe. Select the cumulus cell-oocyte complex with dense cumulus and more than 3 layers of cumulus under a solid microscope, at 39°C, 5% CO 2 , Cultivate for 42-44h in a saturated humidity environment. The cumulus cells were removed by hyaluronidase, and oocytes expelling the first polar body were selected as nuclear transfer recipients.

[0025] 2: Pig ear skin fibroblast culture

[0026] The brief aseptic operation is as follows: Cut the pig ear skin tissue into pieces, wash the tissue pieces with DMEM (Gibco), resuspend with appropriate amount of fetal bovine serum and transfer to a petri dish, keep a...

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Abstract

The invention discloses a method for improving transferring efficiencies of pig somatic cell nucleuses. The method includes the following steps of selecting and cultivating egg mother cells; separating and cultivating donor cells; using a blind aspiration method to remove a mature egg mother cell nucleus and a first polar body, injecting a donor cell to perivitelline space, subjecting a reconstructed embryo to electrofusion, and activating the reconstructed embryo; and placing the reconstructed embryo in a PZM-3 culture solution containing RG108 with concentration of 100-200 mu M and Scriptaid with concentration of 100-500 nM to be cultivated for 14-16 hours, and then placing the reconstructed embryo in a PZM-3 culture solution for cultivation. According to the method, development blastosphere rates and blastosphere qualities of pig cloning early embryos can be obviously improved; and simultaneously, two epigenetic modification of gene methylation and acetylation with close connection and synergistic effects can be adjusted, the method is better in effects than that only by using the Scriptaid for treatment, early genetic expression of embryos can be adjusted to be normal, and finally the transferring efficiencies of the pig somatic cell nucleuses are improved.

Description

technical field [0001] The invention relates to a method for animal cloning, in particular to a method for improving the efficiency of pig somatic cell nuclear transplantation. Background technique [0002] Somatic cell nuclear transfer technology is to transfer donor cells into enucleated oocytes to form reconstituted embryos through micromanipulation and cell fusion technology. After activating the reconstituted embryos, they are cultured and transplanted, and finally achieve the purpose of expanding the population of the same genotype. . Since the birth of "Dolly" sheep in 1997, somatic cell nuclear transfer technology has been successfully used in various mammals one after another. Somatic cell culture, egg activation, embryo in vitro culture and embryo transfer technology have established a set of relatively mature pig somatic cell nuclear transfer procedures. But so far, the efficiency of somatic cell nuclear transfer in pigs is still very low (number of births / numbe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/877
Inventor 吴珍芳贺晓燕许卫华石俊松周荣
Owner 广东中芯种业科技有限公司
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