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A method and application of improving porcine cell reprogramming ability

A reprogramming and cell technology, which is applied in the field of cell transplantation and embryonic development, can solve the problems that GSK126 has not been discovered, improve the efficiency of embryonic development or iPS induction, and achieve the effect of increasing the development rate and reducing the overall level

Inactive Publication Date: 2017-04-05
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has not been applied in the field of reprogramming, and it has not been found that GSK126 can be used to improve embryonic development or the efficiency of iPS induction

Method used

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  • A method and application of improving porcine cell reprogramming ability
  • A method and application of improving porcine cell reprogramming ability
  • A method and application of improving porcine cell reprogramming ability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Mature culture of porcine oocytes

[0031] The ovaries are collected from the slaughterhouse and transported back to the laboratory with 37°C normal saline, and the antral follicles with a diameter of 3-5mm are extracted to collect the cumulus oocyte complex, 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.

[0032] 2. Acquisition and Culture of Porcine Fetal Fibroblasts

[0033] Take 35-day-old pig fetal tissue aseptically, separate tissue cells by conventional 0.25% trypsin (Gibco) digestion method, add 20% fetal bovine serum to DMEM (Gibco) for primary culture, DMEM to add 10% feta...

Embodiment 2

[0039] 1. the mature culture of pig oocyte (with embodiment 1)

[0040] 2. Acquisition and Culture of Porcine Fetal Fibroblasts

[0041] PEF were treated with 0.5μM, 0.75μM and 3μM GSK126 for 48h before nuclear transfer. All the other are with embodiment 1.

[0042] 3. Somatic cell nuclear transfer (same as Example 1)

Embodiment 3

[0044] 1. the mature culture of pig oocyte (with embodiment 1)

[0045] 2. Acquisition and cultivation of pig fetal fibroblasts (same as Example 1)

[0046] 3. Somatic cell nuclear transfer

[0047] The fused embryos were treated with 0.05μM, 0.1μM and 0.15μM GSK126 for 24h. Embryos were then placed in normal PZM-3 for further culture. All the other are with embodiment 1.

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Abstract

The invention discloses a method for increasing the pig cell reprogramming capability and application, and belongs to the technical field of cell transplantation and embryonic development. The method provided by the invention comprises the following steps: obtaining and culturing a pig oocyte and a pig fetus fibroblast, then conducting somatic cell nuclear transplantation, and finally conducting iPs induction and alkaline phosphatase staining. According to the method, a micromolecular medicine GSK126 is adopted for treatment, and the micromolecular medicine GSK126 is used in the following three ways: 1) the pig fetus fibroblast is treated with the micromolecular medicine GSK126 before nuclear transplantation; 2) a fused embryo is treated with the micromolecular medicine GSK126 after the somatic cell nuclear transplantation of the pig fetus fibroblast; 3) the pig fetus fibroblast for iPS induction is treated after being cultured to be mature and before being inoculated through the utilization of the micromolecular medicine GSK126. The method provided by the invention can remarkably increases the developmental rate of cloned embryos and the iPS induction efficiency by 44.7% and 111.3% respectively.

Description

technical field [0001] The invention relates to a method and application for improving the reprogramming ability of pig cells, and belongs to the technical field of cell transplantation and embryo development. Background technique [0002] Somatic cell nuclear transfer technology is a kind of cell reprogramming technology. Since the birth of the cloned sheep "Dolly", this technology has been successful in many mammals, but the cloning efficiency is generally 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 technology procedures, oocyte maturation quality, in vitro embryo culture conditions, screening and pretreatment of nuclear donor cells, etc. Although some progress has been made, the cloning efficiency Still needs to be improved. At present, the most important reason for the low cloning efficiency is the incomplete nuclear reprogramming of somatic cells. So...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/877
Inventor 孔庆然解炳腾刘忠华
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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