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A method for inducing reprogramming of bovine fetal fibroblasts

A fibroblast and reprogramming technology, applied in the cell field, can solve the problems of low cell induction efficiency and cancer induction

Inactive Publication Date: 2016-09-07
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the efficiency of cell induction is low, and it faces the threat of inducing cancer, which seriously restricts its application.

Method used

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  • A method for inducing reprogramming of bovine fetal fibroblasts
  • A method for inducing reprogramming of bovine fetal fibroblasts
  • A method for inducing reprogramming of bovine fetal fibroblasts

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0045] In some specific embodiments, the preparation method of the Xenopus oocyte extract specifically includes:

[0046] Step 1. Collect Xenopus eggs separately in a beaker, pour off the excess water, suck out the waste eggs with a plastic straw, and keep the eggs with clear outline and uniform shape;

[0047] Step 2. After removing excess water and waste eggs, add freshly prepared 2% cysteine, and gently stir the egg-removing film;

[0048] Step 3. Pour off the above solution, wash eggs three times with 0.25×MMR, and once with 1×MMR;

[0049] Step 4. Rinse once with the cell lysate. At this stage, pick out bad eggs with a plastic straw again, and rinse again with the cell lysate;

[0050] Step 5. Transfer the eggs to the cell lysate containing 1mM dimercaptothreitol and 100μL protease inhibitor, pour off the cell lysate after gently stirring and transfer the eggs to a 15mL centrifuge tube, and use a plastic straw after the eggs settle Remove excess solution from the upper ...

Embodiment 1

[0065] Example 1: Superovulation of Xenopus laevis

[0066] Selectively mature female Xenopus laevis, superficially injected 100 μL of freshly prepared PMSG (concentration: 1 IU / μL) into the dorsal lymphatic sac. After injection, Xenopus was placed in 0.1M saline to prevent infection of Xenopus. Inject 100 μL of HCG (concentration: 5 IU / μL) at the same position one day before egg collection (usually 3-30 days after injection of PMSG). Xenopus laevis were placed alone in 0.1M saline after injection. After 16-22 hours, the Xenopus laevis was taken out, the excess water was poured off, and the Xenopus laevis eggs were collected.

Embodiment 2

[0067] Example 2: Xenopus oocyte content extraction

[0068] 1. Xenopus eggs were collected separately in a beaker, poured off the excess water, sucked out the waste eggs with a plastic straw, and kept the eggs with clear outline and uniform shape.

[0069] 2. After removing excess water and waste eggs, add freshly prepared 2% cysteine, and gently stir the egg-removing gelatinous film, the process should not exceed 5 minutes. When the glue film is completely removed, the eggs are usually clumped together, and the washing solution is cloudy and precipitated.

[0070] 3. Pour off the membrane-removing buffer, wash the eggs three times with 0.25×MMR, and once with 1×MMR. The egg washing process should be as fast as possible to avoid the eggs staying in cysteine ​​and MMR for too long.

[0071] 4. Rinse once with the cell lysate. At this stage, use a plastic straw to pick out the bad eggs (very ill-defined, floating, fluffy eggs from animals and plants). Rinse again with the cell...

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Abstract

The invention belongs to the field of cells, and discloses a method for inducing fetal bovine fibroblast to reprogram. According to the invention, the fetal bovine fibroblast is subject to permeabilized treatment by a digitonin solution, and is subject to co-incubation with xenopus oocytes extract; whole medium containing 2mMcaCl2 is adopted to block reversible pores in the cell membrane, extract treated medium is adopted to cultivate and induce the fetal bovine fibroblast to reprogram to obtain fetal bovine fibroblast pluripotent stem cells. The method discloses by the invention is efficient, stable, and safe; the alkaline phosphatase staining of the fetal bovine fibroblast pluripotent stem cells disclosed by the invention shows a positive result; the acetylation degree of histone H3K9 is lowered; Oct-4 protein and pluripotent marker gene Oct-4, Nanog are expressed; therefore the fetal bovine fibroblast undergoes reprogramming, the gene expression mode of cells is changed to show characteristics of stem cell, partial growing totipotent is restored, and the fetal bovine fibroblast can be used as nuclear donor cells to prepare clone embryo for body cell clone.

Description

technical field [0001] The invention belongs to the field of cells, and in particular relates to a method for inducing reprogramming of somatic cells, in particular to a method for inducing reprogramming of bovine fetal fibroblasts. Background technique [0002] Since ancient times, cattle are domestic animals that humans love to raise. Cows have a wide range of uses: meat and milk can be used as food, leather is raw material for tanning, horns and bones are also industrial raw materials, horns can also be used as medicinal materials and handicrafts, and the bezoar inside is a precious medicinal material, and cattle can also be used for agricultural production. etc. to provide labor force and fertilizer. It has long been desired to obtain cattle breeds with certain desirable traits or properties, such as beef cattle breeds with high meat production, dairy cattle breeds with high milk production, long lactation intervals, and disease resistance, among others. Traditional br...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/073
Inventor 杜卫华范宗兴朱化彬
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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