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Ungulate animal cell line for inductively expressing pluripotent maintenance gene and construction thereof

An ungulate and fibroblast technology, applied in the field of genetic engineering, can solve problems such as failure to establish an embryonic stem cell line

Inactive Publication Date: 2013-11-13
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite long-term efforts, scientists have not been able to establish true embryonic stem cell lines from pigs and other ungulates

Method used

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  • Ungulate animal cell line for inductively expressing pluripotent maintenance gene and construction thereof
  • Ungulate animal cell line for inductively expressing pluripotent maintenance gene and construction thereof
  • Ungulate animal cell line for inductively expressing pluripotent maintenance gene and construction thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Embodiment 1, packaging and titer determination of virus for infection

[0087] 1. Amplification, concentration and purification of plasmids for virus

[0088] The vectors Lv-ef1α-EGFP-TRE-Oct4 and Lv-ef1α-rtta-IRES-EGFP were sequenced to identify the correct plasmid transformation competent bacteria GBE180 (purchased from Stance Biological Co., Ltd.) for amplification. The extraction kit (AxyPrep plasmid Maxiprep Kit) was used to extract the medium amount of the lentiviral vector plasmid.

[0089] The extracted plasmid was purified and concentrated in an ultra-clean bench. The steps of purification and concentration are as follows: 1) Add 1 / 10 volume of NaAC (3M) after pumping, then add 2 times the volume of absolute ethanol, and mix well; 2) Centrifuge at 13000rpm for 20min, suck off the supernatant, and use Rinse with (v / v) ethanol once, and absorb absolute ethanol; 3) After drying in the ultra-clean bench, use double distilled water to dissolve the plasmid precipi...

Embodiment 2

[0115] Embodiment 2, cell culture

[0116] 1. 293T cells

[0117] For the culture of 293T cells and 293T-ETD, the cells were digested with 0.25% (w / v) trypsin at 37°C for 5 minutes, and the digestion reaction was terminated with D-MEM containing 10% FBS, and the cell mass was blown away. After making into single cells, passaging or cryopreservation at a ratio of 1:8.

[0118] 2. Porcine fibroblast cell line

[0119] The porcine fibroblast cell line was obtained from the ear fibroblasts of one-month-old pigs and cultured in D-MEM with 10% FBS. During subculture, cells were digested with 0.25% trypsin for 5 min at 37°C, and the digestion reaction was terminated with D-MEM containing 10% FBS.

Embodiment 3

[0120] Example 3, lentivirus infection of pig ear fibroblasts

[0121] Specific steps are as follows:

[0122] 1) Pre-spread gelatin in a T25 bottle, and then seed 500,000 pig ear fibroblasts per well.

[0123] 2) Mix the lentivirus Lv-ef1α-EGFP-TRE-Oct4 and Lv-ef1α-rtta-IRES-EGFP packaged in Example 1 at a ratio of 1:1 (calculated as virus pfu), and infect pigs with MOI (multiplicity of infection) 2 ear fibroblasts.

[0124] 3) The medium was changed on the second day after infection, and the proportion of EGFP-positive cells was observed under a fluorescence microscope.

[0125] The result is as image 3 , the proportion of EGFP-positive cells was about 15%.

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Abstract

The invention relates to an ungulate animal cell line for an inductively expressed pluripotent maintenance gene and construction thereof. The ungulate animal fibroblast cell line for expressing and maintaining pluripotent key gene oct4 by an inducible system (tet-on) is established by the invention. The cell line has the characteristics of low virus copy number, obviously up-regulated oct4 expression and high purity for cell single cell source. The cell line is used as donor cell for nuclear transplantation; and the expression of oct4 is induced by drug doxycycline, thereby facilitating reprogramme of egg cells after the nuclear transplantation is facilitated and finally achieving the object of establishing embryonic stem cells of the ungulate animals, so as to breed various transgenic animals.

Description

technical field [0001] The invention belongs to the field of genetic engineering; more specifically, the invention relates to an ungulate animal cell line inducibly expressing a pluripotency maintenance gene and its construction. Background technique [0002] Pigs are a common domestic animal that share considerable immunological and physiological similarities with humans. Pig organs are very similar to humans in terms of anatomical structure, physiological and biochemical characteristics, and their size is also similar to human organs, and the possibility of human-pig comorbidity is very small. Therefore, pigs are currently the preferred organ donor animals for xenotransplantation. The average lifespan of pigs is 20 years, and the longer life cycle also creates favorable conditions for specific research. Therefore, for a long time, pigs have been very useful experimental animals in many fields of biomedical research such as various diseases and the safety evaluation of ne...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10C12N15/873
Inventor 吴璐
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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