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Bovine induced expansion pluripotent adult stem cells, line establishment method and culture solution

A technology of adult stem cells and bovine fibroblasts, applied in the field of cell biology and molecular biology, can solve the problem of not forming chimeras, and achieve the effect of fast line establishment

Pending Publication Date: 2022-04-19
INNER MONGOLIA UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Wu Xia et al. (Establishment of bovine embryonic stem cells after knockdown of CDX2[J]. Scientific Reports, 2016, 6(1):28343-28343.) obtained bovine expanded pluripotent embryonic stem cells (CDX2 -KD bESCs), with the ability to differentiate in vitro and in vivo, but did not form chimeras

Method used

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  • Bovine induced expansion pluripotent adult stem cells, line establishment method and culture solution
  • Bovine induced expansion pluripotent adult stem cells, line establishment method and culture solution
  • Bovine induced expansion pluripotent adult stem cells, line establishment method and culture solution

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Embodiment 1

[0052] Preparation and culture of feeder cells

[0053] 1.1 Preparation and culture of feeder cells:

[0054] Bovine fibroblasts (BEF) were thawed, and the BEF culture medium was Knockout DMEM medium containing 10% FBS (BI), 1× glutamine, 1× non-essential amino acids, and 1× penicillin-streptomycin. When the confluence of the cells reached more than 80%, the cells were passaged according to the ratio of 1:16. When the confluence of the cells reaches 80% again, add 10 μg / ml mitomycin and culture in the incubator for 2.5-3 hours. After trypsinization, stop the digestion with BEF culture medium, centrifuge and remove the supernatant, resuspend in culture medium containing 10% DMSO, 10% fetal bovine serum and 80% BEF culture medium, and freeze them as feeder layer cells.

[0055] 1.2 Culture of feeder cells:

[0056] The day before the experiment, the feeder cells were thawed and inoculated on a petri dish covered with 0.1% gelatin and cultured with BEF medium.

Embodiment 2

[0058] Establishment, passage and cryopreservation of bovine induced expanded pluripotent adult stem cells

[0059] 2.1 Establishment of bovine induced expanded pluripotent adult stem cells:

[0060] Bovine fibroblasts (BEF) were thawed, and the BEF culture medium was Knockout DMEM medium containing 10% FBS (BI), 1× glutamine, 1× non-essential amino acids, and 1× penicillin-streptomycin. When the confluence of the cells reached more than 80%, the collected cells were counted at 1×10 6 . The exogenous genes Oct4, Sox2, Klf4, cMyc, LIN28, Nanog, LRH1 and RARG were transferred. See SEQUENCE LISTING for the transferred foreign gene sequence. Cultured on feeder cells that had been thawed in advance, the culture conditions were M15+DOX culture medium, M15+DOX culture medium 15% FBS (BI), 1×glutamine, 1× non-essential amino acid, 1× penicillin and streptomycin , Doxycycline Dox Knockout DMEM medium. After the stem cell clones grew out, they were picked into the cytokine culture ...

Embodiment 3

[0076] Detection of bovine induced expanded pluripotent adult stem cells

[0077] 3.1 AP staining of bovine induced expanded pluripotent adult stem cells:

[0078] When the confluence of bovine induced expanded pluripotent adult stem cells reached 80%, the sample cells were fixed with citrate-acetone-formaldehyde, and stained according to the alkaline phosphatase staining kit [theAlkaline Phosphatase]. The operating instructions of the Kit (Sigma-Aldrich)] were strictly followed. React at room temperature in the dark for 30-40 minutes, and examine under the microscope. See the test results figure 2 , AP staining results showed strong alkaline phosphatase activity, the stem cells were in an undifferentiated state.

[0079] 3.2 Bovine induced extended pluripotency adult stem cell pluripotent gene detection:

[0080] The total RNA of bovine induced expanded pluripotent adult stem cells was obtained according to the extraction method of Qiagen's RNeasy Mini Kit, and dissolved...

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Abstract

The invention discloses bovine induced expansion pluripotent adult stem cells, a line establishment method and a culture solution, and the bovine induced expansion pluripotent adult stem cells are obtained by transgenosis and induced culture from bovine fibroblast cells and can be stably subcultured in vitro. The stem cell has totipotency, stability and safety, can be differentiated in an in-vivo and out-of-embryo tissue and an out-of-embryo tissue, and can be applied to various life science and medical fields such as breeding and breeding, gene editing, animal cloning, medical models, drug development carriers, induced production of bovine reproductive gametes and the like.

Description

technical field [0001] The invention belongs to the technical field of cell biology and molecular biology, and in particular relates to a bovine induced expanded pluripotent adult stem cell, a line establishment method and a culture medium. Background technique [0002] Induced pluripotent stem cells (iPSCs) are stem cells obtained by introducing specific transcription factors into somatic cells and inducing their nuclear reprogramming. They are obtained by expressing specific transcription factors in differentiated cells and inducing the reprogramming of somatic cells. Cells capable of continuous self-renewal and multilineage differentiation potential. Induced pluripotent stem cells (iPSCs) are capable of unlimited self-renewal and differentiation into all cell types including the three germ layers, and the biggest feature is that they do not need to use fetuses or embryos, but differentiated cells through the adjustment of gene expression Direct reprogramming to the pluri...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/074
CPCC12N5/0696C12N2506/1307C12N2510/00C12N2501/603C12N2501/602C12N2501/604C12N2501/606C12N2501/608C12N2501/605C12N2500/80C12N2501/385C12N2501/115C12N2501/415C12N2501/16C12N2501/405C12N2500/44C12N2500/38C12N2501/235Y02A50/30
Inventor 李喜和赵丽霞王子馨
Owner INNER MONGOLIA UNIVERSITY
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