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Line establishment method and culture solution for bovine expanded pluripotent embryonic stem cells

A technology of embryonic stem cells and pluripotency, 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

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Problems solved by technology

Wu Xia et al. (Establishment of bovine embryonicstem 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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  • Line establishment method and culture solution for bovine expanded pluripotent embryonic stem cells
  • Line establishment method and culture solution for bovine expanded pluripotent embryonic stem cells
  • Line establishment method and culture solution for bovine expanded pluripotent embryonic stem cells

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Experimental program
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Embodiment 1

[0048] Preparation and culture of feeder cells

[0049] 1.1 Preparation of feeder layer cells:

[0050] 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.

[0051] 1.2 Culture of feeder cells:

[0052] 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

[0054] Line establishment, passage and cryopreservation of bovine extended pluripotent embryonic stem cells

[0055] 2.1 Establishment of bovine extended pluripotent embryonic stem cells:

[0056] The embryos on the sixth day after in vitro fertilization were taken from cattle, washed twice with BO-WASH medium (BIOSCIENCE), and cultured on feeder cells that had been thawed in advance. The culture condition is bovine expanded pluripotent embryonic stem cell cytokine bEPSCM culture medium supplemented with 10 μM Y-27632, which contains 100 × penicillin streptomycin, 0.1 mM 2-mercaptoethanol, 1 μM CHIR99021 (Tocris, cat.no.4423 ), 0.3μM WH-4-023 (Tocris, cat.no.5413), 5μM XAV939 (Sigma, cat.no.X3004) or 5μM IWR-1 (Tocris, cat.no.3532), 50μg / ml vitamin C (Sigma, cat. no. 49752-100G), 10ng / ml LIF (Millipore), 20.0ng / ml Activin A (R&D), mTeSR TM 1 (STEMCELL) medium, in which IWR-1 and XAV939 are a kind of Wnt pathway inhibitor; CHIR99021 is a GSK3i inhibitor; WH-4-023 is an effect...

Embodiment 3

[0078] Detection of Bovine Extended Pluripotent Embryonic Stem Cells

[0079] 3.1 AP staining of bovine extended pluripotent embryonic stem cells:

[0080] When the confluence of bovine extended pluripotent embryonic stem cells reached 80%, the sample cells were fixed with citrate-acetone-formaldehyde and stained according to the Alkaline Phosphatase Kit [theAlkaline Phosphatase Kit]. (Sigma-Aldrich)] instructions 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 that the activity of alkaline phosphatase was very strong, and the embryonic stem cells were in an undifferentiated state.

[0081] 3.2 Bovine extended pluripotency embryonic stem cell pluripotency gene detection:

[0082] The total RNA of bovine extended pluripotent embryonic stem cells was obtained according to the extraction method of Qiagen's RNeasy Mini Kit, and dissolved in RNase-Free ...

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Abstract

The invention discloses a line establishment method and a culture solution of bovine expanded pluripotent embryonic stem cells (bEPSC), and the embryonic stem cell product is obtained by separation and induction from bovine embryos and can be stably subcultured. The embryonic stem cell has totipotency, stability and safety, and can be applied to breeding and breeding of cattle (dairy varieties, meat varieties, dual-purpose varieties or service varieties and the like), gene editing, animal cloning, medical models, drug development carriers, induced production of cattle reproductive gametes and other life science and medical fields. And large-scale production and application can be carried out.

Description

technical field [0001] The invention belongs to the technical fields of cell biology and molecular biology, and in particular relates to a method for establishing a line of bovine expanded pluripotent embryonic stem cells and a culture solution. Background technique [0002] Embryonic stem cells (ESCs) are a type of cells isolated from early embryos (before gastrulation stage) or primitive gonads, which have the characteristics of unlimited proliferation, self-renewal and multi-lineage differentiation in vitro. Whether in vitro or in vivo, embryonic stem cells can be induced to differentiate into almost all cell types in the body. Existing stem cell lines have been very useful for development, disease and treatment research. However, the two currently available stem cell lines -- embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) -- have certain limitations, and it is not yet possible for them to differentiate into every cell type, so when generating certai...

Claims

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Application Information

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IPC IPC(8): C12N5/0735
CPCC12N5/0606C12N2502/13C12N2500/46C12N2501/415C12N2501/998C12N2500/30C12N2500/44C12N2500/38Y02A50/30
Inventor 李喜和赵丽霞王子馨包斯琴刘澎涛曹贵芳
Owner INNER MONGOLIA UNIVERSITY
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