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Application of Bend5 protein to acceleration of induced differentiation of embryonic stem cells to into primitive reproductive progenitor-like cells

A technology of embryonic stem cells and induced differentiation, applied in embryonic cells, peptide/protein components, medical preparations containing active ingredients, etc., can solve the problem of low efficiency of fate transformation and achieve good application prospects

Active Publication Date: 2016-10-12
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in this in vitro induction system described above, the fate transition of embryonic stem cells to germ cell differentiation is very inefficient

Method used

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  • Application of Bend5 protein to acceleration of induced differentiation of embryonic stem cells to into primitive reproductive progenitor-like cells
  • Application of Bend5 protein to acceleration of induced differentiation of embryonic stem cells to into primitive reproductive progenitor-like cells
  • Application of Bend5 protein to acceleration of induced differentiation of embryonic stem cells to into primitive reproductive progenitor-like cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1Bend5 Gene expression in germ-like cells

[0067] 1. Real-time quantitative PCR assay to detect the expression status of Bend5 gene in germ-like cells

[0068] (1) Experimental cells: mouse embryonic fibroblasts (MEF), mouse embryonic stem cells (mES) and primordial germline progenitor cells (PGC).

[0069] (2) Breed Oct4 reporter gene mice, and use the 13.5-day fetal mice to isolate mouse embryonic fibroblasts (MEFs) and primitive genital ridges, and analyze the ratio of GFP-Oct4 by flow cytometry after the primitive genital ridges are digested, GFP-Oct4 positive cells were sorted out. After the three kinds of cells were collected, the total RNA was extracted, and the total RNA of the cells was extracted using the RNeasy kit (Qiagen Company), and the RNA was treated with DNase I (Ambion Company) at 37°C for 30 minutes to digest the DNA. cDNA was synthesized using AffinityScriptqPCR cDNA Synthesis Kit (Stratagene). Using SYBR green PCR mixture kit (App...

Embodiment 2

[0071] Example 2Bend5 Promotes germ-like cell formation

[0072] 1. Construct a stem cell line stably expressing the Stella-GFP reporter gene of Bend5. After removing the trophoblast cells, mercaptoethanol (sigma) containing 15% serum replacement (KSR, purchased from Gibco) at a final concentration of 0.1mM, 1mM Essential amino acids (purchased from Gibco), 1 mM sodium glutamate (purchased from Gibco), streptomycin / penicillin (purchased from Gibco), LIF (purchased from Millipore), 2i (purchased from Gibco) Knock-out DMEM (purchased After two generations of culture in Gibco) medium, adding cytokines such as bFGF and Acvitin A to the induction medium can promote the differentiation of mouse embryonic stem cells (mESCs) into epiblast like cells (EpiLCs). Two days later, the cells were collected for the analysis of the green fluorescent protein (GFP) signal, and the Stella-positive cells were analyzed to detect their EpiLCs induction efficiency. The expression levels of Ste...

Embodiment 3

[0081] Example 3Bend5 Promote Research on the mechanism of primordial germline progenitor-like cells

[0082] The wild-type Bend5 and the BEN domain-deleted Bend5 were constructed respectively, and the mechanism of Bend5 promoting primordial germline progenitor-like cells was studied.

[0083] 1. Experimental method

[0084] (1) EpiLC induction and flow cytometric analysis methods are the same as in Example 2.

[0085](2) Embryoid body induction differentiation experiment: count the stem cells after digestion, and resuspend the stem cells in PBS to about 1000 cells / μl. Add 1000 cells to each well of low cell binding U bottom 96 well plate (purchased from Nunc), supplement with 100 μl LIF-free ES medium, move to the incubator and culture for two days without disturbing the cells. After that, half of the culture medium was changed every day.

[0086] (3) Chromatin immunoprecipitation: cells (approximately 1×10 7 ) for 10 min, and then stop the cross-linking with a fi...

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Abstract

The invention discloses application of Bend5 protein to acceleration of induced differentiation of embryonic stem cells into primitive reproductive progenitor-like cells. An amino acid sequence of the Bend5 protein is shown as SEQ ID NO.1. A primary structure of the protein comprises a Coiled-coil structure domain and a BEN structure domain. Specifically, excessive expression of the Bend5 can accelerate differentiation of mouse embryonic stem cells into similar ectoderm-like cells; the excessive expression of the Bend5 also can accelerate differentiation of mouse embryonic stem cells into primitive reproductive progenitor-like cells. Furthermore, research results show that transcription factor protein Bend5 is combined with Stella gene with dependence on BEN structure domain protein, and Tet1 and Oct4 are recruited to activate expression of the Stella gene, so that the differentiation of embryonic stem cells into primitive reproductive progenitor-like cells can be accelerated. The Bend5 protein has a good application prospect in treatment of infertility.

Description

technical field [0001] The invention belongs to the technical field of biomedicine. More specifically, it relates to the application of protein Bend5 (BEN domain containing protein 5) in improving the induction of embryonic stem cells to differentiate into primordial germline progenitor-like cells. Background technique [0002] Infertility often has a serious impact on families and society. Investigations have shown that the divorce rate of infertile couples is 2.2 times that of the normal population, and has become an important medical and social problem. The etiology of infertility is diverse, among which germ cell defects and deletions are very common factors causing infertility. [0003] Studies have shown that the transformation efficiency of embryonic stem cells into primitive germline progenitor-like cells can be improved through the mediation of transcription factors. Under in vitro conditions, adding cytokines such as bFGF and Acvitin A to the medium can promote t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735A61K38/17A61K45/00A61P15/08
CPCA61K38/17A61K45/00C12N5/0611C12N2506/02C12N2501/60
Inventor 松阳洲黄军就时光
Owner SUN YAT SEN UNIV
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