Uses of estrogen correlated acceptor 2 in maintaining versatility of mouse embryo stem cell

A technology of embryonic stem cells and mouse embryos, applied in the application, introduction of foreign genetic material modified cells, measurement/testing of microorganisms, etc., can solve the problem that the molecular mechanism of stem cells to maintain pluripotency and self-renewal is not very clear, etc.

Inactive Publication Date: 2009-07-29
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Recently, large-scale biological tools (such as microarray nucleoproteomics, RNA interference, etc.) for the analysis of stem cells have generated a large amount of data on regulators of stem cell pluripotency (Boyer, L.A., et al., (2005) Cell 122 (6), 947-956; Assou, S. et al. (2007) Stem Cells 25(4), 961-973; Orkin, S.H. (2005) Cell 122(6), 828-830; Wang J. et al. ( 2006) Nature 444(7117), 364-368; Ivanova, N, et al., (2006) Nature 442(7102), 533-538), but the molecular mechanism of stem cells maintaining pluripotency and self-renewal is not very clear

Method used

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  • Uses of estrogen correlated acceptor 2 in maintaining versatility of mouse embryo stem cell
  • Uses of estrogen correlated acceptor 2 in maintaining versatility of mouse embryo stem cell
  • Uses of estrogen correlated acceptor 2 in maintaining versatility of mouse embryo stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Culture of ES cell lines

[0030] Mouse ES cells (CGR8 ES and E14T ES) were cultured on 0.1% gelatin-coated substrates, supplemented with 20% fetal bovine serum FBS (GIBCO), 100 mM non-essential amino acids (Invitrogen, CA), 0.55 mM β-mercaptoethanol ( Sigma, St.Louis, MO), 2mML-glutamic acid (Invitrogen, CA), and 1000 units / ml human recombinant LIF (CHEMICON, CA) were cultured in a 37°C incubator (see Pan Guangjing et al., (2006) Faseb J 20(10), 1730-1732, Mitsui K et al., (2003) Cell 113(5), 631-642, Chambers, I et al., (2003) Cell 113(5), 643-655 , Pan, G et al. (2005) J Biol Chem 280(2), 1401-1407).

Embodiment 2

[0031] Example 2: Construction of Esrrb vector (SEQ ID NO: 1) and expression of Esrrb vector (pPyCAGIP-Esrrb) in ES cells

[0032] 2ug of total RNA was reverse transcribed using a reaction system with a final volume of 20ul (see Pan, G et al., (2006) Faseb J 20(10), 1730-1732) to obtain a cDNA library. The mouse Esrrb fragment was amplified using the primers in Table 1, and then recovered by agarose gel electrophoresis.

[0033] Table 1: Primers for cloning Esrrb fragments:

[0034] SEQ ID NO

[0035] Plasmids for the generation of ES stable genetic lines were constructed by inserting PCR fragments into the multiple cloning site pme I of the pPyCAGIP vector, that is, the pPyCAGIP vector was digested by pme I for 4 hours at 37°C, and then amplified from embryonic stem cell cDNA The mouse Esrrb fragment was ligated in the presence of ligase at 16°C for 4 hours to obtain the vector pPyCAGIP-Esrrb.

Embodiment 3

[0036] Example 3: Construction of stably transfected cell lines and observation of their morphology

[0037] with 10 3 Feeder-free ES cells (CGR8 and E14T) cultured in u / ml LIF (Chemicon) ES medium were planted in a 6cm dish, and 2ug of vectors expressing pPyCAGIP and pPyCAGIP-Esrrb were transfected. 24 hours after transfection, cells were diluted 1:100 and seeded into new 10 cm dishes for selection. Puromycin (2ug / ml, Invitrogen, CA) was added to the medium during selection. After 10 days of selection, the transfected ES cells were observed under an Olympus inverted microscope, as shown in figure 2 As shown, the stably transfected ES cell line integrated with the pPyCAGIP-Esrrb vector and the stably transfected ES cell line integrated with the PyCAGIP empty vector both showed typical ES cell phenotypes, and both showed strong AP (alkaline phosphatase) positive reaction. However, in the absence of LIF (interleukin growth factor), after 6 days of culture, it was found that...

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Abstract

The invention discloses a carrier construction method of a mouse Esrrb (estrogen-related receptor 2) gene and an application thereof in researches on mouse embryonic stem cells. Lack of the Esrrb in the mouse leads to stem cell differentiation of an epiblast and abnormal embryonic development, the embryo dies within ten and half a days during the development, which indicates that the Esrrb plays a very important role in maintaining normal embryonic development of the mammal. In the invention, the Esrrb is taken as a transcription factor which can maintain pluripotent function of the embryonic stem cells without depending on LIF (Leukemia Inhibitory Factor). The construction method of the recombinant of the invention and the application thereof in embryonic stem cells provide reference for studying how other transcription factors play a role in the embryonic stem cells.

Description

Technical field: [0001] The invention relates to the construction expression vector of Esrrb (estrogen-related receptor 2) and the use in embryonic stem cells. The Esrrb is an estrogen-related receptor family receptor and a transcription factor that plays an important role in the embryonic development process. Background technique: [0002] Embryonic stem cells (ES cells) are a type of pluripotent cells derived from the inner cell mass (ICM-Inner cell mass) of the blastocyst (Blastocyst) stage in the early stage of mammalian embryo development. One of the important characteristics is It still has the ability to develop into three complete germ layers, which is also the basis for the clinical application of embryonic stem cells. Therefore, therapies based on this totipotent or pluripotent property of stem cells are a promising solution for many currently unsolved diseases such as Parkinson's disease. However, before human ES cells can be safely and effectively used to treat ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N5/10C12Q1/68G01N33/53
Inventor 裴端卿张小飞张娟王涛
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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