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Embryonic stem cell specific marker GM-CSFR alpha and application thereof

A GM-CSFR and embryonic stem cell technology, applied in the field of embryonic stem cell-specific marker GM-CSFRα, can solve the problem of limited number of embryonic stem cell membrane surface markers, and achieve high specificity and sensitivity

Inactive Publication Date: 2013-07-10
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the specificity of these markers has certain limitations: they are also expressed in some adult stem cells, normal tissues or cancer cells, and the number of membrane surface markers specific to embryonic stem cells is even more limited

Method used

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  • Embryonic stem cell specific marker GM-CSFR alpha and application thereof
  • Embryonic stem cell specific marker GM-CSFR alpha and application thereof
  • Embryonic stem cell specific marker GM-CSFR alpha and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Preparation of Rabbit Monoclonal Antibody

[0020] Using mouse embryonic stem cells (mES) as the immunogen, each time 1×10 8 A mES was used to immunize 3-month-old New Zealand white rabbits for 4 times, with an interval of 3 weeks between each immunization. Serum was collected after four times of immunization, and 1 × 10 8 3 days after boosting, rabbit spleen cells were isolated and fused with myeloma cell 240E-W2 (purchased from Epitomics), and the hybridoma cell line ZJUESRMAB29 (accession number CGMCC) producing GM-CSFRα rabbit monoclonal antibody was screened out. NO.7302), applied to the identification and isolation of stem cells.

Embodiment 2

[0021] Example 2 Application of GM-CSFRα as a marker for embryonic stem cells

[0022] The expression of GM-CSFRα decreases with cell differentiation and is limitedly expressed in adult tissues, which can be used as a new marker for the identification of embryonic stem cells.

[0023] IHC staining experiment: Prepare the frozen sections of each tissue; place the frozen sections of each tissue in 3% hydrogen peroxide for 10 minutes to inactivate endogenous peroxidase; use blocking solution (containing 10% goat serum and 1% BSA in PBS) at 37°C for 1 hour; add primary antibody (GM-CSFRα rabbit monoclonal antibody prepared in Example 1) and incubate at 37°C for 1 hour; then add goat anti-rabbit-HRP secondary antibody and incubate at 37°C for 1 hour ; Finally, use freshly prepared 3,3'-diaminobenzidine (DAB) to develop color at 37°C for 5 minutes, observe and read the film. The result is as figure 1 shown. The results of IHC staining showed that GM-CSFRα was highly expressed in ...

Embodiment 3

[0024] Example 3 Application of GM-CSFRα as a marker for embryonic stem cells

[0025] ICC staining experiment proved that GM-CSFRα rabbit monoclonal antibody can be applied to the identification and sorting of embryonic stem cells.

[0026] ICC experiment: Cultured cells were fixed with 4% formaldehyde (Sigma) at 37°C for 1.5 hours; then blocked with blocking solution (PBS containing 10% goat serum and 1% BSA) at 37°C for 1 hour; the blocked cells were added with primary antibody ( The GM-CSFRα rabbit monoclonal antibody prepared in Example 1) was incubated at 37°C for 1 hour; washed with PBS; the washed cells were added with secondary antibody and incubated for 45 minutes at 37°C in the dark; finally stained with 0.5 μg / ml Hochest33258 (Sigma) nuclear 5 minutes; observed and photographed under a fluorescent microscope (Olympus IX-70). The result is as figure 2 shown. GM-CSFRα rabbit monoclonal antibody ICC staining results showed that compared with undifferentiated mES c...

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Abstract

The invention provides an embryonic stem cell specific marker GM-CSFR alpha (granular cell-macrophage colony stimulating-factor receptor alpha link) and an application thereof. The embryonic stem cell is directly used as immunogen, the prepared GM-CSFR alpha polyclonal antibody or rabbit antibody GM-CSFR alpha is used as a stem cell identification reagent, the detected specificity and sensitivity are higher, and the embryonic stem cell specific marker GM-CSFR alpha can be used for the separation and activity detection of the embryonic stem cell. An embryonic stem cell detection reagent kit containing the GM-CSFR alpha polyclonal antibody or a rabbit antibody GM-CSFR alpha not only can be used for testing the totipotency of the embryonic stem cell, but also can be used for detecting the activity of the stem cell, so that the function of the detection reagent kit can be enlarged.

Description

technical field [0001] The invention belongs to the field of cell biology and immunology, and specifically relates to embryonic stem cell specific marker GM-CSFRα and its application. Background technique [0002] Embryonic stem cells (ES cells) refer to normal diploid cells isolated from the inner cell mass of embryos, which can be cultured in vitro for a long time and maintain the ability of self-renewal and the potential to differentiate into cells of all three germ layers. The high self-renewal ability and differentiation pluripotency of embryonic stem cells are important characteristics that distinguish it from other cells. Its specific molecular markers are of great significance for the identification, isolation, self-renewal and differentiation mechanism of embryonic stem cells. Currently commonly used embryonic stem cell-specific markers include transcription factors OCT4, NANOG, REX1, etc.; surface markers SSEA-1, SSEA-3, TRA-1-60, CD9, etc. Antibodies corresponding...

Claims

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Application Information

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IPC IPC(8): C07K14/715C12N5/20G01N33/68G01N33/577C12R1/91
Inventor 宋秀丽谭舟张铭顾斌张佳蓉侯倩女
Owner ZHEJIANG UNIV
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