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Method of inducing pluripotent stem cells throuhg c-Jun N terminal deletion and applications

A technology for pluripotent stem cells and pluripotent stem cells, which can be applied to cells modified by the introduction of foreign genetic material, recombinant DNA technology, and the use of vectors to introduce foreign genetic material. question

Inactive Publication Date: 2013-10-16
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the progress in revealing the mechanism of transcription factor-induced reprogramming process is still slow

Method used

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  • Method of inducing pluripotent stem cells throuhg c-Jun N terminal deletion and applications
  • Method of inducing pluripotent stem cells throuhg c-Jun N terminal deletion and applications
  • Method of inducing pluripotent stem cells throuhg c-Jun N terminal deletion and applications

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0071] Experimental Example 1: Construction of c-Jun and its different deletion types, c-Jun truncation promotes SKO-induced reprogramming efficiency.

[0072] Using conventional molecular cloning methods, the c-Jun protein full-length (324 amino acids), and 1-256, 75-324, 170-334, 254-334, 274-334, 170-334 and other 6 different amino acid fragments The nucleic acid sequence was cloned into the pMXs retroviral expression vector. Final cloned fragments such as figure 1 shown.

[0073] After the mouse fibroblasts were digested, 20,000 cells per well were planted in a 12-well plate, and infected with OKS or OKSM and the expression vector constructed in Example 1, respectively. After infection, culture with universal reprogramming medium, replace the medium every day, and count the number of fluorescent clones under a fluorescent microscope on appropriate days.

[0074] like figure 2 As shown, compared with empty load, the full length of C-Jun protein, the 1-256 fragment, and...

experiment example 2

[0075] Experimental example 2: c-Jun-DN promotes the reprogramming efficiency induced by SKO, and can replace the reprogramming factor Oct or Sox2.

[0076] MEF cells were infected with c-JunDN and three-factor SKO or different factor combinations respectively, and cultured in serum or serum-free induction culture system, the medium was changed every day and the reprogramming efficiency was calculated on appropriate days. To observe the effect of c-JunDN on reprogramming efficiency and its substitution effect on reprogramming factors.

[0077] The "serum-free medium SF1" described herein is the iPS-SF1 medium in the invention authorized by China with application number 200910038883.4. The serum medium described herein is a mouse embryonic stem cell culture medium well known to those skilled in the art, and its main components are basal medium supplemented with fetal bovine serum and leukocyte inhibitory factor LIF. The "serum medium" in this example is the mES medium in the i...

Embodiment 3

[0087] Example 3: c-Jun-DN can maintain the pluripotency of stem cells.

[0088] In order to study the role of C-Jun gene in the maintenance of pluripotency of embryonic stem cells, we studied the expression of this gene in the differentiation process of embryonic stem cells, adult cells and embryonic stem cells. Effects on functional maintenance and differentiation.

[0089] RNA was extracted from mouse embryonic fibroblasts (MEF) and embryonic stem cells (ES), and the expression of c-Jun in the two cells was analyzed by RT-PCR.

[0090] like Figure 11 It was shown that the expression level of c-Jun in MEF cells was much higher than that in embryonic stem cells. Indicating that c-Jun factor may be detrimental to the maintenance of pluripotency.

[0091] ES cells were differentiated into embryoid bodies (EBs) in a standard way, samples were collected at different days, RNA was extracted, and the expression of c-Jun in the two cells was analyzed by RT-PCR.

[0092] like ...

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Abstract

The invention relates to the stem cell research in the biological technology field, in particular to a method of inducing pluripotent stem cells throuhg the c-Jun N terminal deletion and applications. The technical scheme aims to provide an application of combined utilization of the c-Jun N terminal deletion and pluripotent stem cell inducing factors in a luripotent stem cell inducing process, and the c-Jun N terminal deletion is a series of clipped amino acid sequences or corresponding nucleotide sequences of amino acids from 170 bits to 334 bits of the c-Jun N protein of AP-1 family protein. According to the method and applications, during the process of pluripotent stem cell inducing, N terminal cut truncated type of the c-Jun N protein of the AP-1 family protein is used, an efficient reprogramming induction system is provided, micro molecules or genes of core reprogramming factors are replaced, and a promoting role of revealing core reprogramming factors in the reprogramming is played.

Description

technical field [0001] The invention relates to stem cell research in the field of biotechnology, in particular to a method for inducing pluripotent stem cells by deleting the c-Jun N-terminal of AP-1 family protein and its application. Background technique [0002] The AP-1 family is a class of transcriptional activator proteins that cells respond to external signals including growth factors, cytokines and extracellular stress. Structurally, it consists of different subunits to form homologous or heterologous dimers. These subunits include Jun, Fos and ATF. Each subunit has a leucine zipper structure, which forms a zinc finger module combined with DNA in pairs. [0003] ES cells and embryonic stem cells are cells that are isolated from the inner cell mass of blastocysts and have the characteristics of in vitro unlimited proliferation, self-renewal and multidirectional differentiation. Whether in vitro or in vivo, ES cells can be induced to differentiate into almost all c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10
Inventor 裴端卿韩庆凯刘晶陈捷凯韦备彭梅秀
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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