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Approaches to Altering Cell Fate

A cell and somatic cell technology, applied in the fields of changing cell chromatin structure, changing cell histone H3 methylation, changing cell heterochromatin protein HP1a expression and localization in cells, achieving good reproducibility and high efficiency , The method is simple and easy to implement

Inactive Publication Date: 2017-07-28
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, how to change cell fate, especially to promote the reprogramming of somatic cells, remains to be further studied

Method used

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  • Approaches to Altering Cell Fate
  • Approaches to Altering Cell Fate
  • Approaches to Altering Cell Fate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: Cloning of Gadd45 protein

[0055] Using the cDNA of the cell as a template, the primers shown in Table 1 were used to amplify the Gadd45a, Gadd45b and Gadd45g fragments respectively, and then recovered by agarose gel electrophoresis. Insert the PCR fragment after PmeI digestion at the multiple cloning site of the pMXs vector, connect, transform, pick bacteria, and sequence, and finally obtain the retroviral packaging plasmids of Gadd45a, Gadd45b and Gadd45g respectively.

[0056] Table 1 Primers for cloning Gadd45 protein fragments

[0057]

Embodiment 2

[0058] Embodiment 2: Gadd45 protein promotes reprogramming experiment

[0059] Retroviruses of reprogramming factors Sox2, Klf4, Oct4 and c-Myc as well as Gadd45a, Gadd45b and Gadd45g were packaged with Plat-E cell line. Collect the virus with a syringe, filter it into a centrifuge tube with a 0.45-micron filter, add an appropriate amount of fresh medium (about 12 ml in a 10 cm dish with a total volume), add polybrene and mix well, and it can be stored at 4 ° C or used immediately Infect. Another 24 hours later, the virus was collected in the same way as secondary infection.

[0060] After secondary infection, MEF cells are replaced with various induction media; commonly used is mESC medium or various compound molecules are added to mESC medium. After that, fresh medium needs to be replaced every day until the end of the experiment. If the efficiency of iPS is to be measured, OG2-MEF (GFP protein coupled to the endogenous Oct4 promoter) should be used. When iPS clones have...

Embodiment 3

[0063] Example 3: Effect experiment of Gadd45a on heterochromatin protein HP1a

[0064] When analyzing the effect of Gadd45a on the heterochromatin protein HP1a, the retrovirus of Gadd45a must be packaged first to infect MEFs (any MEFs are acceptable). After culturing for 3 days, wash with PBS once, and fix with 4% paraformaldehyde at room temperature for half an hour. After fixation, absorb paraformaldehyde and replace with PBS. Wash once with glycine solution, then wash three times with PBS, and then permeabilize and block with a mixed solution of Triton X100 and BSA for 1 hour. After washing 3 times with PBS, incubate the primary anti-HP1a antibody for at least 1 hour; wash 5 times with washing solution and incubate the fluorescent secondary antibody. Finally, the nuclei were stained with DAPI, washed once with PBS, sealed on the glass slide with mounting medium, and then observed and photographed with a laser confocal microscope.

[0065] figure 2 The results of exper...

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Abstract

The invention discloses a method for changing cell fate, a construct, a method for changing expression quantity and positioning of cell heterochromatin protein HP 1a in cell, a method for changing a structure of cell chromatin, and a method for changing the cell histone H3 methylation and cells thereof. The method for changing the cell fate comprises protein selected from one of following amino acid sequences by the cell overexpression: a)an amino acid sequence shown by at least one of SEQ ID NO: 1-3; b)a part of an amino acid sequence shown by at least one of SEQ ID NO: 1-3; and c)an amino acid sequence having at least 80% of homogeneity with the amino acid sequences shown in the a) or b). The method can be used for using cell overexpression Gadd45 family protein for effectively open the cell chromatin, so that the cell fate can be conveniently and effectively changed, and especially reprogramming of somatic cell can be promoted.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically, the present invention relates to a method for changing cell fate, a construct, a method for changing the expression level and location of heterochromatin protein HP1a in cells, and a method for changing the structure of cell chromatin A method, a method for altering histone H3 methylation in a cell, and a cell. Background technique [0002] With the gradual elucidation of the regulatory mechanism of the core transcription factors (Oct4, Sox2, Nanog) of embryonic stem cells, Japanese scientist Yamanaka successfully used four transcription factors Oct4, Sox2, Klf4 and c-Myc to transform mouse fibroblasts into induced multiple Potential stem cells (iPSCs) have pointed out a new direction for obtaining pluripotent stem cells from specific individuals. In just a few years, the iPSCs neighborhood has made rapid progress. Reprogramming of various somatic cells in various spe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12N5/10C12N15/63
CPCC07K14/00C12N15/63
Inventor 刘兴国裴端卿陈可实龙琪王涛
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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