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Method for changing cell fate and applications thereof

A cell fate and cell technology, applied in the biological field, achieves the effects of high efficiency, improved expression, and convenient and effective changes

Inactive Publication Date: 2015-07-15
刘兴国
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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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  • Method for changing cell fate and applications thereof
  • Method for changing cell fate and applications thereof
  • Method for changing cell fate and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: Cloning of Srebp-1 protein

[0047] Using the cDNA of mouse embryonic fibroblast (MEF) cells as a template, the Srebp-1 fragment was amplified using the primers shown in Table 1, 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 plasmid of Srebp-1.

[0048] Table 1 Primers for cloning Srep-1 protein fragments

[0049] Primer name

Embodiment 2

[0050] Example 2: Srebp-1 protein promotes reprogramming experiment

[0051] Monotropic (Plat-E) retrovirus packaging cell line is used to package the retroviruses of reprogramming factors Sox2, Klf4, Oct4, c-Myc and Srebp-1. The specific process is as follows: Plat-E cells are inoculated into culture It can be transfected in the dish for 12 hours or overnight, and the cell density can be observed before transfection, and it can be 70%-90%. When transfecting, first add DNA and OPTI-MEM, mix well, then add PEI, mix well, vigorously invert and mix well, then let stand for 13-15 minutes, then add the mixture to the Plat-E that has been changed cells, place the cells in the incubator. After 8-10 hours, the cells were replaced with MEF medium. 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 for a cell culture dish with a diameter of 10 cm), add polybrene (polybrene) and mix well, ...

Embodiment 3

[0054] Example 3: Experiment of the influence of Srebp-1 on the binding of c-Myc to the Oct4 promoter level

[0055] When analyzing the influence of Srebp-1 on c-Myc binding to the Oct4 promoter level, the retrovirus of Srebp-1 should first be packaged to infect mouse embryonic fibroblast (MEF) cells, wherein, it should be noted that, Any MEF can be a recipient cell for virus infection. MEF cells infected with empty vector were collected as control and MEF cells expressing Srebp-1 virus, fixed with formaldehyde at a final concentration of 1%, and terminated with glycine at a final concentration of 0.125M. After washing twice with PBS, resuspend the cells with an appropriate amount of PBS, and distribute 10 million cells per tube. Centrifuge to remove supernatant. Place the cell pellet on ice, add 1 ml of lysis buffer A, wherein the composition of lysis buffer A is as follows: 50mM HEPES-KOH pH7.3; 140mM NaCl; 1mM EDTA pH8.0; 10% glycerol; 0.5% NP -40; 0.25% Triton-100; prot...

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Abstract

The invention discloses method for changing the cell fate. In the provided method, the cells over-express the protein containing the following amino acid sequences: (a) an amino acid sequence represented by the SEQ ID No.1; (b) a part of the amino acid sequence represented by the SEQ ID No.1; (c) an amino acid sequence having at least 80% homology of the amino acid shown in (a) and (b), preferably the homology is at least 90%, more preferably the homology is at least 95%, and the most preferably the homology is at least 99%. Through the method the Srebp-1 family proteins can be over-expressed by the cells, thus the combination of c-Myc and multifunctional gene promoters such as Oct4, and the like is effectively promoted, and the expression of multifunctional gene is improved, so that the cell fate can be effectively changed, and especially the reprogramming of somatic cells is 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, and a construct and cells obtained by the method. Background technique [0002] With the deepening of stem cell research, scientists have discovered that terminally differentiated mammalian cells are also totipotent, and adult cells can be reprogrammed to change their gene expression profiles to change their fate, regain pluripotency or directly transform differentiation into another cell. There are two main methods for early somatic cell reprogramming: nuclear transfer and cell fusion, but these two methods still need to involve embryos, which are greatly restricted in ethics. In 2006, the Japanese Yamanaka laboratory obtained breakthrough research results in the field of reprogramming, and found that mouse fibroblasts can be reprogrammed into pluripotent stem cells by exogenously expressing four transc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12N5/10
Inventor 刘兴国邬毅陈可实刘西银
Owner 刘兴国
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