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Method for improving inductive generation efficiency of induced pluripotent stem cells

A technology for pluripotent stem cells and stem cells, which is applied in the field of improving the efficiency of inducing and generating pluripotent stem cells, can solve problems such as low induction efficiency, and achieve the effects of improving efficiency, improving quality and high induction efficiency.

Inactive Publication Date: 2013-06-26
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

[0013] However, the combination of transcription factors that can be reprogrammed currently includes Oct4, Klf4, Sox2, c-Myc; Oct4, Nanog, Lin28, Sox2; Sox2, Klf4 and Lrh1; Oct4, bmi1 and other combinations and esrrb, tbx3 and so on Reprogramming-related genes, the combination of transcription factors required by the existing reprogramming method needs to import up to three or four transcription factors, and the induction efficiency is low, how to reduce transcription factors and maintain a high reprogramming efficiency is crucial for reducing The accumulation of reprogramming cell mutations is of great significance to improve the operability of reprogramming technology

Method used

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  • Method for improving inductive generation efficiency of induced pluripotent stem cells
  • Method for improving inductive generation efficiency of induced pluripotent stem cells
  • Method for improving inductive generation efficiency of induced pluripotent stem cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] 1. Construction of vectors comprising Jhdm1a and Jhdm1b coding regions:

[0089] a. Cloning primer design

[0090] Sequence information for cDNA of Jhdm1a and Jhdm1b was obtained from http: / / www.ncbi.nlm.nih.gov / pubmed. The sequence of the Jhdm1a cDNA cloning region is SEQ ID NO: 1, the sequence of the Jhdm1b cDNA cloning region is SEQ ID NO: 2, and the coding sequences of Jhdm1a and Jhdm1b are amplified by designing specific primers.

[0091] The nucleotide sequence of the upstream primer of Jhdm1a is shown in SEQ ID NO: 3;

[0092] The nucleotide sequence of the downstream primer of Jhdm1a is shown in SEQ ID NO: 4;

[0093] The nucleotide sequence of the upstream primer of Jhdm1b is shown in SEQ ID NO: 5;

[0094] The nucleotide sequence of the downstream primer of Jhdm1b is shown in SEQ ID NO:6.

[0095] b. Amplification of coding sequences by RT-PCR

[0096] Total mRNA was extracted from isolated ICR mouse embryonic fibroblasts (MEFs) and human H1 embryonic ste...

Embodiment 1

[0130] Identification of induced pluripotent stem cells obtained in Example 1:

[0131] like image 3 as well as Figure 6 to Figure 10 As shown, a series of identification experiments were performed on Oct4 and Jhdm1b-induced pluripotent stem cell clones to prove whether they were iPS cells (induced pluripotent stem cells). Identification experiments included: quantitative PCR, immunofluorescence analysis of their surface markers, priming Sub-methylation level analysis, karyotype identification, chimera formation, etc.

[0132] Quantitative PCR experiment:

[0133] All quantitative PCR experiments were completed on the CFX-96 quantitative PCR instrument of Biorad Company using Takara’s kit, the reaction conditions used were 95°C for 2 minutes; 95°C for 10 seconds, 60°C for 30 seconds, read the fluorescence value, and repeat 40 loops.

[0134] Analysis of the methylation status of the promoter region

[0135] The analysis was determined by the sodium bisulfite sequencing ...

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Abstract

The invention relates to a method for improving the inductive generation efficiency of induced pluripotent stem cells, and especially relates to a method for improving the inductive generation efficiency of the induced pluripotent stem cells through using modified histone gene jhdm1a. Jhdm1a and a stem cell induction factor are utilized to improve the induction efficiency and the induction quality of the induced pluripotent stem cells. The stem cell induction factor can be an Oct4 and Klf4 combination, or an Sox2, Oct4 and Klf4 combination, or an Oct4 and Sox2 combination, or individual Oct4. The method also comprises a step that the cells are exposed to vitamin C, so the induction efficiency of the induced pluripotent stem cells is higher than that of methods which do not use vitamin C. The method uses less stem cell induction factor, so the latent carcinogenicity is reduced, a high induction efficiency is obtained, and the induced pluripotent stem cells having a reproduction system transmission capability and a high quality can also be obtained.

Description

[0001] The present invention is a divisional application of a Chinese invention patent application with the patent application number 201110323779.7 and the filing date of October 21, 2011, entitled "A Method for Improving the Efficiency of Inducing Pluripotent Stem Cells". technical field [0002] The invention relates to a method for improving the efficiency of inducing and generating pluripotent stem cells. In particular, it relates to a method for improving the efficiency of inducing pluripotent stem cells by modifying histone gene jhdm1a. Background technique [0003] my country is a country with a large population in the world. Every year, organ defects, failures, and dysfunctions caused by trauma, disease, aging, and genetics also rank first in the world. Therefore, the research on stem cells and regenerative medicine has attracted the general attention of quite a few scientific research institutes and all walks of life. [0004] Cell transplantation therapy is an imp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10
Inventor 裴端卿王涛
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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