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Novel ips cell inducing method

A cell and inducer technology, applied in the field of cell engineering, can solve the problems of low reprogramming efficiency and low stemness of iPS stem cells

Pending Publication Date: 2019-06-21
ALLIFE MEDICAL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the inefficiency of reprogramming and the low stemness of iPS stem cells have always been a headache for scientists.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1, preparation of reprogramming factor carrier

[0055] Eight reprogramming factors were used in reprogramming: Oct3 / 4, Klf4, Sox2, P53, L-Myc, Lin28, RARg, Lrh-1, and the expression vector was episomal vector.

[0056] The expression vector is an episomal vector, specifically pCXWB, purchased from addgene.

[0057]Reprogramming factor vector A is a vector obtained by inserting fragment A between the pCXWB vector ECoRI restriction sites, expressing Oct3 / 4, Klf4, Sox2, P53, L-Myc, Lin28 genes;

[0058] The nucleotide sequence of fragment A consists of Oct3 / 4 gene sequence (sequence 1), T2A nucleotide sequence (sequence 9), Klf4 gene sequence (sequence 2), P2A nucleotide sequence (sequence 10), Sox2 gene sequence (SEQ ID NO: 3), T2A nucleotide sequence (SEQ ID NO: 9), P53 gene sequence (SEQ ID NO: 6), IRES sequence (SEQ ID NO: 12), L-Myc gene sequence (SEQ ID NO: 4), E2A nucleotide sequence (SEQ ID NO: 11) and Lin28 gene sequence (sequence 5), and each sequen...

Embodiment 2

[0062] Example 2, reprogramming of iPS pluripotent stem cells

[0063] 1. Reprogramming of iPS pluripotent stem cells

[0064] 1. Culture human skin fibroblasts (purchased from promocell, article number: C-12302);

[0065] 2. Transfer the reprogramming factor carrier A, reprogramming factor carrier B and EBNA1 (purchased from addgene, Plasmid#41857) obtained in Example 1 into isolated human skin fibroblasts or peripheral blood at a mass ratio of 1:1:1 In monocytes, cells expressing reprogramming factors are obtained; details are as follows:

[0066] a. The fibroblasts obtained in the above step 1 cover about 90% of the bottom of the T25 flask;

[0067] b. The day before the experiment, spread Matrigel on a 6-well plate. Before electroporation, 2 mL of fibroblast culture medium was added to each well in advance.

[0068] c. Digest the cells with Trypsin-EDTA (0.05%), neutralize, centrifuge, and suck up the supernatant. Select a 100uL system electroporation kit and resuspend...

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Abstract

The invention discloses a novel ips cell inducing method and provides application of inducing genes Oct3 / 4, Klf4, Sox2, P53, L-Myc, Lin28, RARg and Lrh-1 in inducing isolated mammal somatic cells to generate iPS multipotent stem cells. By an iPS cell establishing method, a method for inducing human iPS stem cells free of exogenous serum, trophoblast cells and animal source is established on the basis of no insertion. The method shortens reprogramming time of the iPS multipotent stem cells and improves cloning generation efficiency, and obtained iPS cells are higher in dryness.

Description

technical field [0001] The invention relates to the field of cell engineering, in particular to a new method for inducing ips cells. Background technique [0002] In 2006, Shinya Yamanaka's team invented a "cocktail" method composed of four transcription factors, OCT4, SOX2, KLF4 and c-Myc, which can successfully reprogram terminally differentiated skin fibroblasts into differentiated pluripotent cells stem cells, which are called induced pluripotent stem cells (induced pluripotent cells). These stem cells have similar differentiation potential to embryonic stem cells, and can form the three most basic germ layers of human development, and eventually form a variety of adult cells. This breakthrough invention breaks through the ethical limitations of using human embryonic stem cells in medicine, and using this technology can obtain patient-specific iPSCs, which can solve the problem of immune rejection in cell transplantation therapy, and greatly expand the clinical applicat...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/867C12N5/10
Inventor 顾雨春吴理达
Owner ALLIFE MEDICAL SCI & TECH CO LTD
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